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.[158] 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,[159] 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.[160]
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.
Of _Astragalus mollissimus_ from Imperial, Nebr., 200 grams were ashed
in a platinum bowl and the ash treated with acetic acid water. After
freeing from acid, one half of the solution and emulsion was fed one day
and the second half fed the following day. The rabbit at the time of
feeding weighed 1,275.7 grams. Fourteen days later the animal died,
weighing 1,105.6 grams. No autopsy.
A similar extract of the ash from between 100 and 150 grams of the same
dried plant produced death in a rabbit weighing 1,190 grams in two hours
and fifty-eight minutes.
The acetic acid extract of the ash of 125 grams of a mixture of the
dried _Astragalus mollissimus_ and _Aragallus lamberti_ received from
Hugo, Colo., June, 1907, after freeing from acid, was fed to a rabbit
weighing 1,304 grams on July 29. On July 30 it weighed 1,332.4 grams.
August 1 it weighed 1,219 grams, and it died the same day. The stomach
was reddened and showed ulcers.
A similar extract from 250 grams of the same dried plants on boiling
gave a heavy precipitate, but this precipitate was inactive, while the
filtrate killed a rabbit in four hours.
Of dry _Aragallus lamberti_ collected in September, 1906, 200 grams were
extracted with water and fed to a rabbit weighing 1,516.7 grams. Two
days later the animal weighed 1,360 grams and died the same day.
The ash from 200 grams of the same dried plant was extracted with acetic
acid, and after evaporating off the acid this was fed to a rabbit
weighing 2,045.3 grams. Seven days later the animal weighed 1,729.3
grams, having lost 316 grams in weight.
The ash from 250 grams of the same species of plant, after similar
treatment with acetic acid, induced death in a rabbit weighing 2,069
grams in 2 hours and 20 minutes. The stomach was inflamed.
* * * * *
FOOTNOTES:
[158] Work is now being done by the writer on the inorganic
constituents of various plants.
[159] Scattered throughout the veterinary literature one
finds cases of poisoning in animals with symptoms similar to
those occurring in locoed animals which are attributed to
eating plants grown on a peculiar soil, as in Oserow, Ueber
Krankh. d. Pferde, welche Aehnlichkeit mit der Cerebro-spinal
meningitis haben, aber durch Vergiftungen mit Gräsern von
Salzgründen (Salzmooren) verursacht werden, Journ. f. Allgem.
Veterinär-Medicin, St. Petersburg, p. 486, 1906. Abstract in
Jahresber. über d. Leistungen auf dem Gebiete d.
Veterinär-Medicin, vol. 26, p. 226, 1906.--Compare also Étude
sur Quelques Plantes Vénéneuses des Regions Calcaires, Bul.
Soc. Cent. de Méd. Vét., vol. 48, p. 378. 1894.
[160] After completing this work the writer found that Sayre
had said that he "had the suggestion that the harm coming
from this plant is due to the inorganic constituents; this
clue has been followed up, but like the others has brought us
no nearer to the solution of the problem." Kans. Acad. Sci.
Trans., vol. 18, p. 144. 1903.
=EFFECT OF THE AQUEOUS EXTRACT OF ASHED LOCO PLANTS.=
The filtrate from the ash from 200 grams of dried _Astragalus
mollissimus_, from Imperial, Nebr., after similar treatment with acetic
acid water and freed from free acid, killed a rabbit in several hours.
Hydrochloric acid also rendered the toxic agent of the ash soluble in
water, but proved unsuitable for our work, as it was found impossible to
obtain neutral residues by mere evaporation on the bath. At first one of
the heavy metals or members of the H_{2}S group[161] was suspected, but
on passing H_{2}S into the slightly acid extract of the ash no active
precipitate resulted, but the filtrate remained active.[162] A special
Marsh test was, however, made for arsenic and antimony with negative
results. A test for tungsten with zinc and hydrochloric acid proved
negative.
