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Analytical Chemistry, Volume I: Chromatography; Plenum
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Dialkyl Phosphate Residues in Urine. J . Anal. Toxicol. 1981,
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drolytic Products of Organophosphorus Pesticides Chemicals
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Exposure to Organophosphorus Pesticides. A Modified Pro-
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Alkyl Phosphate Metabolites in Urine. J Agric. Food Chem.
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Received for review September 19,1988. Accepted March 9,1989.
Composition of Jimson Weed (Datura stramonium ) Seeds
Mendel Friedman* and Carol E. Levin
Bulk commercial grain, such as soybeans and wheat, may be contaminated by nongrain impurities,
including jimson weed seeds, that coexist with the crop to be harvested. The present study was undertaken
to determine the content of the major alkaloids of jimson weed seeds, atropine and scopolamine, as well
as protein, carbohydrate, fat, mineral, hemmagglutinin, and tannin. Combined GC-MS analysis of a
jimson weed seed extract revealed the presence of atropine and scopolamine plus possibly three additional
tropane-like alkaloids. An improved HPLC procedure showed that the alkaloid concentration in samples
obtained from different parts of the United States varied by as much as 50%: 1.69-2.71 mg/g for atropine
and 0.36-0.69 mg/g for scopolamine. The presence of a strongly fluorescent green compound of unknown
structure is also described. Baking experiments with jimson weed seed fortified wheat flour showed
that atropine and scopolamine largely survive bread-baking conditions. Jimson weed seeds do not contain
protease or amylase inhibitors. These observations provide a rational basis for relating seed composition
to biological effects in animals and for assessing the possible significance of low levels of the seeds in
food-producing animals and in the human diet.
The plant Datura stramonium was grown in England
in the 16th century from seeds that came from Constan-
tinopole, Turkey (Claus, 1961). The English presumably
imported the plant to the American colonies, as evidenced
by the fact that when English soldiers, who were sent to
quell Baconâ??s rebellion at Jamestown in Colonial Virginia,
inadvertently ate the plant as part of a salad in 1676, some
of them became ill and died. The name jimson weed or
Jamestown weed derives from this episode of fatal poi-
soning (Claus, 1961; Duke, 1984 Feenghaty, 1982; Oâ??Grady
et al., 1983). This and related reports of poisonings by
jimson weed seeds demonstrate that the plant exerts
pharmacological and toxicological effects in animals and
humans. In fact, Klein-Schwartz and Oderda (1984)
suggest that jimson weed abuse is a potentially serious
form of substance abuse in adolescents and young adults.
The most common symptoms of jimson weed ingestion are
altered perception of the environment, visual hallucina-
tions, mydriasis (dilation of the eye pupils), and tachy-
cardia (increase in heart rate) (Oâ??Grady et al., 1983). High
Western Regional Research Center, US. Department
of Agriculture-Agricultural Research Service, 800 Bu-
chanan Street, Albany, California 94710.
levels may cause depression of the central nervous system,
with symptoms ranging from lethargy to coma (Klein-
Schwartz and Oderda, 1984). Antidotes include the use
of the anticholinesterase drug physostigmine, charcoal to
slow down absorption, magnesium citrate to speed passage
through the intestinal tract, and ipecac to induce vomiting
(Om, 1975; Oâ??Grady et al., 1983).
The literature on jimson weed covers a variety of aspects
including agronomic and botanical (Broekaert et al., 1988;
Hagood et al., 1981; Kilpatrick et al., 1984; van De Velde
et al., 1988; Weaver, 1986), chemical and pharmaceutical
(Cordell, 1981; Duez et al., 1985; List and Spencer, 1976;
List et al., 1979), and medical toxicological (Day and
Dilworth, 1984; El Dirdiri, 1981; Fangauf and Vogt, 1961;
Feenghaty, 1982; Flunker et al., 1987; Gururaja and Khare,
1987; Keeler, 1981; Levy, 1977; Mahler, 1975; Mikolich et
al., 1975; Nelson et al., 1982; Shervette et al., 1979; Testa
and Fontanelli, 1988; Urich et al., 1982; Weintraub, 1960;
Williams and Scott, 1984; Worthington et al., 1981).
The objectives of this study were to (a) develop an im-
proved HPLC procedure for the analysis of atropine and
scopolamine in jimson weed seeds, (b) to demonstrate the
presence of known and unknown alkaloids in the seeds by
GC-MS analysis, (c) to measure the nutrient and antinu-
trient composition of the seeds, and (d) to assess the
This article not subject to U.S. Copyright. Published 1989 by the American Chemical Society
Composition of Jimson Weed
variation in the range of toxicant levels as affected by
growing locale i n order to estimate worst case concentra-
tions.
MATERIALS AND METHODS
Atropine and scopolamine were obtained from Sigma Chemical
Co. (St. Louis, MO). Apoatropine came from Adams Chemical
Co. (Round Lake, IL). Seed samples were obtained from the
Federal Grain Inspection Service (Kansas City, KS) and from
Valley Seed Co. (Fresno, CA). Samples were first picked through
to clean out any debris. They were then ground on a Wiley mill
using a coarse screen (No. 10).
Proximate Composition. Analyses for nitrogen, moisture, fat,
fiber, ash, carbohydrate, and mineral content were carried out
by standard procedures (AOAC, 1975).
