Industrial Crops and Products 21 (2005) 1–7

Alkaloid profiles of Mexican wild lupin and an effect of alkaloid

preparation from Lupinus exaltatus seeds on growth and
yield of paprika (Capsicum annuum L.)
Jacek K. Przybylak a , Danuta Ciesiołka b , Waleria Wysocka a,∗ ,
Pedro M. Garcı́a-López c , Mario A. Ruiz-López c ,
Wojciech Wysocki b , Krzysztof Gulewicz b
a Department of Alkaloids Chemistry, Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, Poznań 61-780, Poland
b Institute of Bioorganic Chemistry, Polish Academy of Science, Z. Noskowskiego 12/14, Poznań 61-704, Poland
c Centro Universitario de Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, Las Agujas,

Nextipac, Zapopan, Jal. Km 15.5, Carretera Guadalajara-Nogales, Mexico

Received 7 March 2003; accepted 4 December 2003

Abstract

With the use of GC–MS, the resolution and identification of alkaloids occurring in the seeds of six species of wild Mexican
lupins has been achieved. From among 46 detected compounds it was possible to identify unambiguously 24 of them. Most of
identified alkaloids are from the lupanine group. The percentage content of particular alkaloids in comparison with the total
alkaloid pool (ranged from 2.0 to 3.5%) in the species tested has also been determined. Alcoholic extract from Lupinus exaltatus
seeds (ALP) with low RFOs and sucrose content, introduced in different doses (80, 320 and 1600 mg d.w./pot.) to soil has
increased paprika fruit yield (for doses 320 and 1600 mg/pot) and its vegetative mass (for all applied doses). The experiments
were carried out in greenhouse in controlled conditions within a period of March–December 2001.
© 2003 Elsevier B.V. All rights reserved.

Keywords: Mexican wild lupin; Paprika; Alkaloids; GC–MS; Biological activity

1. Introduction American ones, with the exception of variable lupin

(Lupinus mutabilis, sweet one), have got fine seeds
The genus Lupinus comprises above 200 species. and are in majority mountain perennial. In recent years
From this number only a few species have become do- great interest in wild lupin species has been observed.
mesticated and cultivated on a large scale. The main It is related not only to a high protein content in these
region of wild species of lupin is the South, North and plants but also to numerous compounds they contain
Central America. Contrary to European species the showing biological activity like: alkaloids, raffinose
family oligosaccharides (RFOs), phenolic compounds
∗ Corresponding author. Fax: +48-61-865-80-08. and others (Kinghorn and Balandrin, 1984; Błaszczyk
E-mail address: wysocka@amu.edu.pl (W. Wysocka). et al., 1994; Gulewicz et al., 2002). The main limiting

0926-6690/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.indcrop.2003.12.001
2 J.K. Przybylak et al. / Industrial Crops and Products 21 (2005) 1–7

