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TEXI; AGRIIEIEIILTURAI. EXPERIMENT STATIUN

A. B. OONNER, DIRECTOR
COLLEGE STATION, BRAZOS COUNTY, TEXAS

BULLETIN NO. 537 NOVEMBER, 1936

DIVISION OF CHEMISTRY

LOCOINE, THE POISONOUS
PRINCIPLE 0F LOCO WEED

Astragalus Earlei

  

E "I B R A R Y
Agricultural & -‘~(i.'2,_f»1a11‘=3a§ ‘Q-Jlfiege {of Texas
Cnflege Staiiun, Texas.

AGRICULTURAL AND MECHANICAL COLLEGE OF TEXAS
T. O. WALTON, President

This bulletin contains an account 0f the separation and
isolation of the toxic principle of the loco weed Astragalus
earlei. The toxic principle has been given the name locoine.
It has been isolated by a long series of chemical separations,
each separation being tested by feeding to cats. It is a strong
base, very soluble in water and alcohol, but only slightly soluble
in ether, petroleum ether, and chloroform. It also forms an
acetyl compound. It contains about 8.8% nitrogen and forms
salts with acids such as hydrochloric, tartaric, and oxalic acids.
It is fairly stable toward reagents and is not afiected by boiling
with dilute acids or alkalies. The most concentrated of our
preparations required a feeding of only 1 gram over a. period
of fifty days to loco a cat. The toxic base gives reactions with
many of the reagents used to test for alkaloids.

CONTENTS

Previous Work

Manifestations of the loco disease
Care of the cats used in testing the loco extracts _________________________________ _-

Isolation of the toxic principle

Progress of concentration of the locoine
Preparation of the toxic base, locoine
Acetyl-locoine

Preparation of the tartrate, citrate and other salts _____________________________ -_

Some properties of the locoine

Summary

5
6
7
7
11
13
14
14
16
17

BULLETIN NO. 537 NOVEMBER, 1936

LOCOINE, THE POISONOUS PRINCIPLE OF LOCO WEED,
ASTRAGALUS EARLEI

By G. S. Fraps, Chief, and E. C. Carlyle, Assistant Chemist,
Division of Chemistry.

This bulletin contains an account of the isolation of the toxic principle
_of the loco weed, Astragalus earlei.

The isolation was made by various chemical methods of fractionating
extracts of the plant. The different fractions were fed to cats to ascer-
tain whether or not they contained the loco poison. Further separations
were made and these tested. The preparations became stronger and
stronger until the concentrated extract was secured.

Cats were found by Couch (3) to respond to the loco poison, giving
symptoms very like those shown by locoed livestock. Goldfish, rats,
and chicks were also tried in the work here reported but these animals
did not seem susceptible to the poison.

Some work was done with Astragalus wootoni. Since fractions from
this plant behaved similarly to the same fractions from Astragalus earlei,
no further work was done with the weed, attention being confined to
Astragalus earlei.

Previous Work

Crawford (4) found barium in loco plants gathered in the vicinity
of Hugo, Colorado. He fed barium acetate to rabbits and reported
symptoms of poisoning similar to loco poisoning. His report contains
many references to work done by others in an effort to find an alkaloid
or some other organic poison that could be traced back to the loco weed.
None of these efforts appeared to be successful. Marsh (5) found a
close analogy between symptoms of barium poisoning and those result-
ing from feeding extracts of certain of the loco plants. He also found
that some loco plants which contained no barium were inactive. In a
later publication Marsh (6) reported that feeding barium to animals
produced an acute poisoning but the symptoms were not like those of
the loco weed disease. Some of his animals received as much as 50
grams per day of barium chloride. The symptoms and pathology of
barium poisoning differed in a marked degree from those of loco poison-
ing. Alsberg and Black (1) also concluded that the loco poison is not
barium. Many plants in the western states which contained as much
barium as loco weeds were not harmful to livestock. Barium was found
Lin plants from Virginia, where loco weed disease is unknown, and these
plants were also harmless. The investigators found enough salts of
(potassium, calcium, and other basic elements in the extracts from 250
;pgrams of dried plant, the usual toxic dose for rabbits, to account for
lritheir death. Marshall (7) concluded that there are several diseases of

6 BULLETIN NO. 537, TEXAS AGRICULTURAL EXPERIMENT STATION

western livestock masquerading under the name of the loco weed disease.
One hundred per cent of the locoed sheep he studied were not suffering
from locoism but from under-feeding combined with a parasitic infection.
He considered the existence of a pure bona fide loco poison to be doubtful
and he considered it certain that the heavy losses attributed to loco weed
disease were in a large measure due to other causes which could usually
be ascertained by careful study.