Amino Acid Composition. Three analyses with samples
containing about 5 mg of protein (N X 6.25) were used to establish
the amino acid composition of the jimson weed seed protein: (a)
standard hydrolysis with 6 N HC1 for 24 h in evacuated sealed
tubes (Friedman et al., 1979); (b) hydrolysis with 6 N HC1 after
performic acid oxidation to measure half-cystine and methionine
content as cysteic acid and methionine sulfone, respectively; (c)
basic hydrolysis by barium hydroxide to measure tryptophan
content (Friedman and Cuq, 1988).
Tannin Content. Tannin content was determined by the
vanillin assay (Price et al., 1978) with 100 mg of jimson weed
samples extracted with 5 mL of MeOH in capped vials with
stirring for 20 min. The extracts were then centrifuged in a
Beckman microfuge. The assay was carried out a t 30 "C with
reagents previously warmed to this temperature. The tannin
content was calculated with the aid of a catechin standard curve.
Hemagglutination Assay. The assay for the presence of
hemagglutinins (lectins) was carried out with 150-mg samples of
jimson weed or soybean seed samples and human red blood cells,
as described previously for lima bean and soybean flours (Wallace
and Friedman, 1985; Friedman and Gumbmann, 1986).
Trypsin, Chymotrypsin, and a-Amylase Inhibition Assays.
Titration by previously described techniques (Friedman and
Gumbmann, 1986; Buoncore and Silano, 1986) demonstrated the
absence of inhibitors of digestive enzymes in jimson weed seed
extracts.
GC-MS Analysis of Jimson Weed Seed Extracts. The
samples were defatted by extracting with hexane in a Soxhlet
extractor for 18 h. The seed meal was air-dried and ground to
a fine powder on a UDY cyclone mill. The defatted samples (100
mg) were extracted with methanol in a Soxhlet extractor for 18
h. The volume was reduced to a few milliliters, which was then
filtered through a 0.45-wm membrane (Schleicher and Schuell).
The extract was bought up to a volume of 10 mL with methanol
and stored under refrigeration in an air-tight flask.
Preliminary studies showed that the same extracts of jimson
weed seeds used for HPLC analysis described below were suitable
for GC-MS analysis provided any HCl present was neutralized
as follows: 0.5 mL of the methanol solution was dried under a
stream of nitrogen. The residue was dissolved in 1.0 mL of 0.05
N NaOH. This solution was then mixed with 1 mL of chloroform,
the two phases were vigorously stirred, and the chloroform phase
containing the alkaloids was separated and used for analysis. The
neutralization step was necessary because any HC1 present was
found to degrade the packing of the GC capillary column.
To minimize the appearance of large numbers of peaks asso-
ciated with fatty acids and esters in the GC-MS chromatograms,
samples were defatted twice with hexane before extraction with
methanol.
HPLC Assay for Atropine and Scopolamine in Jimson
Weed Seeds. An Alumina A Sep-Pak (Waters) was conditioned
with 5 mL of chloroform. A 2-mL portion of the methanol extract
followed by 5 mL of chloroform was then passed through the
Sep-Pak. The eluants were combined and evacuated to dryness
with an aspirator. The residue was then taken up in 1 mL of
methanol containing 0.2 mg/mL cystamine as an internal
standard. This solution was used for analysis by HPLC.
A Beckman Instruments (San Ramon, CA) 334 HPLC system
with a 427 integrator and a 165 UV-vis variable-wavelength de-
tector was used. The column was a C8 Beckman Ultrasphere (4.6
X 250 mm) with a Beckman CB precolumn.
J. Agric. Food Chem., Vol. 37, No. 4, 1989 999
The mobile phase consisted of W36 water-methanol containing
0.02 M phosphate buffer (pH 3; 0.87 mL of 87% phosphoric acid
plus 0.77 g of monosodium phosphate/L) and 0.01 M dibutylamine
as a counterion. Solvent flow rate was 0.8 mL/min. Dibutylamine
was chosen as a counterion because it has lower absorptivity a t
the wavelength used (200 nm) and produced a more stable base
line than other amine modifiers, such as ethanolamine. The
mixture of water and methanol proved to be stable on the column
compared to other solvents such as acetonitrile. The low ab-
sorbtivity in the UV and the relatie low toxicity of this mobile
phase were additional benefits.
The following compounds were evaluated as possible internal
standards for the analysis of atropine and scopolamine by HPLC:
benzylamine, cystamine, diaminopropionic acid, histamine, hy-
d r o ~ yethylamine, hydroxytryptamine, nicotinamide, nicotinic acid,
nicotinic acid methyl ester, penicillamine, theobromine, theo-
phylline, and tyramine. Cystamine was selected as the internal
standard because its elution position, peak shape, and linear
relationship of peak areas of concentration were superior to the
other compounds tested.
Relative recovery of alkaloids was determined by spiking the
ground jimson weed samples with atropine and scopolamine
standards. The samples were then extracted and analyzed by
HPLC.
Baking Experiments. Unbleached, unbrominated, malted,
and enriched white wheat flour (Mellow Judith) was obtained from
Con Agra Inc. (Oakland, CA). Fresh cake yeast (Fleischman's)
was obtained a t a local market.