factor of using the wild lupin as protein source are and Rzedowski (2001). Voucher specimens were de-
alkaloids that in toxic doses cause neuromuscular posited at the IBUG herbarium.
blockage, respiratory depression, cyanosis, cramps
and cardiac arrest (Culvenor and Petterson, 1986). The 2.2. Extraction of alkaloids for GC–MS analyses
presence of lupin alkaloids in fodder results also in a
bitter taste, which limits its consumption by animals. Extraction of lupin seeds was carried out according
In order to prepare alkaloid-rich lupins for con- to the modified method of Harris and Wilson (1988).
sumption the seeds are subject to the debittering Finely ground and dried lupin seeds (0.5 g) were ex-
process. Extracts obtained during this process con- tracted with 5 ml of 5% trichloroacetic acid (TCA) in
tain many valuable natural compounds and show ultrasonic bath (Snorex Super AK 103H) three times
insecticide, bacteriostatic and fungistatic activities for 20 min. The crude TCA extract mixture was cen-
(Wink, 1984a, 1984b, 1988; Krzymańska et al., 1988; trifuged at 3100 × g for 30 min. The supernatant was
Wyrostkiewicz et al., 1996; Sas-Piotrowska et al., pipetted into a separatory funnel and made basic with
1996; Folkman et al., 2002). 1 ml of 10 M NaOH. The aqueous basic layer was ex-
It has been found that bitter lupin extracts have tracted three times with 15 ml of methylene chloride
beneficial effect on the growth and yield of vari- (DCM). Combined DCM extracts were dried (anhy-
ous cultivated plants (Kant and Hijazi, 1987, 1991; drous Na2 SO4 ) and filtered. The solvent was removed
Cwojdziński et al., 1989; Gulewicz et al., 1997; under vacuum. The residue was dissolved in 1 ml of
Wysocki et al., 2001). methanol and 100 ␮l of caffeine (1 mg/ml solution)
In Mexico there are about 80 wild Lupinus species was added. 1 ␮l of this mixture was injected into cap-
(McVaugh, 1987; Rzedowski and Rzedowski, 2001). illary gas chromatograph.
Their alkaloid content has been hardly known so far.
In this aspect the analysis of alkaloids in Mexican 2.3. Gas chromatography
lupins is very important. It is also essential taking
into consideration the application of the alkaloid Measurements were performed on a 5890 II
lupin preparations in the nearest future for cultivation Hewlett-Packard gas chromatograph coupled with a
and biological protection of agaves, against pests, 5971 A mass selective detector. Separation of the al-
which is presently a great problem in Mexico. This kaloids was achieved on a DB-5 fused silica capillary
paper reports the first stage of research in the area. column (30 m × 0.25 mm i.d., J&W, Folsom, CA,
We were going to compare alkaloid profiles of some USA). Helium was the carrier gas at a flow rate of
wild lupins: L. elegans, L. exaltatus, L. madrensis, 1 ml/min. The injector temperature was 290 ◦ C; the
L. mexicanus, L. montanus, L. rotundiflorus growing detector temperature was 300 ◦ C. The injections were
in Mexico and examine the influence of the alkaloid performed in the split (1:20) mode (Stobiecki et al.,
preparation obtained from L. exaltatus on the growth 1997). The temperature program was 180 ◦ C 2 min
and yield of paprika (Capsicum annuum L.). In the isothermal, 180–300 ◦ C at 5 ◦ C min−1 , 300 ◦ C 10 min
second step we plan to use various alkaloid prepara- isothermal.
tions as natural yield-increasing factors and pesticides Caffeine was used as internal standard. Retention
in agave protection. indices were estimated by co-chromatography of the
authentic sample with a homologous series of n-hydro-
carbons according to Wehrli and Kovats (1959), and
2. Material and methods were corrected with the use of McReynolds constants
(McReynolds, 1970; Supelco Bulletin 880).
2.1. Plants
2.4. Preparation of L. exaltatus extract for
The seeds of each wild lupin species were collected greenhouse experiments
during 1997 and 1998 in Jalisco State, Mexico. All
specimens were taxonomically verified following the Extraction of L. exaltatus seeds was carried out with
botanical guides of McVaugh (1987) and Rzedowski 50% of ethanol according to the method described
 J.K. Przybylak et al. / Industrial Crops and Products 21 (2005) 1–7 3

previously by Gulewicz (1991). After precipitation of 3. Results and discussion

sucrose and raffinose family oligosaccharides (RFOs)
from the extract by means of 98% ethanol (Gulewicz 3.1. Alkaloid composition of lupin species
et al., 2000) the supernatant was condensed by evap-
oration under vacuum. The so obtained extract, called The paper reports results of determination of the to-
by us “alkaloid lupin preparation” (ALP), was used in tal alkaloid content and the alkaloid composition of the
greenhouse experiments. extracts obtained from seeds of six species of Mexican
wild lupins. The extracts were examined as a whole,
without separating particular compounds from them.
2.5. Greenhouse experiments
Using the GC–MS system, it was possible to detect
46 compounds. Twenty-four of them were identified
Biological activity of the L. exaltatus extract (ALP)
unambiguously trough the comparison of their mass
was determined by its effect on the growth and yield
spectra and retention indices (collected in Table 1)
of paprika (Capsicum annuum L.) cv. Koźlak. The
with the literature data. The structure of the rest of
experiments were carried out in greenhouse in con-
the compounds remains unclear since it was impos-
trolled conditions within a period of March–December
sible to identify the molecular ions in the mass spec-
2001 using garden soil containing: 320 mg/l N; 50 mg/l
tra, and also because of the spectra’s illegibility that
P; 390 mg/l K; 2870/l Ca and micronutrients like Cu,
was often caused by overlapping with the spectra of
Fe, Mn, Mo, Zn, Mg, B. The seeds of paprika were
the substances that had left the column a while be-
sowed to the soil on March 26. The seedlings of pa-
fore (the tiling effect). However, most of them seem
prika were transplanted to pots of 12 cm diameter on
to be esters of hydroxylupanine. The attempts to re-
May 18. After 17 days the paprika was transplanted
solve the structures of these compounds are still under
once to 22 cm diameter and 5 l capacity pots. The ex-
way.
periments were performed according to the following
The total alkaloid content in the extracts was com-
scheme:
paratively high and ranged from 2.0 to 3.5%. The alka-
loid patterns of all seeds examined are diverse. Apart
1. ALP was added to the soil in doses: 80, 320 and
from gramine (an indole alkaloid) and ammodendrine
1600 mg d.w./pot.
(a dipiperidine alkaloid), both found in L. mexicanus,
2. The control combinations were treated with water
in most cases the compounds are all from the lupa-
only.
nine group. The presence of piperidine-quinolizidine
alkaloids such as angustifoline and tetrahydrorhomb-
Each combination was made with five plants si-
ifoline has also been detected.
multaneously. One pot containing one plant made
Lupanine is the main alkaloid in the seeds of L.
one repetition. ALP was added to soil of experimen-
rotundiflorus, L. exaltatus and L. mexicanus. It was
tal combinations on June 14 in volume 200 ml. To
found in considerable amounts in L. montanus and L.
control combination 200 ml of water was added.The
madrensis. In L. elegans only its traces were noted.
fruits of paprika were collected continuously dur-
Actually, in the L. elegans the presence of only four
ing vegetation time and then their weight and yield
compounds has been recorded, which is the lowest
was determined. The termination of experiment was
number within the extracts examined.
on the 17th of December, and the total yield of the
The analysis of the alkaloid content of the L. elegans
vegetative parts of paprika (leaves, pods, roots) was
seeds had been done before by Wink et al. (1995).
determined.
However, he proved the presence of six major alka-
loids: sparteine (19%), 3␤-hydroxylupanine (12%),
2.6. Statistical analysis hydroxytetrahydrorhombifoline (5%), ammodendrine
(3%), 5,6-dehydrolupanine (2%), 17-oxolupanine
Statistical calculations were carried out in support (2%) and 19 other minor alkaloids, which differs
for one way analysis of variance of balanced design significantly from the results of our investigation.
according to the method of Elandt (1964). The differences must stem from the various methods
 4
Table 1
Mass spectra, retention indices, and percentage content of alkaloids in given lupin species
Compound RIa Percent of total alkaloid content Mass spectra
Rotundiflorus Montanus Exaltatus Mexicanus Elegans Madrensis M+ 100% Most abundant ions
Gramine 1620 0.04 174 130 174 131 77 44 103
Sparteine 1785 0.20 16.79 234 137 98 193 110 234 84
Ammodendrine 1865 0.42 208 109 165 191 179 136 94 208
n.i. 4 1979 1.64 ? 260 148 55 112 82 136 231
Epiaphyllidine 2020 0.49 246 97 246 135 55 218
Tetrahydrorhombifoline 2050 13.64 248 58 207 112 108 84 148
Epiaphylline 2057 11.93 1.68 248 136 137 248 55 96 220