Mathews (8) found both Astragalus earlei and Astragalus wootoni
to be about equally toxic to livestock. He found that green loco weed
weighing about 90 per cent of the weight of cattle was required to pro-
duce the first visible symptoms, while 320 per cent of the weight of the
cattle produced death. When concentrates were fed with the loco weeds,
a smaller amount of the plant was required to produce the same results.
Feeding 30 per cent of the weight of horses produced death. He gives
descriptions of the loco plants. '

Couch (3) prepared from the loco weed Oxytropis lambertii gathered
in the vicinity of Raton, New Mexico, a highly toxic fraction in the fil-
trate from silicotungstic acid. He was unable to purify it further or to
crystallize it. He held that there is one specific substance in Oxytropis
lambertii capable of producing the loco weed disease and that the chen1i-
cal studies of this substance indicate it to be a stable, non-basic,
nitrogenous, and highly hydroxylated organic substance, not a glucoside,
acid, ester, or ether, and free from barium compounds.

Diseases ascribed to the loco plant in some localities appear really to
be due to selenium (2). The plants referred to are varieties of Astra-
galus different from those here discussed.

Manifestations of the Loco Disease

According to Mathews (8) the symptoms of the loco disease manifest
themselves-mostly as a nervous disturbance, such as rearing and shying
in horses and plunging when they are startled. In cattle there is a
marked trembling of the head, disturbed vision, and a tendency to
fright. In the last stages emaciation is'common. In sheep, the most
prominent symptoms are cerebral depression and lassitude; less fre-
quently there is a shaking of the head. In goats, paralysis of the hind
legs is the most prominent symptom. Abortion of pregnant cattle
frequently follows the consumption of loco Weed, although the fertility
of the cattle is not affected.

In cats, the symptoms vary somewhat with different animals. The
head usually shakes up and down, the vision seems distorted in advanced
cases, and the animal seems to be unable to judge the distance to its
food, frequently beginning to lap its milk when two or three inches from
the dish. The eyes frequently assume a staring expression, and there is
often weakness or paralysis in the hind legs, the left leg seeming to be

LOCOINE, THE POISONOUS PRINCIPLE OF LOCO WEEI) 7

affected more than the right. Some of the cats in the advanced stages
of the disease became very rigid when\picked up; others fed on the
same extract remained limp. The usual symptoms, however, were the
shaking head and staring eyes.

Care of the Oats Used for Testing the Loco Extracts

As already stated, the poisonous preparations were tested by feeding
them to cats until it was ascertained whether or not they would loco
the cats. The cats used in the loco work are kept in cages about
3 x 3 x 2 feet. In the morning they are fed milk made from powdered
whole milk. In the afternoon they are fed suitable canned dog food
five times a week and salmon twice a week. The loco extract was usually
mixed with the milk although sometimes it was added to the other food.

Figure 1. Locoed cat and healthy cat.

 Water was provided all the time. Occasionally the cats were let out
for exercise. The cages and pens were kept thoroughly clean. The cats
first secured were almost all infected with hook worm or tape worm
or suffered from enteritis, and had to be treated to remove the parasites
a or inoculated with enteritis serum, and brought to a state of good health
 before they could be used. The cats later secured did not seem to be as
1;; unhealthy as those secured at first.

5.

Locoedand healthy cats are shown in Fig. 1.
Isolation of the Toxic Principle

 The loco weed was at first extracted with boiling water. Considerable
iquantities of materials other than the toxic substance were dissolved.
;After the poisonous principle was found to be very soluble in alcohol,
,the plant was extracted with cold 95 per cent alcohol. The alcohol

    

8 BULLETIN NO. 537, TEXAS AGRICULTURAL EXPERIMENT STATION

extracted more of the toxic principle than it did of other materials, so
that smaller amounts of reagents were required and much smaller non-
toxic precipitates were encountered.

The process of separating and concentrating the locoine was long and
tedious. Various reagents and different methods were tried for the
purpose of separating the extracts into two or more preparations. After
each separation had been made, it was necessary to test the preparations
by feeding them to cats, in order to ascertain Whether the procedure
separated the poisonous compound from the other substances, and which
of the preparations contained it. As the test with cats required from
two to four months, this procedure was time-consuming.

