INDUSTRlALCROPS

ANDPRODUCTS
ANINTERNATIONALJOURNAL

ELSEVIER Industrial Crops and Products 2 (1994) 273-280

Herbicide-like effect of Lupinus alkaloids

Mercedes Muzquiz av*,Celia de la Cuadra a, Carmen Cuadrado a, Carmen Burbano a,
Rosa Calvo b
aArea de Tecnologiade Alimentos, CIT-INL4,Aptdo. 8111, Madrid 28080, Spain
b Centro de C&lculo,INIA, Aptdo. 8111, Madrid 28080, Spain
Received 22 October 1993; accepted 29 March 1994

Abstract

To ascertain the antigermination effect of lupine alkaloids, sparteine, gramine, lupanine and lupinine, germination
tests were performed with seeds of the following species: pea (Pisum sutivum L.), tomato (Lycopersicon sculentum
Mill.), wheat (Triticum aestivum L.), lamb’s_quarters (Chenopodium album L.), vetch (vi& viflosu Kunth), and
winter wild oat (Avena steri1i.s L.). Crude alkaloid extract from Lupinus ulbus (L.) and L. hispunicus (Boiss et
Reut) containing mainly lupanine and lupinine, respectively, were tested. The individual alkaloid effects of lupanine,
lupinine, sparteine and gramine at different concentrations were also analyzed. The results indicated a particularly
high antigermination effect for lupanine. A. sterilisshowed the greatest sensitivity to alkaloids. Lupanine, sparteine
and gramine also affect postgermination stages. The possible herbicide effect of Lupines alkaloids is discussed.

Keywords: Lupinus ulbus; Lupinus hispanicus; Ttiticum uestivum; Pisum sutivum; Lycopersicon sculentum; Viciu villosu;
Avenu sterilis; Chenopodium album; Germination; Alkaloids, lupanine, lupinine, sparteine and gramine

1. Introduction the seed are also in progress (Schafer-Menuhr,

Lupines, like other grain legumes, are attract-
ing attention throughout the world as a potential
source of high quality protein and fat (Gpez-
Bellido and Fuentes, 1986). One factor which
could limit the acceptance of lupine seeds is the
presence of toxic alkaloids. These compounds pos-
sess antinutritional properties, give lupines a bit-
ter taste, and influence the use of lupine plants in
the nutritional economy (Ciesiolka et al., 1988).
Processes for removal of alkaloids from lupines
are being investigated (Wink, 1984; Hill, 1985).
Breeding programs to reduce alkaloid levels in
* Corresponding author. Fax: (1) 3572 293.
1990).
However, there is evidence that alkaloids con-
stitute an element of the lupine plant’s resistance
against herbivores and pathogens (Wink, 1990).
Lupin alkaloids inhibit the germination of lettuce
and grass seeds (Wink and Twardowski, 1991) and
preliminary experiments showed that lupin plants
can reduce competing weeds under experimental
conditions (Wink, 1985). On the other hand, it
has been observed that sweet lupines have lower
fitness and natural resistance than the wild types.
These sweet varieties are useful for consumption
but have the ecological disadvantage that they
need more fences and pesticides (Wink, 1990).
In such cases, the continued cultivation of

0926-6690/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved.