Members of the ammonium sulphid group were then suspected, but while
ammonium hydrate alone gave a heavy white precipitate, this precipitate,
as also the black one with ammonium sulphid, proved inactive save when
not thoroughly freed from acid (used for solution). The action of this
ammonium sulphid precipitate on rabbits was watched for sixteen days,
but without result. Nevertheless, the writer still suspected some of the
rare earths.[163]
Sestini[164] had found that if certain plants were nourished with a
solution of a beryllium salt, in the ash of these plants could be shown
the presence of beryllium.
Two grams of beryllium chlorid were fed in aqueous solution to a rabbit
weighing 1,800.2 grams. In four days this animal lost 241 grams and
died. The stomach showed the same general pallor seen in chronic locoed
rabbits, but no ulcers. The tests for beryllium by Sestini's method,
however, failed to show beryllium in the active loco plants examined.
Thorium chlorid, cerium chlorid, and lanthanum chlorid in 2-gram doses
and zirconium chlorid in 3-gram doses produced no chronic symptoms in
rabbits or, in fact, any disturbance. Titanium chlorid, 2.5 grams,
evaporated in the air and then fed in an emulsion to a rabbit, also
proved inactive, but this inactivity may have been due to its
insolubility.
Thallium nitrate c. p., in aqueous solution, in 2-gram doses, killed a
rabbit weighing 2,154.6 grams in two hours and fifteen minutes. The
stomach in this case, while pink, was not hemorrhagic.
Zirconium chlorid has an astringent taste, and if fed repeatedly will
cause the metallic astringent action. On boiling an acetic acid solution
of the ash with sodium acetate a precipitate formed.[165]
The presence of zirconium was thus suspected and Dr. E. C. Sullivan, of
the United States Geological Survey, estimated it to be present in the
ash of a sample of _Aragallus lamberti_ in about 0.01 per cent zirconium
oxid, with also 0.1 per cent titanium dioxid.[166]
Zirconium chlorid, 3 grams, was fed in aqueous solution to a rabbit
weighing 850.5 grams. This rabbit lost 96 grams in seven days, and was
then fed 3 grams more of the same solution and the following day 2 grams
more. It died eight days later, weighing 656 grams. The stomach and
intestines were contracted, but showed no ulcers. However, 4 grams
killed a rabbit in two hours and thirty-two minutes.
The filtrate, after treating an active solution of the ash with hydrogen
peroxid, proved active, thus showing that zirconium was not entirely
responsible for the poisonous action.
Yttrium, while not found in the plant, was administered as yttrium
chlorid to a rabbit weighing 1,530 grams in 2-gram doses in solution.
This animal gained 113.4 grams in five days.
Didymium chlorid c. p., in 3-gram doses, was fed to a rabbit weighing
1,020 grams. This rabbit lost 70 grams in four days.
The administration of manganese acetate[167] in 2-gram doses was
followed by a gain in weight of a rabbit of 42.5 grams, while a dose of
3 grams killed a rabbit weighing 1,077 grams in two hours and thirty
minutes. Wohlwill[168] has emphasized the fact that the members of the
iron group owe their comparative harmlessness to not being absorbed by
the gastro-intestinal tract.
No zinc was found in the plant.[169]
It is well recognized that potassium salts given hypodermically are
decidedly toxic and that ammonium salts given per os will kill, so that
the writer considered the possibility of other members of the group
being responsible for the injurious action. The fact that the alkaline
distillate of the plant proved inactive eliminated the ammonium salts.
Cæsium chlorid c. p., 2 grams, was fed in aqueous solution to a rabbit
weighing 1,077.2 grams. In six days this animal lost 255 grams in
weight, when it died.[170]
A second rabbit, weighing 1,020.5 grams, was fed with 2 grams of the
same solution and lost 368 grams in twenty-one days. The spectroscopic
test, however, failed to show cæsium in the ashed plant. Rubidium
chlorid c. p., in 2-gram doses, proved inactive. The platinum chlorid
precipitate from the extract of the plant proved inactive.