The recipe for one loaf of bread consisted of 183.1 g of flour,
106.8 g of water, 3.5 g of salt, 6.1 g of yeast, and 25 g (12% of dry
weight) of jimson weed seeds in the final mix. The jimson weed
seeds were mixed whole with the flour, and the mixture was then
ground in a Wiley mill. This overcame the difficulty of finely
grinding the fatty seeds alone. The ingredients were combined,
and the resulting dough was kneaded on a Hobart Model ClOO
kneader (Troy, OH) for a total of 8 min. The dough was allowed
to rise twice in a fermentation chamber (National Co., Lincoln,
NE) at 37 "C and 90% humidity for 45 min. The dough was
placed in 14.6 X 7.6 X 7.4 cm nonstick-coated pans and baked
35 min at 215 "C. The crust was separated from the crumb with
an electric knife. Both the crust and crumb were sliced, lyo-
philized, and ground in a Wiley mill with a 1-mm screen.
Green Fluorescent Compounds. In working with extracts
of jimson weed seeds, it was noted that there was present a
substance that under UV light (365 nm) exhibits an intense green
fluorescence in neutral or acidic solutions and a bright yellow
fluorescence in basic solution. The substance is present in such
quantity that the interior portion of freshly ground seeds fluoresce
without purification. The material is easily extracted with several
portions of methanol.
The material was chromatographed on thin layer precoated
silica gel plates (E. Merck). Twenty microliters of a methanol
extract (1 g extracted with 10 mL of hot methanol) was spotted
on plates 2 cm from bottom and allowed to run until the solvent
front was 15 cm from the origin. In the solvent ethyl acetate-
methanol-water (100:13.5:10), a large spot a t R, 0.05-0..10 was
observed. In the solvent ethyl acetate-formic acid-glacial acetic
acid-water (100:11:11:25), this green spot resolved itself into two
components, a smaller spot a t Rf 0.35 and a larger one at Rf 0.43.
RESULTS AND DISCUSSION
Seed Morphology and Contaminants. Seeds of the
25 species of Datura appear morphologically indisti-
guishable (J. Effengerger, Department of Food and Ag-
riculture, S t a t e of California, Sacramento, private com-
munication, May 16,1985). D. stramonium (jimsonweed)
and Datura ferox (Chinese thornapple), for example, have
seeds that are very similar i n appearance. Generally, a
genus name rather than that of a species should be used
unless a grow-out test is carried out with the seed i n
question.
The nature of t h e contaminants found t o be present i n
pure Datura spp. collected i n the field are summarized i n
Table I. These need to be removed manually, as was done
i n our studies, before the seeds can be used for composi-
1000 J. Agric. Food Chem., Vol. 37, No. 4, 1989 Friedman and Levin
Table I. Contaminant Seeds in Datura Jimson Weed Seeds
Collected in the Field"
no. of
seeds %
Datura spp. seeds, pure 1303 78.78
contaminant
broken seeds of Glycine max (soybean),
caryopses of Triticum spp. (wheat), soil
particles from microscopic to 7 mm long 243 14.69
possibly G. max. 39 2.36
Polygonum pennsylvanicum (Bigweed 24 1.45
Triticum spp. (wheat) 5 0.30
Sida spinosa (prickly mallow) 1 0.06
Digitaria sanguinalis (crabgrass) 1 0.06
Atriplex: s p p . (saltbush) 1 0.06
1654 100.00
"Test results by count. Some counts were made with an elec-
immature shriveled seeds of Fabaceae,
Abutilon spp. (Indian mallow) 35 2.12
ladysthumb)
Amaranthus spp. (pigweed) 2 0.12
tronic seed counter and are approximate.
Table 11. Comparison of Composition (%) of Jimson Weed
and Some Commonly Contaminated Grains
unpurified
jimson weed defatted full-fat whole wheat
material seed flour soy flour soy flour pastry flour
nitrogen 3.1 8.0 5.4 2.2
H20 7.7 11.2 9.0 11.1
fat 18.1 0.9 20.7 0.6
fiber 17.8 1.5 4.5 1.7
ash 6.6 5.9 4.9 1.8
carbohydrate 31.9 37.8 31.5 81.6
starch 1.1 0.7 0.6 57.5
sugar 2.1 13.9 10.6 2.4
reducing sugar 0.3 0 0 0
glucose 0.16 0.13 0.12 0.06
" Detection limit 0.1%.
tional and toxicological studies.
Proximate Composition. Table I1 shows the nitrogen,
moisture, fat, fiber, carbohydrate, and ash content of jim-
son weed seed flour, full-fat and defatted soy flour, and
whole wheat flour for comparison. The value for nitrogen
of 3.1 %, corresponding to a protein content of about 20%
(3.1 X 6.251, compares to 2.2% and 5.4% for wheat flour
and soy flours, respectively. Thus, protein content of
jimson weed is greater than that of wheat flour but much
lower than of soy flour. The table also shows that the fat
content of 18.1% in jimson weed seeds is similar to the
20.7% in soy flour. Wheat flour has a very low fat content.
The crude fiber content of jimson weed seed flour was
17.8% compared to 4.5% for soy and 1.7% for wheat flour.