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274 M. Muzquiz et al. lha’ustrial Crops and Products 2 (1994) 273-280
alkaloid-rich bitter varieties with subsequent post-
harvest debittering processes remain attractive.
For such processes to be economic, and to en-
able lupine protein to compete with alternative
products, it is necessary that the alkaloid-rich by-
products be used, for example as a raw material
for herbicides and natural agrochemicals and this
should avoid or greatly reduce the problems of
alkaloid wastes as an industrial pollutant.
This paper reports the quantitative analysis of
lupanine and lupinine alkaloids in different Lupi-
nus species and the study of the antigermination
properties of alkaloid extracts from lupins and
pure alkaloids on some plant seeds under labora-
tory conditions. We have used different crops and
weeds to test the species-specific effect of these
alkaloids.
2. Materials and methods
2.1. Plant material
The following lupine species were used. L. al-
bus: var. multolupa (Chile); var. llaima (Chile); cv.
1444 (Spain) and cv. AL (Spain). L. angustifolius:
var. ritter (Brazil) and var. chittick (Australia). L.
mutabilis: Ecotype (Peru). L. luteus: var. amarello
(Brazil); var. aurea (Chile) and Ecotype (Spain).
L. hispanicus: Ecotype (Spain).
The weeds Vicia villosa, Avena sterilis and
Chenopodium album were selected. They affect
the cultivation of different crops. Seeds of Kvillosa
were provided by Instituto National de Semillas
y Plantas de Vivero, A. sterilis from the experi-
mental farm La Canaleja of Instituto National de
Investigation y Tecnologia Agraria y Alimentaria
(INIA) in Madrid, and C. album from Laboratory
of Virology of the INIA.
The crops selected were Ttiticum aestivum (var.
anza) from INIA (El En&r), Lycopersicum scu-
lentum (var. marmande) from Mazarron (Murcia),
and Pisum sativum from Valladolid.
2.2. Analytical methods
Quantification of alkaloids
Extraction procedure. The
alkaloid extraction
method was the same as used by Muzquiz et
al. (1982, 1989). The acidic extracts were collected
and neutralized with 4 N sodium hydroxide, ex-
tracted with chloroform and evaporated to dryness
in vacua at 40°C. The residue was dissolved in 1
ml of methanol. An aliquot of 0.5 ml of this solu-
tion was used and 0.5 ml of a solution of caffeine
(Sigma, St Louis, MO., USA) in methanol (5 mg
ml-‘) was added as an internal standard.
Gas chromatography. A gas chromatograph with
an integrator was used (Perkin Elmer Sigma 1B).
A 25 m by 0.5 mm inner diameter BP-l(530) col-
umn with nitrogen as carrier gas was used (flow-
rate 6 ml min-‘).
The operating conditions for L. albus, L. an-
gustifolius and L. mutabilis were: injection tem-
perature 260°C; detector temperature 300°C; oven
temperature program 17O”C, increase 5°C min-’
to 235°C final hold 5 min. For L. luteus and L.
hispanicus the chromatographic conditions were:
injection temperature 240°C; detector tempera-
ture 260°C; oven temperature 160°C for 15 min.
The alkaloid standards were: lupinine from
Serva (Heidelberg, FRG), and lupinine isolated
in this laboratory from bitter L. hispanicus (Eco-
type). Lupanine was isolated from the seeds of
bitter L. albus (AL). Sparteine and gramine were
purchased from Sigma. Calibration curves were
prepared for lupanine and lupinine. The response
was linear with a determination coefficient of 0.99.
Purification of alkaloids
Lupanine. Lupanine perchlorate was isolated from
L. albus (AL) seeds. 800 g of flour were ex-
tracted according to the method of Muzquiz et
al. (1982) with some modifications. The aqueous
phase was evaporated to 200 ml and neutralized
with 4 N sodium hydroxide. The alkaloids were
extracted with chloroform (4 x 30 ml) and the
combined extracts were collected and evaporated
to dryness. The product was dissolved in 20 ml
of methanol and adjusted with a perchloric acid
solution in methanol (1: 1) to pH 6 to 7, accord-
ing to Perkowska et al. (1979). The white crystals
obtained were recrystallized from methanol.
Lupinine. Lupinine was isolated from L. hispani-
cus (Ecotype) seeds. The extraction was as above
for lupanine perchlorate. The aqueous phase was
evaporated to 200 ml and then made alkaline
 M. Muzquizet al. /Industrial Crops and Products2 (1994) 273-280
with 4 N sodium hydroxide. The alkaloids were
extracted with diethyl ether (4x30 ml), the ex-
tracts were combined and evaporated to dryness.
The crystals obtained were recrystallized from
petroleum ether, which separates lupinine from
sparteine (Cromwell, 1955).
Lupanine and lupinine crystals were dissolved
in methanol and the purity determined by gas
chromatography, as described above. Mass spectra
were recorded using a combined MS/GLC system
(Hewlett Packard Mod. 5992), equipped with a
quadrupole mass analyzer.
2.3. Germination experiments
The antigermination properties of whole alka-
loid extracts from L. albus, containing mainly lupa-
nine, and whole alkaloid extracts from L. hispani-
cus, containing mainly lupinine, were tested. Pure
alkaloids, lupanine and lupinine, purified from
the above alkaloids extracts as well as sparteine
and gramine from a commercial source were also
tested
Firstly, different solutions of L. albus alkaloid
extract were made in distilled water containing 0,
5, 10, and 15 mM of lupanine. Different solutions
of L. hispanicus extract were also prepared con-
taining 0, 5, and 10 mM of lupinine because the
levels in the seeds are lower than those of the lupa-
nine alkaloids. The concentrations of lupanine and
lupinine in the extracts were calculated according
to the previously analyzed alkaloid content of the
seeds used.
Filter paper discs (9 cm diam.) were placed in
Petri dishes and soaked with 5 ml of solution. All
the seeds, with exception of those of winter wild
oat, were incubated at 20°C in the dark and moist-
ened as needed. Winter wild oat was incubated
at 10°C according to the method of de la Cuadra
(1990).
To ascertain the antigermination effect of the
individual alkaloids, further studies were carried
out in the same way with the purified lupanine
and lupinine as in the previous test with alka-
loid extracts of L. albus and L. hispanicus. In this
case only those species and alkaloid concentra-
tions were included for which the extract results
were positive. Furthermore, different concentra-
215
tions of sparteine and gramine (0, 5, 10 and 15
mM) were tested.
Twenty-five seeds were placed in each Petri
dish and eight dishes were used for each species
and alkaloid. In accordance with the results of
preliminary tests where the seeds were incubated
in distilled water, the percentage of germinated
seeds was measured daily during 1 week for wheat,
pea, lamb’s_quarters and vetch because of their
rapid germination. Observations were made 3 days
a week for 3 weeks for winter wild oat and tomato.
Seeds were scored as they germinated.
When the seeds incubated with alkaloid solu-
tions began to germinate they were transferred
to another Petri dish containing distilled water to
observe if the development of the seedling was
normal or not. When the seedling showed abnor-
mal growth (i.e.: cessation of growth, dark spots
on root, no development of the root, etc.), obser-
vations and measurements were made.
2.4. Statistical analyses
A three-way analysis of variance was conducted,
with alkaloid type, alkaloid concentration and
species as factors, using the BMDP-7D (ANOVA)
programm (W.J. Dixon, BMDP Statistical Soft-
ware, Software Release, 1990). Mean values were
compared using Duncan’s multiple range test.
3. Results and discussion
The lupanine and lupinine content of the dif-
ferent Lupines species analyzed by GLC is shown
in Table 1. It is apparent that lupanine occurs
only in L. albus, L. angustifolius and L. mutabilis
samples, whereas lupinine appears in L. luteus and
L. hispanicus. These results are in agreement with
those of Muzquiz et al. (1982). Lupanine and lupi-
nine are the major alkaloids in all of these species
although other, minor, compounds also occur. For
example L. angustifolius contains angustifoline, L.
mutabilis contains sparteine, L. luteus contains
sparteine and gramine, and L. hispanicus contains
gramine. Table 1 also shows that L. albus cv. AL,
has the highest lupanine content (2.7%), repre-
senting 75% of the total alkaloid content, whereas
L. hispanicus is richest in lupinine content (l.O%),
276 M. Muquiz et aL I Industrial Crops and Products 2 (1994) 273-280