The fact that the filtrate after precipitation of the phosphates by tin
and nitric acid and H_{2}S was active excluded the phosphoric acid
radical, and the filtrate after treatment with BaCO_{3} and AgO being
active excluded the H_{2}SO_{4} and HCl radicals as the toxic body.
Fluorine was proved to be absent.
A radio-active substance was suspected, but Dr. L. J. Briggs, Physicist
of Bureau of Plant Industry, reported that the dried plant showed no
special amount of radio-activity.[171]
Power and Cambier, Sayre, and Kennedy had previously called attention to
the abundance of calcium in the plant, and the writer's investigations
confirm this. Pharmacologists are averse to believing calcium given per
os poisonous. The writer has, however, fed 5 grams of the acetate of
calcium in solution to a rabbit weighing 652 grams. This animal died in
two hours, with marked irritation of the stomach, the result being due
to the so-called "salt action." Much larger amounts were fed in divided
doses, but without injury. Calcium phosphate and calcium sulphate in
2-gram doses proved harmless to a rabbit weighing about 1,400 grams.
Three grams of magnesium acetate[172] were fed in solution for five
successive days to a rabbit weighing 1,417 grams, but without apparent
effect.
Strontium acetate c. p., in 2-gram doses, likewise caused no
disturbance.[173] No strontium in any amount recognizable by chemical
tests was proved in the plant. So that by a process of exclusion the
writer was forced to think of barium as the main cause of the trouble.
The writer noted that if the ashed plant was extracted with H_{2}SO_{4}
water and this extract freed from sulphuric acid with PbCO_{3} and
H_{2}S the solution proved inactive to rabbits and also that after this
extraction the acetic acid extract of the ash failed to kill. In other
words, the sulphate of our body was insoluble in water. At times in
passing H_{2}S into active solutions of the ashed plant freed from the
acetic acid by evaporation the filtrate and likewise the precipitate
were inactive. Noyes and Bray[174] have noted that if H_{2}S is passed
into certain solutions in the presence of an oxydizing agent, such as
ferric iron, H_{2}SO_{4} would be formed, which would throw any barium
out of solution.
In one blood-pressure record made with a dog (vagi nerves cut), a rise
in blood pressure (a characteristic physiological action of barium) was
seen to follow the intravenous injection of the aqueous extract of the
plant, in spite of its normal acid reaction.
Accidentally the writer found that Sprengel[175] had reported the
presence of barium in _Astragalus exscapus_, a closely allied plant.
Barium has also been found in the vegetable world by Scheele in 1788,
and later by Eckard,[176] who found it in beech, while Forchhammer[177]
proved it in birch, and Lutterkorth found it in the soil of the same
area in which Eckard worked. Dworzak[178] noted the occurrence of traces
of this element in wheat grown along the Nile, and Knop[179] found it in
the soil. Doctor Balfour, of Khartum, Egypt, informed the writer that he
knew of no cases in which this barium in wheat had produced poisoning.
Hornberger[180] found barium both in the red beech grown in Germany and
in the soil on which these trees grew. It has also been claimed that
various marine plants may take up barium from the sea.[181]
Hillebrand[182] has called attention to the fact that the igneous rocks
of the Rocky Mountains showed a higher percentage of barium than rock
from other portions of the United States, so that under these conditions
one might expect the presence of barium in plants growing in this
region. A sample of _Aragallus lamberti_ and one of _Astragalus
mollissimus_ were sent to the Bureau of Chemistry for spectroscopic
examination for various elements and they reported traces of barium in
each.[183]
With these arguments the writer felt sure of the presence of barium, and
the matter was discussed with Dr. E. C. Sullivan, of the United States
Geological Survey, and he kindly corroborated the conclusions reached as
to the presence of barium, controlling its presence by means of the
spectroscope, and estimated it roughly as 0.1 per cent BaO in the ash of
a sample of _Aragallus lamberti_ (6.3 milligrams BaSO_{4} in 4 grams
ash). This determination was made by Hillebrand's method.