By trying various procedures, incorporating those which were success-
ful into the method of isolation, and eliminating those which gave little

progress, it was possible to devise a method which gives a highly con-

centrated substance, not yet entirely pure, which we term locoine.

It is not necessary to give a full description of the many methods
which were tested and the tests which were made in arriving at the final
method. Some of them will be given in outline.

The aqueous extract was precipitated with basic lead acetate. After
the lead had been removed, the precipitate was found to be non-toxic
to cats and the filtrate was toxic.

Ether, chloroform, fu1ler’s earth, and carbon black did not remove
the toxic substance.

The aqueous extract was treated with phosphotungstic acid. After the
phosphotungstic acid precipitate had been decomposed by barium hydrox-
ide and the barium removed, the precipitated material and the filtrate
were tested by means of cats. The precipitated material was toxic; the

filtrate was not.

A concentrated solution of material precipitated by phosphotungstic
acid was treated with alcohol; the precipitate was not toxic, the filtrate

was toxic to cats.

The partly purified material was precipitated with silver nitrate; the
precipitate did not contain the toxic substance. Mercurous nitrate failed
to precipitate the toxic substance.

The insoluble substance produced by reaction with benzoyl chloride
was not toxic; the filtrate was toxic. Alcoholic mercuric chloride did
not precipitate the toxic compound, nor did tannic acid.

Table 1 gives an outline of some of the procedures followed.

LOCOIN-E, THE POISONOUS PRINCIPLE OF LOCO WEED

TABLE 1. Loco‘ extracts from Astragalus earlei
Labora- Serial
tory Description number of Effect
Number preparation on cats
32597 Water extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Toxic
32598 Filtrate from treatment of water extract No. 1 with
lead acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Toxic
32720 Alcohol-soluble preparation from No. 2 shaken with
ether, chloroform, fullers earth, and carbon black,
evaporated to a paste and taken up with alcohol. . . . Toxic
32721 Alcohol-insoluble residue from No. 3 dissolved in water 4 Toxic
33281 Water extract from Extract No. 1, precipitated with
12% phospho-tungstic acid . . . . . . . . . . . . . . . . . . . . . . . 5 Toxic
33282 Alcohol precipitate No. 2 evaporated and precipitated
with 4 volumes 95% alcohol . . . . . . . . . . . . . . . . . . . . . . 6 Not toxic
33283 Acetone insoluble from filtrate from No. 6, freed of
alcohol and precipitated with 4 volumes acetone. . . 7 Toxic
33284 Acetone soluble from filtrate from No. 7 (soluble in _
alcohol and acetone) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Slightlyjoxic
33897 Precipitate soluble in alcohol and acetone, filtrate from
phosphotungstic acid precipitate No. 5 treated with
lead acetate and basic lead acetate and the filtrates
precipitated with alcohol and acetone . . . . . . . . . . . . . . 9 Slightly toxic
33898 Lead precipitate. Acetone insoluble from No. 9 pre-
cipitated with basic lead acetate . . . . . . . . . . . . . . . . . . . 10 Not toxic
33899 Silver precipitate with silver nitrate from the lead free
filtrate from No. 10 . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 11 Not toxic
33900 Mercurous nitrate precipitate from filtrate from silver
precipitate No. 11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Not toxic
33901 Acetone insoluble not precipitated by lead, silver, or
mercury filtrate from No. 12 . . . . . . . . . . . . . . . . . . . . . . 13 Toxic
34213 Sulphuric acid precipitate of phosphotungstic acid Ex-
tract N o. 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Not toxic
34214 Filtrate from 14- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Toxic
34875 Phospho-tungstic acid of extract No. 5 purified with
basic lead acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Toxic
35661 Filtrate from No. 16 precipitated with additional
phospho-tungstic acid. Precipitate . . . . . . . . . . . . . . . . 17 Toxic
-- 35662 Filtrate from No. 17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Very slightly
toxic l
35665 Efiect of hydrolysis. Filtrate from alcohol precipitate H
No. 6 boiled with 5 cc 1.115 hydrochloric acid in
200 cc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Toxic
35666 Effect of nitrous acid. Preparation No. 6 treated with
sodium nitrate and hydrochloric acid . . . . . . . . . . . . . . . 2O Very toxic
35690 Carbamate precipitate, from lead-purified water ex-
tract N0. 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Not toxic
35691 Filtrate from No. 21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Very toxic
i5 ‘ 35901 Benzoyl chloride precipitate of lead-purified water ex-
 tract N o. 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Not toxic
 35902 Filtrate from No. 2s ............................. .. 24 Toxic