L. hispanicus

01.7

4
,
I
,

I
I
+t
a
:
C

Time (min) Time (min)

Fig. 1. Gas chromatogram of crude alkaloid extracts of L. albus (AL)-and L. hispanicus (Ecotype). Peaks: A = Iupanine; B =
lupinine; C = caffeine (internal standard).

representing 70% of the total alkaloid content
(Fig. 1). These species were therefore selected as
sources to obtain pure lupanine and lupinine.
After purification, the gas chromatogram of lu-
panine and lupinine crystals dissolved in methanol
revealed purities of over 97%. Mass spectral data
for lupanine and lupinine were identical to those
reported for the same compounds by Wink et al.
(1980).
The total percentage of germination and the
necessary time scale for each species involved
in this experiment are listed in Table 2, which
shows the results obtained when the seeds were
incubated in water. The data indicate that lamb’s-
quarter, vetch, pea and wheat have a rapid pat-
tern of germination whereas winter wild-oat and
tomato have a slow pattern. A lower viability of
the tomato seeds can also be observed. The per-
centage of germination obtained with winter wild
oat seeds indicated that the population used had
overcome its dormancy, so interference between
the action of the alkaloids and the dormancy of
the seed was not expected.
The analysis of variance applied to the data of
germination revealed that of the three factors an-
alyzed (alkaloid type, alkaloid concentration and
 M. Muzquiz et at! /Industrial Crops and Products 2 (1994) 273-280 277