The carbohydrate content of jimson weed seeds (31.9%)
is identical with that of soy flour (31.5%) but less than half
that of whole wheat flour (81.6%). A separate analysis for
starch content revealed that both jimson weed and soy
flours contain negligible amounts of this polysaccharide
compared to a 57.5% content in whole wheat flour. These
considerations suggest that, from a nutritional standpoint,
jimson weed seeds are a good source of protein and an
excellent source of fat and fiber.
Table I11 summarizes the content of minerals in jimson
weed seeds, full-fat raw soy flour, defatted soy flour, and
whole wheat flour. The data show that (a) the toxic trace
elements cadmium, mercury, and selenium were not de-
tected in any of the flours; (b) the iron content of jimson
weed seed flour is about 40 times greater than in wheat
flour and 14 and 17 times greater than in defatted and
full-fat soy flour, respectively; (c) the chromium and
Table 111. Mineral Content (ppm) of Jimson Weed Seeds
and Other Grains
unpurified
jimson weed defatted full-fat whole wheat
mineral seed flour soy flour soy flour pastry flour
cadmium" 0 0 0 0
calium 2310 2680 2250 480
chromium 2.2 0.22 0.22 0.10
copper 16.4 16.0 14.5 4.4
iron 1305 76.9 91.2 33.9
magnesium 2890 2890 2500 1630
magnanese 134 36.4 28.0 40.7
mercuryb 0 0 0 0
potassium 6540 2260 18700 4710
seleniumb 0 0 0 0
sodium 175 221 271 54
zinc 59.5 63.1 60.6 38.8
'Detection limit 0.05 ppm. *Detection limit 0.5 ppm.
Table IV. Free Amino Acid Content of Jimson Weed Seeds
elution content
amino acid mg/100 g mg/16 g N time, min
unknown" 51.0 230.0 40.2
histidine 8.3 37.0 56.0
arginine 12.4 5.6 77.0
y-aminobutyric (GABA) 16.6 74.6 53.5
a Calculated as leucine equivalents.
Table V. Amino Acid Content (g/16 g of N) of Defatted
Jimson Weed Seed Flour, Defatted Soy Flour, and
Commercial Wheat Flour"
amino jimson soy wheat
acid weed seed flour flour FAO'
ASP 7.74 11.74 4.11
Thr 3.14 3.58 2.54 4.0
Ser 4.03 4.90 4.39
Glu 13.05 18.59 33.1
Pro 3.32 5.17 11.5
G1Y 3.87 4.04 3.48
Ala 3.51 4.05 2.80
Val 3.62 5.20 4.07 5.0
Metc 1.38 1.19 1.62 3.5'
Ile 3.22 4.66 3.53 4.0
Leu 5.31 7.74 6.73 7.0
TYr 2.55 3.42 3.20 6.08
Phe 3.47 5.05 4.78
His 1.84 2.47 2.09
LYS 3.19 5.82 1.85 5.5
'4% 6.54 7.27 3.65
Tryd 0.51 1.13 0.55
Cysb 2.00 1.14 2.20
N content, %: defatted jimson weed seed flour, 3.56; defatted
soy flour, 8.00; wheat flour, 2.18. bDetermined as cysteic acid after
performic acid oxidation. Determined as methionine sulfone after
performic acid oxidation. d T ~ o separate determinations by ion-
exchange chromatography after hydrolysis by barium hydroxide.
eProvisional amino acid scoring pattern for an ideal protein (FAO,
1973). fCys + Met. gTyr + Phe.
manganese levels in jimson weed seeds are also greater
than in the three grain flours; and (d) copper, zinc, mag-
nesium, and calcium levels is the three flours do not differ
significantly.
Free and Protein Amino Acid Contents. Table IV
lists the free amino acid content of jimson weed seeds. The
seeds contained only histidine and arginine in the free form
along with two unknown amino acids. One of these eluted
in the same positions as y-aminobutyric acid. The second
elutes in the vicinity of cystine. It does not appear to be
y-glutamyl-L-aspartic acid that Ungerer et al. (1988) found
in jimson weed seeds. This dipeptide elutes in the same
position as methionine on our column. Elucidation of the
Composition of Jimson Weed J. Agric. Food Chem., Vol. 37, No. 4, 1989 1001
Table VI. Lectin Content of Jimson Weed Seeds Measured
by Agglutination
jimson activityb activity 2 years
weed seedsn (n = 2) later (n = 3)
0.63 0.63 f 0
0.80
2.50
0.63 1.7 & 0.3
1.25
0.08 5.6 f 1.0
0.10
0.10
0.04
See Table IX for origin of seeds. Minimum amount of sample
required to agglutinate human red blood cells (pg/50 pL). The
lower the number, the greater the hemagglutinating activity.
structure of the second ninhydrin-positive compound
awaits further studies.
Table V lists the amino acid composition of acid hy-
drolysates of defatted jimson weed seed flour, defatted soy
flour, and commercial wheat flour. The values of the
amino acid scoring pattern of the essential amino acids for
an ideal protein, as defined by the Food and Agricultural
Organization of the United Nations (FAO, 1973), are also
shown for comparison. The data show that the amino acid
pattern of jimson weed seeds, especially the essential amino
acids, falls between those of a legume such as soy and a
cereal such as wheat. The results show that jimson weed
seeds meet the provisional requirements for the sulfur
amino acids (Cys + Met) and for Tyr + Phe, but values
for Thr, Val, Ile, and Lys are below those of the FA0
pattern. In spite of these deficiencies, the amino acids of
jimson weed could contribute significantly to protein nu-
trition, if it were possible to remove the toxic alkaloids
either by chemical inactivation or through plant genetics.