10 BULLETIN NO. 537, TEXAS AGRICULTURAL EXPERIMENT STATION

TABLE 1. Loco extracts from Astragalus earleL-Continued
Labora- Serial
tory i Description number of Effect
Number preparation on cats
36155 Effect of alkali. Lead purified extract No. 2 evapor-
ated from 1000 to 100 cc with excess sodium hydrox-
ide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Toxic
35940 Concentrated phospho-tungstic acid preparation from
lead-purified water extract N0. 2 . . . . . . . . . . . . . . . . . . 26 Not fed
36236 No. 26 shaken with ether, chloroform, and fullers earth,
evaporated to dryness, boiled 30 minutes with
absolute alcohol. Alcohol solution . . . . . . . . . . . . . . . . . . 27 Toxic
36239 Residue from No. 27 boiled 12 times with 100 cc por-
tions alcohol. Extracts, 2 to 11 combined . . . . . . . . . . 28 Not toxic
36237 12th extraction of insoluble from No. 27 with absolute
alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Not toxic
36238 Material insoluble in alcohol from Nos. 27-28-29,
soluble in water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Not toxic
36240 Soluble in hot absolute alcohol Nos. 27-28-29, but
separates on cooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Not toxic
36295 Extract N0. 26 evaporated to dryness and extracted
with absolute alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Not fed
36296 Insoluble residue from No. 32 boiled 2 times with
absolute alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Not toxic
36339 No. 32 freed of alcohol, precipitated by mercuric
chloride in alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Not toxic
36340 Filtrate from No. 34 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Toxic
36345 Tannic acid precipitate of No. 32 . . . . . . . . . . . . . . . . . . . . 36 Not toxic
36346 Filtrate from No. 36 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Toxic
36893 Material separated from cold alcohol and that precipi-
tated from alcohol-soluble by ether from No. 32. . . . 38 Very slightly
toxic
37107 Filtrate from ether precipitate of No. 38 evaporated
and precipitated with hydrochloric acid . . . . . . . . . . . . 39 Not toxic
37108 Filtrate from No. 39. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Toxic
_ Both cats died
37263 Loco Weed extracted by hot barium hydroxide . . . . . . . . 41 thelsame day,
no oco
37366 Separates on cooling from hot absolute alcohol extract symptoms
of lead purified water extract No. 2 . . . . . . . . . . . . . . . . 42 lightly toxic
37367 Precipitated by ether from filtrate from No. 42 . . . . . . . . 43 Toxic
37368 Filtrate from ether precipitate No. 43 . . . . . . . . . . . . . . . . 44 Toxic
37535 Insoluble in absolute alcohol, from lead purified alco-
hol extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Not toxic
37537 First water extract of phosphotungstic acid precipitate
from 48 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Not toxic
37538 Second water extract phosphotungstic acid of pre-
cipitate from 48 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Not toxic
37539 Phosphotungstic acid precipitate after 2 extractions
with water Nos. 46 - 47 . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Toxic
37536 Filtrate from phosphotungstic acid precipitate No. 48 49 Slightly toxic