Table 1 Winter wild oat (A. stetilis) is one of the most

Lupanine and Iupinine content (% dry matter) determined by pervasive weeds in Spain and is closely related
gas-chromatography
to the well-known North European and North
Samples Alkaloids American wild oat (A. fatua L.) (Peters and Wil-
lupanine lupinine
son, 1980). It is important to find control methods
that kill the seeds rather than merely extend their
L. albus multolupa 0.06 zt 0.010 -
dormancy (de la Cuadra, 1990). However, the
L. albus llaima 0.01 rt 0.002 -
L. albus 1444 2.40 f 0.010 - decrease in germination of the wheat seeds incu-
L. albus AL 2.70 f 0.029 - bated together with lupanine extract represents an
L. angustifolius ritter 1.52 f 0.080 - obvious problem as winter wild oat grows in cereal
L. angust~olius chittick 0.01 f 0.003 - crops.
L. mutabilis Ecotype 2.27 f 0.150 -
- When considering the data on the effects of
L. luteus amarelio 0.02 f 0.005
L. 1uteu.saurea - 0.01 rt 0.009 pure lupanine (Fig. 3a) it is evident that the
L. luteus Ecotype - 0.80 f 0.010 above antigermination effect in wheat is absent
L. hispanicus Ecotype - 1.00 f 0.080 (93% of germination at 15 mM). The observed in-
Mean X!Zstandard deviation of eight determinations for each hibitory effect on the germination of wheat seeds
sample. could be due to the presence of other compo-
nents in the crude extract of L. albus or perhaps,
Table 2 to the occurrence of a synergistic effect among
Maximum percentage of germination and the number of days the alkaloids present in the extract. The greater
to attain it, of the species studied when they were incubated effect of the lupanine extract as compared with
with distilled water
that of pure lupanine on winter wild oat (3% vs.
Species Germination Time 23% of germination at 15 mM) and vetch (68%
(%) (days) vs. 94% of germination at 15 mM) supports this
Lamb’s_quarters 100 2 idea, in agreement with the results of Korcz et al.
Vetch 93 4-8 (1987).
Winter wild oat 91 19-21 The lupinine results are rather less notable, as
Pea 91 3
the only species responding to the whole alkaloid
Tomato 71 17-21
Wheat 97 3-5 extract of L. hispanicus containing mainly lupi-
nine, is tomato (Fig. 2b). Here the effect of pure
lupinine (Fig. 3b) is greater (62% of germination
species) the alkaloid type (F = 82.67) and the at 10 mM) than that of the lupinine extract (75%
species assayed (F = 242.73) showed values sig- of germination at 10 mM). Although there are
nificant at the 0.1% level. The combined effect of significant differences between the control seeds
both factors (F = 34.49) as well as the triple inter- and those incubated with alkaloids, the reduced
action alkaloid-concentration-species (F = 2.33) vitality of the tomato control seeds (Table 2) leads
was also significant at the same level. to questioning the true biological significance of
The results of the germination experiments with the data.
crude alkaloid extracts from L. albus and L. hirpan- Finally sparteine (Fig. 4a) and gramine
ices are presented in Figs. 2a and 2b, respectively. (Fig. 4b) had no effect on the germination of
The extract of L. albus, containing mainly lupa- seeds according to the analysis of variance. The
nine, has an antigermination effect on winter wild effect of these alkaloids on tomato and winter
oat, wheat and vetch. However, though the effect wild oat seeds should be carefully considered be-
on winter wild oat (3% of germination at 15 mM cause of the reduced viability of the control seeds.
concentration) suggests the possibility of using this Therefore, mean comparisons were not made.
alkaloid extract as a herbicide, the antigerminative Previous data reported by Wink (1983) in-
effect on wheat (45% of germination at 15 mM dicated that germination of Lactuca sativa was
concentration) diminishes this hope. inhibited by mixtures of lupin alkaloids. 13-
278 M. Muzquiz et al. /Industrial Crops and Products 2 (1994) 273-280

Lupanine extract Lupinine extract

20
10
0
Vetch W. oat tambaq. Wheal TOmal Vetch W. oat Lambaq. Wheat Tomato Pea

??
Control ? ?
5 mM 010 mM al5 mM ??
Control Ei5 mM 010 mM

a b
Fig. 2. Antigermination effect of crude alkaloid extract of L. albus (a) and L. hispanicus (b) extracts. Each bar represents mean of

:
eight replications. Means with different letters are significantly different at P > 0.01.