Thus, it may be possible in the future to develop jimson
weed cultivars in which the genes controlling the biosyn-
thesis of the alkaloids are suppressed or eliminated. The
new varieties might then displace the ones currently
growing in the field. A possible approach would be to use
molecular biological techniques to insert DNA constructs
capable of generating antisense RNA specific for genes
involved in alkalid biosynthesis, resulting in blocked ex-
pression in the jimson weed plant (Walder, 1988). Instead
of presenting a problem to the consumer, the new cultivars
would contribute to the nutritional value of the grain. At
this stage, more information is needed about specific en-
zymes involved in the biosynthesis of the alkaloids (Cor-
dell, 1981) before it would be possible to control, via an-
tisense RNA or otherwise, the genes coding for these
compounds.
Hemagglutinin (Lectin) Content. Lectins, present
in many plant seeds, are carbohydrate-binding proteins
with differing sugar specificities. Normally, lectins are
thermally unstable and are partly or f d y denatured during
cooking of foods. Lectins from different sources may,
however, differ in heat stability. Inadequately cooked
legumes containing lectins may cause gastrointestinal
disturbances and adverse nutritional and toxic effects in
humans (Reaidi et al., 1981; Liener, 1988).
D. stramonium seeds are reported to contain a lectin
visualized by an immunocytochemical technique and
quantified by agglutination assays (Broekaert et al., 1988).
These authors also demonstrated a possible physiological
function for the jimson weed seed lectin in the plant, in-
volving mediation of cell-cell interactions.
Our agglutination assay of the jimson weed seed samples
listed in Table VI revealed that the minimum amount of
jimson weed seed flour required to cause agglutination of
Table VII. Tannin Content of Jimson Weed Seeds
mg tannin/ mg tannin/
samplea g seedb samplea g seedb
2.9 & 0.05 1 1.7 & 0' 4
2 1.4 f 0 5 2.3 f 0
3 1.9 f 0 7 4.5 f 0.02
bExpressed as catechin.
Average f standard deviation from two separate determinations.
human red blood cells ranged from 0.04 to 2.5 pg/50 pL
(the smaller the number, the more potent the hemagglu-
tinating activity).
The reason for the wide variation in lectin content is
unknown. An assay of the same three samples after a
2-year interval indicated an unchanged value for one and
a lower content for two (Table V). The lectins of jimson
weed seeds might be slowly inactivated during storage and
exposure to heat and sunlight under field conditions, ac-
counting for the variable results.
Tannin Content. Table VI1 lists the tannin contents
of seven jimson weed samples obtained from different
locations. The values ranged from 1.6 to 5.6 mg/g of seed.
The reasons for the more than &fold variability in tannin
content is not immediately apparent. Soil and climatic
conditions may affect the biosynthesis of tannins (Desh-
pande et al., 1984).
Tannins adversely affect the nutritional quality and
safety of foods (Griffiths, 1986; Deshpande et al., 1984),
presumably through chelation of essential trace elements
such as iron and through inhibition of proteolytic digestive
enzymes such as trypsin and chymotrypsin.
Atropine and Scopolamine Content. In their review
of toxic weed seed contamination in soybeans during
harvest, transport, and storage, List et al. (1979) state that
jimson weed seeds are probably the more prevalent and
most toxic compared to others such as castor (Ricinus
communis), cocklebur ( X a n t h i u m strumarium), corn
cockle (Agrostemma githago), cow cockle (Saponaria
uaccaria), crotolaria (Crotolaria spp.), morning glory
(Ipomoea spp.), nightshade (Atropa belladonna), and
pokewood (Phytolacca americana). These authors also
suggest that, aside from toxicity aspects, weed seed con-
tamination may also adversely affect the appearance, or-
ganoleptic, functional, and nutritional properties of grain.
The major toxic principles present in jimson weed seeds
are the alkaloids atropine and scoplamine. These sola-
naceous alkaloids are present in a number of other plants,
including Atropa belladonna (nightshade) and Hyo-
scyamus niger. The alkaloids (see structures) have a bi-
cyclic structure in which a five-membered pyrrolidine ring
is fused to a six-membered pyridine ring. Atropine
probably exists in nature as the optically active hyoscya-
mine isomer, which racemizes to the DL form (atropine)
during isolation. The major difference between scopola-
mine, also known as hyoscine, and atropine is the prescence
of an epoxide group in cis position to the N-bridge (Cordell,
1981):
'See Table IX for origin of seeds.
CH *OH CH 2 OH
ATROPINE (d 1 ) SCOPOLAMINE (d 1 )
HYOSCYAMINE ( 1 ) HYOSCINE ( 1 )
J. Agric. Food Chem., Vol. 37, No. 4, 1989 Friedman and Levin
1
1002
IO0
no
1 5 60
VI
40 z ,
MASS SPECTRUM of
PURE ATROPINE
M+ j ...
LI I! Pb 289 I
I I
? I !
0 -, , , , , I , ,. ~ , I ,
20 60 100 100 180 220 260 300
rn /z
Figure 1. Mass spectrum of atropine.