LOCOINE, THE POISONOUS PRINCIPLE OF LOCO WEED 11

TABLE 1. Loco extracts from Astragalus earleh-Continued

Labora- Serial
tory Description number of Effect
Number preparation on cats
37686 Phosphotungstic acid precipitate from No. 45 . . . . . . . . . 50 Toxic
37687 F iltrate from No. 50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Toxic
37722 Extract No. 48, not absorbed by fullers earth 52 Toxic
37723 Extract No. 48, absorbed by fullers earth . . . . . . . . . . . . . 53 Not toxic
38567 Extract No. 48 phosphotungstic acid preparation
further purified by carbamate and hydrochloric
acid precipitate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Not fed
39636 Chloroform solution of acetyl product of extract No. 5 . 55 Not fed
41443 Chloroform solution of acetyl product by alcohol ex-
tract phosphotungstic acid precipitate No. 48 . . . . . . . 56 Not fed
39043 Chloroform-soluble acetyl product No. 56 saponified. . 57 Toxic
39044 Water-soluble acetyl product from No. 57 saponified. . 58 Not toxic
39058 Carbamate precipitate from No. 54 . . . . . . . . . . . . . . . . . . 59 Not toxic
39059 Hydrochloric acid precipitate from filtrate of N0. 59.
50000 gm plant in 200 cc . . . . . . . . . . . . . . . . . . . . . . . . . 60 Not toxic
 39077 F iltrate from hydrochloric acid precipitate No. 60. . . . . 61 Not fed
 39142 Phosphoric acid precipitate of No. 61 . . . . . . . . . . . . . . . . 62 Toxic
 39143 Filtrate from No. 62 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Toxic
39806 Acetic acid precipitate of No. 60 . . . . . . . . . . . . . . . . . . . . 64 Not toxic
39807 Filtrate from No. 64 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6S Toxic
40917 Tartaric acid precipitate from alcohol solution of No.
57. Base recovered by barium hydroxide . . . . . . . . . . 66 Toxic
40918 F iltrate from No. 66. Base recovered by barium hydrox-
ide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Toxic
41086 Citric acid precipitate of free base from No. 48. Base
recovered by barium hydroxide . . . . . . . . . . . . . . . . . . . . 68 Toxic
41087 Filtrate No. 68. Base recovered by barium hydroxide 69 Toxic
41293 Ether precipitate of alcohol solution of No. 57 . . . . . . . . 70 Very slightly
41294 Filtrate from No. 70 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 
41344 Alcohol-insoluble material from No. 57 . . . . . . . . . . . . . . . 72 Not toxic
41525 Crude tartrate from No. 48 after carbamate precipitate 73 Toxic
41578 No. 73 crystallized from acetone . . . . . . . . . . . . . . . . . . . . . 74 Not fed
41651 Crude citrate from No. 48 after carbamate precipitate 7S Not fed
41667 Crude oxalate from No. 48 after carbamate precipitate 76 Not fed

Progress of Concentration of the Locoine

     

The progress of concentration of the locoine is shown in Table 3.
In this table an estimate of the quantity of the preparations fed during
“the time required to loco the cats is given. These estimates are not
texact, because there was a variation in the cats and the cats were not

12'

all carried to the same stage of the disease.
more of the material than was necessary may have been fed.

As much as 70 to 114 egrams of organic matter in the original extract
was at first required to loco the cats. The concentration progressed with

the various preparations. Toward the end about 1 gram fed during 50

days was sufficient definitely to loco the cats.

Loco extracts from Astragalus Wootoni

BULLETIN NO. 537, TEXAS AGRICULTURAL EXPERIMENT STATION

Moreover, in some cases

TABLE 2.
Labora-
tory Description Serial Effect
Number number on cats
32176 Water extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Toxic
33256 No. 77 purified by basic lead acetate . . . . . . . . . . . . . . . . 78 Toxic
34871 Phosphotungstic acid precipitate of No. 77 . . . . . . . . . . . 79 Toxic
34872 Filtrate of No. 79 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Slightly toxic
TABLE 3. Amounts of several preparations required to loco cats
Laboratory Serial Cat Organic Plant
Number number No. matter Gm. Gm.
32597 Water extract . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 114.9 887
4 69 . 6 537
32598 Lead purified water extract No. 1 . . . . . . . . . 2 9 77.9 910
25 68.5 77S
33281 Phosphotungstic acid precipitate . . . . . . . . . . 5 a 20 25.4 4200
35 18.0 3000
47 14 . 3 2375
48 13 . 8 2325
36236 Purified phosphotungstic acid preparations
soluble in absolute alcohol . . . . . . . . . . . . . . 27 59 2. 1 1520
6O 2 . 1 1520
37539 Phosphotungstic acid preparation from
alcoholic extract of plant . . . . . . . . . . . . . . . 48 86 1.6 2460
87 2 . 9 3400
92 2.8 3400
94 1 . 8 2500
40917 Tartaric acid precipitate of chloroform-
soluble ficetyl compound, saponified. . . . 66 111 1. 8 10500
1 12 1 . 6 9700
40918 Filtrate from the tartaric acid preparation 67 113 .6 5550
114 . 7 6700
41086 Citric acid precipitate of the base . . . . . . . .. 6s 105 40.6 e900
2. 6 5700
41525 Tartrate of free base. Fed 1.2 gm of base
in 30 days . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 121 1.7
122 1. 7
41528 Fed 0.6 gm. of base in 30 days . . . . . . . . . . . . 73 123 1.0
124 1.0