Kobert has anticipated this result, saying that "all plants are in the
position occasionally to take up barium combinations from the soil," and
"the plants which thus contain barium may act injuriously to men and
animals."[184]
* * * * *
FOOTNOTES:
[161] Swain, R. E., and Harkins, W. D. Arsenic in Vegetation
Exposed to Smelter Smoke. Journ. Amer. Chem. Soc., vol. 30,
p. 915. 1908.--Harkins, W. D., and Swain, R. E. The Chronic
Arsenical Poisoning of Herbivorous Animals. Journ. Amer.
Chem. Soc., vol. 30, p. 928, 1908.
[162] A similar extract was sent to the Bureau of Chemistry,
and that Bureau also reported an absence of the elements of
the H_{2}S group.
[163] Bachem, C. Pharmakologisches über einige Edelerden.
Arch. Internat. de Pharmacodyn., vol. 17, p. 363. 1907.
[164] Sestini, F. Esper. di Vegetaz. del Frumento con
Sostituz. della Glucina alla Magnesia. Staz. Sper. Agrar.
Ital., vol. 20, p. 256. 1891.--Di alcuni Elementi Chimici
Rari a Trovarsi nei Vegetabili. Staz. Sper. Agrar. Ital.,
vol. 15, p. 290. 1888.
NOTE.--The ammonium sulphid precipitate was very small if the
phosphates were first removed with tin and nitric acid.
[165] Böhm, C. R. Darstellung d. seltenen Erden, vol. 1, p.
40. 1905.
[166] Wait, C. E. Occurrence of Titanium. Journ. Amer. Chem.
Soc., vol. 18, p. 402. 1896.
NOTE.--There seem to be no records of any study of the
pharmacological action of titanium.
[167] Compare Jaksch, R. v. Ueber Mangantoxikosen und
Manganophobie. Münch. Med. Woch., p. 969. 1907.
[168] Wohlwill, F. Ueber d. Wirkung d. Metalle d.
Nickelgruppe. Arch. f. Exper. Path., vol. 56, p. 409. 1907.
[169] Laband, L. Zur Verbreitung des Zinkes im
Pflanzenreiche. Zeits. f. Untersuch. d. Nahrungs u.
Genussmittel, vol. 4, p. 489. 1901.
[170] Cæsium occurs in various plants and the possibility of
poisoning by this element must be considered. It is hoped
that the writer may be able to undertake a more thorough
pharmacological study of this element.
[171] Acqua, C. Sull'accumulo di Sostanze Radioattive nei
Vegetali. Atti della Reale Accad. dei Lincei, 5 s, vol. 16,
sem. 2, p. 357. 1907.
[172] Compare Meltzer, S. J. Toxicity of Magnesium Nitrate
When Given by Mouth. Science, vol. 26, p. 473. 1907.
[173] Burgassi, G. Modificaz. del Ricambio per Azione dello
Stronzio. Archiv. di Farmacol., vol. 6, p. 551. 1907.
[174] Noyes, A. A., and Bray, W. C. System of Qualitative
Analysis for the Common Elements. Journ. Amer. Chem. Soc.,
vol. 29, pp. 168, 172, and 191. 1907.
NOTE.--Barium sulphate is nontoxic on account of its
insolubility. Orfila fed 16-24 grams to dogs without causing
any disturbance. Bary, A. Beitr. z. Baryumwirkung. Dorpat,
1888, p. 25.
[175] Sprengel, C. Von den Substanzen der Ackerbrume und des
Untergrundes, Journ. f. Techn. u. OEkon. Chem., vol. 3, p.
313. 1828.
[176] Eckard, G. E. Baryt, ein Bestandtheil der Asche des
Buchenholzes. Annal. der Chem. u. Pharm., n. s., vol. 23, p.
294. 1856.