__ __ 94 100
I38 no
42 I
I MASS SPECTRUM of
PURE SCOPOLAMINE
- s v 60
108 z I ,
VI
? I
z 1 5 1 ' 136
P 1 &?- I A ! , 303
57 7 7
20
29
27
- - 2 $ y , ~ 1 , , , 0 1 - _ - -
20 60 100 140 180 220 260 300
m /z
Figure 2. Mass spectrum of scopolamine.
Figures 1 and 2 show the mass spectral fragmentation
patterns for authentic atropine and scopolamine obtained
from commercial sources. The figures show the parent
molecular ion peaks for both compounds: M+ = 289 for
atropine and M+ = 303 for scopolamine. The presence of
a parent peak in a mass spectrum generally defines the
molecular weight of a compound (Friedman, 1977). The
GC-MS chromatogram of a methanol extract of jimson
weed seeds is shown in Figure 3. Alkaloids are present
in the extract along with several other peaks associated
with fatty acid esters. Note the clear separation of the
atropine and scopolamine peaks a t scans 374 and 422,
respectively. These results indicate that GC-MS is useful
for demonstrating the presence of atropine and scopola-
mine in plant extracts. However, this technique was found
to be useful only for qualitative measurements. The me-
thod needs further study to assess its potential for a
quantitative assessment of the alkaloid content.
In addition to atropine and scopolamine, Figure 3 sug-
gests the presence of small amounts of three other akaloids
designated as X, Y, and Z. X appears to be atropine-like
in nature. Its molecular weight of 271 corresponds to the
molecular weight of atropine minus 18 (H,O), possible
apotropine. The mass spectrum of authentic apoatropine
was identical with that of X. Y, also atropine-like, has a
molecular weight of 303, which corresponds to the mo-
lecular weight of atropine plus 14 (CH, group). Z may be
scopolamine-like. Its molecular weight corresponds to that
of scopolamine plus 14 (CH,.group).
Extensive studies were carried out to develop a reliable
HPLC procedure for measuring atropine and scopolamine
in jimson weed seed extracts. Beneficial features of this
I
I GC-MS of EXTRACT of
JIMSON WEED SEEDS
0 200 400 600 BOO 1000
4: 1 9:16 1432 19:49 25:6
SCANS and ELUTION TIME in MINUTES
Figure 3. GC-MS analysis of a jimson weed seed extract showing
linear plots of elution time on the gas chromatograph and scan
numbers (scan rate 0.63 scans/s).
0 I I 1 I I
0 0.05 0.1 0.2 0.3 0.4 0 5
ALKALOID CONCENTRATION (rng/ml)
Figure 4. Plots of atropine and scopolamine concentrations
against the ratio of areas of the HPLC peaks for the two alkaloids
to the area for the internal standard cystamine.
method include (a) high sensitivity due to the use of a low
detection wavelength (200 nm), (b) use of a solvent system
with a stable base line even at this low wavelength, (c) use
of a new internal standard that is well resolved from the
complex matrix, and (d) rapid sample preparation using
solid-phase extraction (Sep-Pak). Since tropane alkaloids
are known to hydrolyze in aqueous solution, solid-phase
extraction has an additional advantage over the traditional
liquid-liquid extraction that utilizes acid-base solubility
(List and Spencer, 1976).
Figure 4 shows that the HPLC column responds linearly
to both atropine and scopolamine in the concentration
range shown. As little as 20 ng of alkaloids was detected
on the column. However, the response was not always
linear with concentration below 200 ng of total alkaloids.
Figure 5-7 demonstrate the clear separation of atropine
and scopolamine from each other and from the internal
standard cystamine. Spiking experiments, in which known
amounts of a mixture of atropine and scopolamine were
added to jimson weed flour and then reextracted, revealed
that the recovery of atropine ranged from 92 to 97% of
the amount added and of scopolamine from 87 to 92%
(Table VIII).
Table IX shows that the atropine content of seven seeds
obtained from different parts of the United States ranged
from 1.69 to 2.71 mg/g seed, with an average f SD = 2.27
Composition of Jimson Weed J. Agric. FoodChem., Vol. 37, No. 4, 1989 1003
I -
h E c
0
0
cy v
E
s
9
2
z
pc
Y
Y I e
i
n K
- E
E
0
0
cy
Y
B
s
s1
2
z
pc
0 5 10
ELUTION TIME (minutes)
Figure 5. Typical HPLC chromatogram for atropine and sco-
polamine applied to the column as a mixture. Conditions: 0.05
mg of atropine + 0.05 ma; of scopolamine + 0.2 mg of cyst-
amine/mL. A 20-pL sample of this solution was injected onto
the column. Flow rate: 0.8 mL/min. Solvent: 0.02 M phosphate
buffer; 0.01 M dibutylamine; 36% methanol; pH 3.0.
h C I
Y w l
I
c -
h ' P f r
0 5 10
ELUTION TIME (minutes)
Figure 6. Typical HPLC chromatogram of a jimson weed seed
extract. Conditions as in Figure 5.
f 0.36 mg/g. The corresponding range for scopolamine
was from 0.36 to 0.69 mg/g, with an average f SD = 0.53
f 0.13 mg/g. The atropine and scopolamine values varied
b y as much as 50'70, depending on origin. The cause of
t h i s variation is presently unknown but needs to be es-
tablished. These results are similar to those observed b y
List and Spencer (19761, List et al. (1979), and Mirzamotov
and Luftulin (1986) (who also report no significant dif-
ferences in the biosynthesis rate of the two alkaloids in D.
stramonium at different seasons of growth).