LOCOINE, THE POISONOUS PRINCIPLE OF LOCO WEED 13
Preparation of the Toxic Base, Locoine

The method of preparation of the concentrated toxic base finally
adopted was as follows: 500 grams of dry loco weed was mixed in a
large evaporating dish with 1,500 cc of cold 95 per cent alcohol and well
stirred several times during thirty minutes. The alcohol was pressed
out by means of a screw tincture press, the residue returned to the dish
and mixed well with 500 cc of alcohol and pressed again. The residue
was discarded and the combined alcohol extracts were evaporated under
diminished pressure to about 200 cc. The solution was transferred to
an evaporating dish, 200 cc water added, and the liquid evaporated to
about 200 cc to remove alcohol. An excess of basic lead acetate was
added in small amounts, small filtered portions being tested from time
to time until no more material was precipitated. About 50 cc was

' required.

The precipitate was filtered off and discarded. The filtrate was freed
of lead with hydrogen sulfide, evaporated to about 5O cc, filtered into
an Erlenmeyer flask, and evaporated to dryness under diminished pres-
sure. The residue was boiled with absolute alcohol for thirty minutes,
being shaken well several times during boiling so as to mixthe insoluble
material with the boiling alcohol, and allowed to cool, and the insoluble

 matter was then discarded. The alcoholic extracts from 5,000 grams of
' plant were combined, and the alcohol was distilled off until the residue
f had a volume of about 100 cc. It was transferred to an evaporating

dish and 200 cc water was added; it was then evaporated to 200 cc and
filtered. The filtrate was cooled; to it was added 5 cc concentrated sul-
phuric acid and then an excess of a 25 per cent solution of phosphotunrg-

s. stic acid dissolved in 4 per cent sulphuric acid. About 350 cc was re-
 quired. After standing over night, the precipitate was filtered off. The

precipitate was washed back into the precipitation flask, shaken vigor-

is ously with 500 cc water, and filtered. The shaking with 500 cc water
fand filtering was repeated. The precipitate was washed back into the
 precipitation flask with a mixture of 150 cc acetone and 200 cc water,
T» and shaken well several times, or until all the dark-colored material

had gone into solution. An excess of a solution of barium hydroxide,

 saturated at 50°, was added. Small filtered portions were tested with
* sulphuric acid for an excess of barium. The mixture was shaken vigor-

.~.-

ously every half hour for two hours to assist in decomposing the ‘phos-

1 photungstate of the base. It was then filtered by suction and the pre-
iicipitate discarded. The filtrate was saturated with carbon dioxide and
fi the barium carbonate filtered off. The filtrate was evaporated to about
f 50 cc and allowed to cool. Any material that separated was filtered off.
1 Then 50 cc of cold saturated solution of barium hydroxide was added and
‘this mixture saturated with carbon dioxide. The carbamate was pre-

n r‘

  

A cipitated by the addition of 400 cc of 95 per cent alcohol and filtered ofi.
'_,The alcoholic solution was evaporated to about 50 cc, cooled, and filtered.
gThe solution was tested for barium by means of sulphuric acid, and

14 BULLETIN NO. 537, TEXAS AGRICULTURAL EXPERIMENT STATION

when barium was found, the quantity was determined in an aliquot. The
barium was then removed by adding the exact quantity of a standard
solution of sulphuric acid required to precipitate all of it. The solution
contained the crude locoine.

Acetyl-locoine

The base as prepared above forms a toxic acetyl product with acetic
anhydride. The directions for making the acetyl compound are as
follows:

Neutralize the crude locoine with acetic acid, evaporate to dryness in
an acetylation flask, and dry for an hour in the vacuum oven. Add 0.5
gram of anhydrous sodium acetate and 10 cc of acetic anhydride. Warm
on the water bath until the material is in solution, then boil for thirty
minutes. Pour the contents of the flask into an Erlenmeyer flask and
boil for an hour with 100 cc Water. Transfer to a separatory funnel
and extract the water-insoluble material with chloroform. Discard the
water solution. The chloroform solution contains the chloroform-soluble
acetyl product.

The acetyl product was saponified before feeding to cats by evapo-
rating to dryness, adding an excess of N/2 sodium hydroxide, about
18 cc for every gram of the product, and boiling for an hour. The
solution was then neutralized with hydrochloric acid, evaporated to
dryness, taken up with absolute alcohol, and filtered from the inorganic
salts. The alcohol was evaporated and the toxic principle taken up
with water.