[177] Forchhammer, J. G. Ueber den Einfluss des Kochsalzes
auf die Bildung der Mineralien. Annal. d. Physik u. Chemie,
vol. 5, p. 91. 1905.--Lutterkorth, H. Kohlensäurer Baryt, ein
Bestandtheil des Sandsteines in der Gegend von Göttingen.
Annal. d. Chem. u. Pharm., n. s., vol. 23, p. 296. 1856.
[178] Dworzak, H. Baryt unter den Aschenbestandtheilen des.
Ægyptischen Weizen. Landw. Versuchs.-Stat., vol. 17, p. 398.
1874.
[179] Knop, W. Analysen von Nilabsatz. Landw.
Versuchs.-Stat., vol. 17, p. 65. 1874.--Compare also
Demoussy, E., Absorption par les Plantes de Quelques Sels
Solubles, Thése, Paris, 1899.--Knop, W., Einige neue
Resultate der Untersuchung über die Ernährung der Pflanze,
Ber. ü. Verhandl. d. königl. sächs. Gesells. d. Wissens. zu
Leipzig, Math. Phys. Cl., vol. 29, p. 113, 1877.--Suzuki, U.,
Can Strontium and Barium Replace Calcium in Phænogams? Bul.
Coll. Agric. Tokio Imp. Univ., vol. 4, p. 69, 1900-1902.
[180] Hornberger, R. Ueber d. Vorkommen d. Baryums in d.
Pflanze und im Boden. Landw. Versuchs.-Stat., vol. 51, p.
473. 1899.
[181] Roscoe, H. E., and Schorlemmer, C. Treatise on
Chemistry, vol. 2, p. 455. 1897.
[182] Hillebrand, W. F. Analysis of Silicate and Carbonate
Rocks. Dept. Interior, U. S. Geol. Survey, Bul. 305, p. 18.
1907.
[183] This report came from the Plant Analysis Laboratory of
the Bureau of Chemistry, a different one from that which
later controlled the writer's tests quantitatively and
qualitatively. In other words, the conclusions of the writer
as to the presence of barium were controlled by three
separate individuals.
[184] Kobert, R. Kann ein in einem Pflanzenpulver gefundener
abnorm höher Barytgehalt erklärt werden durch direkte
Aufnahme von Baryumsalze durch die lebende Pflanze aus dem
Boden? Chem. Zeit., vol. 10, p. 491. 1899.
NOTE.--The writer has also found barium in entirely different
botanical families from the loco-weed, and it is hoped a
report can shortly be made of some of these.
NOTE.--The first sample of ash analyzed by the Bureau of
Chemistry had 0.21 per cent Fe_{2}O_{3}, 0.92 per cent
Al_{2}O_{3}, 0.98 per cent CaO, 0.37 per cent MgO, 5.50 per
cent SiO_{2}. The second lot was only examined for certain
constituents, and gave K_{2}O, 2.25 per cent; CaO, 1.20 per
cent; MgO, 0.41 per cent; P_{2}O_{5}, 0.52 per cent; and
SO_{3}, 0.24 per cent.
=TOTAL ASH DETERMINATIONS OF LOCO PLANTS.=
The reports of the ash analyses of the loco plants show marked
variations in the total amount of the ash. Thus, from _Aragallus
lamberti_ Dyrenforth obtained 4.32 per cent and O'Brine 13.52 per cent
of ash. The Bureau of Chemistry analyzed two different samples of this
dried plant and reported in one case 11.15 per cent and in the second
11.64 per cent of ash. O'Brine[185] obtained 13.52 per cent of ash from
the same species. The writer's analysis[186] gave in one sample of
_Aragallus lamberti_, collected at Hugo, Colo., in 1907, 18.8 per cent
of ash; a second lot (1907), 12.44 per cent; a third (1906), 11 per
cent, and a fourth (May, 1905) gave 37.3 per cent of ash.[187] One lot
from Woodland Park, Colo. (October, 1906), gave 6.4 per cent. One lot
from Hugo, Colo. (October, 1907), yielded 9.6 per cent.