0 5 10
ELUTION TIME (minutes)
Figure 7. HPLC chromatogram of a methanol extract of a
defatted diet containing 4% jimson weed seeds. Conditions as
in Figure 4. This diet was used in animal feeding studies to assess
the toxicity of jimson weed seeds.
Table VIII. Recovery of Atropine and Scopolamine Added
to Jimson Weed Seed Floura
atropine scopolamine
material mg/g 70 rec mg/g % rec
jimson weed seed flour, 2.69 f 0.16 0.75 f 0.04
100 mg of jimson weed 3.09 f 0.13 97 0.90 f 0.04 88
control
seed flour + 0.51 mg
of atropine, 0.27 mg
of scopolamine
seed flour + 1.01 mg
of atropine, 0.54 mg
of scopolamine
seed flour + 2.02 mg
of atropine, 1.08 mg
of scopolamine
100 mg of jimson weed 3.55 f 0.21 96 1.19 f 0.06 92
100 mg of jimson weed 4.31 f 0.17 92 1.59 f 0.05 87
Average f standard deviation from two separate determina-
tions.
Table IX. Atropine and Scopolamine Content of Jimson Weed
Seeds Obtained from Different Locations
ratio of
mg/g jimson weed seeds to sample
no. location
1 Indianapolis, IN
2 Peoria, IL
3 Belle Chase, LA
4 Indianapolis, IN
5 Belle Chase, LA
6 Cedar Rapids, IA
7 Fresno, CA
atropine scopolamine scopolamine
1.69 f 0.09 0.36 f 0.03 4.7
2.07 f 0 0.59 f 0.02 3.5
2.09 f 0.06 0.51 f 0 4.1
2.26 f 0.08 0.51 f 0 4.4
2.41 f 0.05 0.69 f 0.02 3.5
2.68 f 0.04 0.39 f 0.05 6.9
2.71 f 0.02 0.66 f 0.05 4.1
av f SD 2.27 & 0.36 0.53 i 0.13 4.5 & 1.16
(I Average from two separate determinations f standard deviation.
Since jimson weed seeds contaminate soybeans, corn,
and wheat, i t is of paramount interest to find out whether
the tropane alkaloids survive the processing conditions to
which these grains may be subjected before consumption.
To obtain information on this question, we added jimson
weed seed flour to wheat flour, which was then baked into
bread. The bread was then separated into crust and crumb
1004 J. Agric. Food Chem., Vol. 37, No. 4, 1989 Friedman and Levin
Release upon Imbibition. Plant Physiol. 1988, 86, 569-574.
Buoncore, V.; Silano, V. Biochemical, Nutritional, and Toxico-
logical Aspects of a-Amylase Inhibitors from Plant Foods. Adv.
Exp. Med. Biol. 1986, 199, 483-507.
Claus, E. P. Pharmacognosy; Lea and Febiger: Philadelphia, 1961;
p 312.
Cordell, C. A. Introduction to Alkaloids-A Biogenetic Approach;
Wiley-Interscience: New York, 1981; p 94.
Crowley, J. F.; Goldstein, I. J. Datura Stramonium Lectin.
Methods Enzymol. 1982,83, 368-373.
Day, E. J.; Dilworth, B. C. Toxicity of Jimson Weed Seed and
Coca Shell Meal to Broilers. Poultry Sci. 1984, 63, 466-468.
Deshpande, S. S.; Sathe, S. K.; Salunkhe, D. K. Chemistry and
Safety of Plant Polyphenols. In Nutritional and Toxicological
Aspects o f Food Safety; Friedman, M., Ed., Plenum: New
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Hagood, E. S., Jr.; Bauman, T. T.; Williams, J. L., Jr.; Schreiber,
M. M. Growth Analysis of Soybean (Glycine max.) in Com-
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Pigs from Sows Ingesting Toxic Levels of Jimsonweed During
Gestation. Vet. Hum. Toxicol. 1981, 23, 413-415.
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Jimsonweed with Soybean. Agron J. 1984, 75, 833-836.
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948-954.
Table X. Effect of Baking on Atropine and Scopolamine
Content of Jimson Weed Seed Fortified Bread
atropine scopolamine
mg/g mg/g
material mixed flour % rec mixed flour % rec
wheat flour + 12% 0.408 100 0.115 100
jimson weed seed
flour, unbaked
bread crumb 0.304 75 0.100 87.
bread crust 0.335 82 0.083 72
fractions. These were freeze-dried and milled into flours.
Table X shows the following recovery of atropine and
scopolamine from these flours compared to an unbaked
control of wheat plus jimson weed flours: for atropine,
bread crumb 75%, bread crust 82%; for scopolamine,
bread crumb 87%, bread crust 72%. The findings show
that both alkaloids in jimson weed seed flour largely
survived the high temperature of bread-baking, in both the
crumb and crust.