The water-soluble acetylated portion was fed over a long period of
time with no toxic effects, while the chloroform-soluble material prepared
as above for feeding produced typical loco symptoms in the cats.

Preparation of the Tartra-te, Citrate, and Other Salts

The tartrate of the base can be prepared from the acetyl product as
prepared for feeding. The dry material was taken up in as little absolute
alcohol as possible, filtered, and cooled. A slight excess of a saturated
solution of tartaric acid in absolute alcohol was added so as to be acid
to bromphenol blue. It was allowed to stand in the refrigerator over
night, filtered, washed with a little alcohol, and dried in a vacuum
desiccator. The precipitate was prepared for feeding by dissolving in
water, adding a slight excess of barium hydroxide, and filtering. Most
of the barium was removed from the filtrate with carbon dioxide. Barium
was determined in the filtrate and removed quantitatively with N/2
sulphuric acid. The preparation proved toxic to cats. The filtrate from
the tartrate freed of tartaric acid as described above also was toxic to
some extent, showing that the tartrate was soluble under the conditions
of the experiment.

LOCOINE, THE POISONOUS PRINCIPLE OF LOCO WEED 15
The tartrate can also be prepared from the base, according to the
following directions:

Take up the base prepared from 5,000 grams of plant with a small
amount of absolute alcohol; about 25 cc will be sufficient. Heat under

Figure 2. Crystals of locoine tartrate, highly magnified.

a reflux condenser for half an hour and centrifuge from any insoluble
matter. Precipitate with tartaric acid as described above. The tartrate
prepared in this manner was fed to cats and proved to be toxic.

The tartrate precipitated from alcohol is not crystalline. If the tar-
trate is dissolved in a small amount of water and an excess of acetone
added, microscopic rhomboidal crystals separate. The citrate was also

prepared in a similar way.

The chloride was prepared by adding a slight excess of hydrochloric
acid to a solution of the base and evaporating it to dryness.

16 BULLETIN NO. 537, TEXAS AGRICULTURAL EXPERIMENT STATION
Some Properties of the Locoine

The crude locoine is a dark brown material and is very hygroscopic.
It has a strong alkaline odor and contains 8.8% nitrogen. It forms
salts with acids, the chloride containing 12.2% chlorine. Most of the
salts are soluble in water and alcohol and are not separated from fairly
concentrated solutions by immiscible solvents. The phosphate was also
prepared, and is very easily soluble in water.

The locoine was tested for selenium by the Robertson method and
none was found.

A solution of the base was boiled with dilute hydrochloric acid for
one hour. The neutralized product was still toxic to cats.

A solution was also evaporated from 1,000 cc to 100 cc in the presence

of 1 gram of sodium hydroxide. It was still apparently as toxic to‘

cats as before the treatment.

Another portion was treated with nitrous acid and hydrochloric acid.
After removing the excess of nitrous acid and neutralizing, the solution
was highly toxic to cats.

When a few drops of the base dissolved in 50 cc of water and a few
drops of this solution on a watch glass were tested with some alkaloidal
reagents, the following results were secured:

Froehde’s sulphomolybdic acid reagent (50 milligrams of molybdic acid
dissolved in l0 cc concentrated sulphuric acid) gave a heavy precipitate
but no change of color.

Wagner's iodine-potassium iodine reagent (1 gram iodine and three
grams potassium iodine dissolved in 50 cc of water) gave a heavy
precipitate.

Wormley’s reagent (strong hydrobromic acid saturated with bromine)
gave a heavy amorphous precipitate, and the bromine was decolorized.

Hydrobromic acid alone gave a heavy precipitate.

Alcoholic picrolonic acid (0.264 grams in 100 cc alcohol) (Autenreith;
Detection of Poisons, page 313) gave no precipitate.

Mayer’s mercuric chlorideepotassium iodide reagent (.338 ‘grams mer-
curic chloride and 12.5 grams potassium iodide dissolved separately in
water and diluted to 100 cc) gave no precipitate.

Bromine water alone gave no precipitate but the bromine was slightly
decolorized. Ammonia was added and there was no change.

Hager’s reagents (saturated aqueous picric acid) gave no precipitate.

Formaldehyde-sulphuric acid (5 cc formaldehyde in 25 cc concentrated
sulphuric acid) gave a heavy precipitate.