::.
:.:
::
Pure lupanine

::.
:1:::i
::
y.1.

1: .:
: :
Pure lupinine

1::.

::

:
.._

Wild oat Wheat Tomato Vetch Tomato

El Control El 15 mM l3Control Cal0 mM

a
b
Fig. 3. Antigermination effect of pure lupanine (a) and pure lupinine (b). Each bar represents mean of eight repetitions. Means
with different letters are significantly different at P > 0.01.

tigloyloxylupanine and lupanine exhibited the The observations during post-germination

strongest inhibition, whereas sparteine showed a
weaker inhibition effect, in agreement with the
present results. The author also found that the
length of the radicle was affected in a similar man-
ner as germination. No evidence of a lupinine or
gramine effect on germination has been repor-
ted.
growth indicated that some alkaloids also affect
to post-germination stages. For example, the seeds
of lamb’s_quarter incubated with lupanine extract
germinated and grew until 1 or 2 mm, whereas
in the control sample they continued their normal
development. Four days after the total germina-
tion, the fresh weight of the controls was 2.21 g/
 M. Muzquiz et al. JIndusrrial Crops and Produch 2 (1994) 273-280
Sparteine
” Vetch W.oat Lambaq. Wheat Tomato
? C?ontrol Cl5 mM IZllO mM t3Il5 mM
Fig. 4. Antigexmination effect of sparteine (a) and gramine (b). Each bar represents mean of eight repetitions.
100 seeds, but only 0.696 g/100 seeds for the batch
incubated with 10 mM crude alkaloid extract of L.
‘albus. A similar effect was observed with tomato
when the lupanine concentration reached 15 mM.
Gramine and sparteine had similar effects on
lamb’s_quarters and tomato. In a latter study on
lamb’s_quarters both alkaloids produced a phyto-
toxic effect characterized by the appearance of
brown stains on the roots, showing sometimes a
poor development. It has also been observed that
the seeds ceased to grow when they were incu-
bated in gramine or sparteine. For example the
length of the lamb’s_quarter seedlings 6 days af-
ter the germination in different concentrations of
sparteine were 30 to 40 mm (5 mM), 25 to 35 mm
(10 mM), and 15 to 20 mm (15 mM); the control
seedlings were 60 to 70 mm long. Therefore, there
is a clear effect of sparteine on post-germination
stages inhibiting the growth of seedlings.
From these results is important to emphasize
that the effect of these alkaloids may be minimized
if only the percentage of germination is evaluated.
4. Conclusion
In summary we should emphasize the following
points.
279
Gramine
Pea Votch W. oat kmbsq. Wheat Tomato Pea
? l?
Control lZl5 mM 010 mM EIlS mM
(1) The alkaloid with the highest antigermina-
tion effect was lupanine.
(2) Lupanine was most inhibitory to winter wild
oat, one of the most invasive weeds in Spain.
Lupanine also affects the later development of
the seedlings. The potential use of lupanine as a
herbicide should be investigated in the future.
(3) Sparteine and gramine do not affect germi-
nation of the seed involved at concentrations of
up to 15 mM, but they produce a clear inhibition
effect on the post-germination stages.
So, it seems to be important to continue the
research in this way to evaluate the possible role
of both alkaloids as natural agrochemicals.
Acknowledgements
The authors are grateful to Dr. G.R. Fenwick,
AFRC Institute of Food Research, Norwich for
assistance and encouragement. The aid of Dr. J.C.
Tello, Mrs. T. Tirado and Dr. A. Ramos is also
greatly appreciated.
This work is partly funded by INIA SC93-162
Project and is part of the PEA Project, supported
by the Commission of the European Communities
ECLAIR Programme, and coordinated by UNIF’,
JII and IFR
280 M. h&quiz et al. llndustrial Crops and Products 2 (1994) 273-280
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