In a related study, List and Spencer (1976) describe the
fate of the two alkaloids during processing of jimson weed
contaminated soybeans. They report that extraction of
a 5050 mixture of soybeans and jimson weed seeds with
petroleum ether produced a meal and crude oil fractions.
Analyses of these fractions by a gas chromatographic
procedure showed that virtually all of the atropine and
scopolamine remained in the meal. They also found that
alkali refining effectively removed atropine added to soy-
bean oil.
Subchronic 90-day toxicity studies with laboratory rats
were conducted to establish safe levels of jimson weed
seeds (Dugan et al., 1989). Dose-related adverse effects
were observed at 0.5% or more in the diet. An unanswered
question is whether toxicological manifestations of jimson
weed seeds can be predicted on the basis of their atropine
and scopolamine contents or whether additional constit-
uents, such as lectins, unknown alkaloids, and the de-
scribed fluorescent compound(s), contribute to biological
activity. If the former is true, then a rapid assay for tro-
pane alkaloid content by the described HPLC method may
be useful for predicting the safety of jimson weed seeds.
Moreover, since dietary constituents and the process of
digestion and metabolism can be expected to modify the
adverse manifestations of atropine and scopolamine
(Fuahiki et al., 1987), the described compositional infor-
mation should contribute to a better understanding of
toxicological manifestations of jimson weed seeds when fed
as a part of a complete diet. These studies are expected
to help provide a basis for establishing maximum tolerance
levels in food and feed and in setting official grain stand-
ards.
ACKNOWLEDGMENT
We thank J. C. Halvorson and colleagues of the Federal
Grain Inspection Service (FGIS) for collecting the seeds
and for helpful advice, R. England and W. F. Haddon for
the mass spectra, F. F. Bautistia and G. McDonald for
excellent technical assistance, and M. R. Gumbmann for
valuable contributions.
Registry No. GABA, 56-12-2; His, 71-00-1; Arg, 74-79-3; ni-
trogen, 7727-37-9; starch, 9005-25-8; glucose, 50-99-7; cadmium,
7440-43-9; calcium, 7440-70-2; chromium, 7440-47-3; copper,
7440-50-8; iron, 7439-89-6; magnesium, 7439-95-4; manganese,
7439-96-5; mercury, 7439-97-6; potassium, 7440-09-7; selenium,
7782-49-2; sodium, 7440-23-5; zinc, 7440-66-6; atropine, 51-55-8
scopolamine, 51-34-3.
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Received for review September 1,1988. Accepted January 9,1989.
Red Squill Modified by Lactobacillus acidophilus for Rodenticide Use
Anthony J. Verbiscar,* Thomas F. Banigan, Rex E. Marsh, and Allen D. Tunberg
Red squill (Urginia maritima, Liliaceae) bulb and root preparations were treated with three strains
of Lactobacillus acidophilus, fortifying the cultures with dry milk and oat, wheat, and rice flour.
Lactobacillus growth with the production of a @-glucosidase converted bitter glucoside scilliroside to
its tasteless aglycon scillirosidin. These products were blended into rat diets at 0.03% scillirosidin levels,
and 95% of the female rats died. Although male rats usually ate more bait than the females, none ate
enough for a lethal dose of scillirosidin. The rats learned to avoid the baits if they did not die after
initial ingestion of these fast-acting rodenticides. Technical scillirosidin mixed into rat diets had a toxic
effect on female rats similar to the L. acidophilus treated red squill products.
Red squill is being investigated as a new economic crop
for the southwest United States where it grows well
(Gentry et al., 1987). The bulb, roots, and other plant parts
contain scilliroside [6@-(acetyloxy)-3@-(@-D-gluco-
pyranosyloxy)-8,14-dihydroxybufa-4,20,22-trienolide], a
highly toxic, emetic, and bitter bufadienolide glucoside
(Verbiscar et al., 1986a, b). Because of high toxicity and
the emetic safety factor, dried bulb powders have been
used in rat baits for centuries. However, rats and mice,
which are unable to vomit, may not eat a lethal amount
of red squill baits when first exposed, resulting in formu-
lation problems.
Anver Bioscience Design, Inc., Sierra Madre, California
91024 (A.J.V., T.F.B.), and Wildlife and Fisheries Biology,
University of California, Davis, California 95616 (R.E.M.,
A.D.T.).
Our initial attempts to improve acceptability involved
conversion of scilliroside to its aglycon scillirosidin, which
is tasteless and equally toxic. The aerobic fungus As-
pergillus niger was used as a source of @-glucosidase to
elicit this cleavage (Verbiscar et al., 1987). A. niger was
grown in extracts of red squill, producing the enzyme
necessary for the hydrolysis of scilliroside to scillirosidin.
The resulting aglycon extracts were administered orally
to Charles River rats. The scillirosidin aglycons were found
to be more toxic to female rats than to males, which is also
the case for scilliroside.
In addition to the A . niger study we tested 12 strains
of Lactobacillus bulgaricus and Lactobacillus acidophilus
on hand from a jojoba detoxification project. It seemed
reasonable that because Lactobacilli cleave lactose, a ga-
lactosylglucose, the active enzyme could also cleave the
glucose from scilliroside. The Lactobacilli are nontoxic and
microaerobic, which facilitates processing. The Lactobacilli
0021-8561/89/1437-1005$01.50/0 @ 1989 American Chemical Society