LOCOINE, THE POISONOUS PRINCIPLE OF LOCO WEED '17

Ten per cent potassium ferrocyanide gave no precipitate.
Ten per cent potassium ferricyanide gave no precipitate.

Tetra-chlor-iodic acid (5 grams iodine dissolved in 27 cc concentrated
hydrochloric acid and saturated with iodine) gave a heavy precipitate.

The tartrate precipitated in an absolute alcohol solution of the base
of the saponified acetyl product is of a light brown color and slightly
hygroscopic. It is very soluble in water, a little less so in 95 per cent
alcohol. It is insoluble in ether or acetone.

Tested with alkaloidal reagents, the tartrate gave the following results:
1.10 sulphuric acid: no precipitate.

Froehde’s reagent: no precipitate, no color change.

Wagner's reagent: heavy amorphous precipitate.

Wormley’s reagent: heavy amorphous precipitate. One drop of the
test solution diluted with 4 cc water still agave a precipitate.

Hydrobromic acid alone: no precipitate.

Picrolonic acid: slight amorphous precipitate on standing,
Mayer’s reagent: no precipitate.

Hager’s reagent: slight amorphous precipitate on standing.
Formaldehyde sulphuric acid: no precipitate or color change.

While many of the alkaloidal reagents gave precipitates with the toxic
base and its tartrate, the fact that it was not removed from either acid
or alkaline solutions with immiscible solvents indicates that the substance
is different from the ordinary alkaloids. It is probably closely related
to the alkaloids.

The formation of the chloroform-soluble acetyl product would indi-
cate that the product contains amine groups or some hydroxy base group.
The acetyl compound is a. dark brown, almost black substance, not
hygroscopic. It is soluble in alcohol but insoluble in acetone, ether, or
water. It contains about 29.9% of the acetyl group calculated from the
saponification value.

SUMMARY

The toxic principle of the loco weed, Astragalus earlei, has been
isolated in comparatively pure form and has been named locoine.

It is precipitated by phosphotungstic acid and can be recovered from
the precipitate by treatment with barium hydroxide.

The base can be acetylated by acetic anhydride and the toxic principle
recovered by extracting the water-insoluble material with chloroform,
evaporating off the chloroform, and saponifying with sodium hydroxide.

18 BULLETIN NO. 537, TEXAS AGRICULTURAL EXPERIMENT STATION

The base forms salts with acids. The tartrate, citrate, chloride, and
oxalate have been prepared. The tartrate and citrate were toxic to cats;
the oxalate was not tested.

When precipitated by alcohol, the citrate and tartrate were not
crystalline. They could be crystallized by dissolving in a little water
and adding about four volumes of acetone. Small microscopic rhom-
boidal crystals were formed.

Locoine is not precipitated by silver or mercury salts.

The results of tests with a large number of alkaloidal reagents are
described.

The locoine was tested for selenium and none was found.

Further work is being done to determine the chemical nature of the
compound. *

BIBLIOGRAPHY

1. Alsberg, C. L., and Black, O. F. Laboratory studies on the relation of
gariturlri togllgco Weed disease. Bur. Plant Industry Bulletin 246:
ar . .

2. Beath, O. A., Eppson, H. F., and Gilbert, C. S. Seleniums and other toxic
gaienersitilagéin soils and vegetation. Wyoming Agr. Exp. Sta. Bull.

3. Couch, James Fitten. A contribution to the study of locoism. Jour.
Pharm. and Exp. Therapeutics. 31:55. 1929.

4. Crawford, A. C. Barium, a cause of loco weed disease. Bur. Plant In-
dustry Bull. 129. 1908.

5. Marsh, C. D. Loco weed disease of the plains. Bur. Animal Industry
Bulletin 112. 1909.

6. Marsh, C. D. A field study of the relation of barium to loco weed disease.
Bur. Plant Industry Bulletin 246: Part I. 1912.

7. Marshall, H. H. Summary of studies on loco weed disease of sheep. Bul-
letins 25:234. Johns Hopkins Hospital Exp. Sta. Rec. 31:781. 1914.

8. Mathews, A. P. Locoism in domestic animals. Texas Agr. Exp. Sta.
Bull. 456. 1932.

9. Robinson, W. O. Determination of selenium in wheat and soils. Jour.
Ass. Off. Agr. Chem. 16:423. 1933.

ETBRARY

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