J.K. Przybylak et al. / Industrial Crops and Products 21 (2005) 1–7

17-Oxosparteine 2067 0.45 248 97 248 110 207 122 84 136
5,6-Dehydro-␣-isolupanine 2070 0.10 1.16 246 98 246 97 55 191 105
Angustifoline 2079 0.66 5.07 234 193 112 55 150
␣-Isolupanine 2107 18.60 10.59 0.28 248 136 248 150 55 98
n.i. 12 2111 3.71 ? 246 136 96
Aphyllidine 2127 1.53 246 98 246 136 96 149 55
5,6-Dehydrolupanine 2132 0.79 246 98 136 106 149
n.i. 15 2134 1.38 ? 98 244 106 246
Lupanine 2165 62.22 18.67 47.32 82.75 0.01 5.54 248 136 248 150 55 110 98
n.i. 17 2168 0.67 ? 209 82 210 244 248
Aphylline 2178 0.68 5.35 2.54 1.03 248 136 248 97 149 220 110
11,12-Dehydrolupanine 2193 0.59 1.09 0.63 246 246 134 55 231 148
n.i. 20 2226 1.21 7.84 0.28 25.96 6.98 246 246 136 96 82 55
11,12-Dehydrooxosparteine 2239 0.25 0.54 0.14 246 134 246 148 110 55 82
3␤-Hydroxylupanine 2255 7.69 1.09 7.13 8.13 6.68 264 264 136 150 98 84 55
n.i. 23 2261 0.28 ? 136 264 134 150 148 165
n.i. 24 2263 0.82 ? 262 136 97 134 122 150
n.i. 25 2289 0.86 0.33 ? 98 262 136 245 134
n.i. 26 2316 0.37 ? 152 55 110 246 264
Multiflorine 2320 0.56 0.47 2.62 29.52 246 134 246 148 110 217
17-Oxolupanine 2373 0.70 0.72 0.33 262 150 110 234 136
n.i. 29 2408 21.41 264 136 97 236 264 208 193
13␣-Hydroxylupanine 2410 24.57 10.95 264 152 165 264 246 134
n.i. 31 2425 0.27 ? 260 136 148 150 207
n.i. 32 2437 51.60 ? 262 97 136 98 219 234 245
n.i. 33 2457 0.25 7.35 ? 152 264 236 134 245 207
n.i. 34 2512 0.97 21.78 ? 262 152 243 244 134 136
3␤,13␣-Dihydroxylupanine 2530 2.70 280 152 280 165 262 134
n.i. 37 2545 0.70 ? 136 265 94 152 207
n.i. 38 2562 0.71 ? 165 276 150 207 112 281
n.i. 39 2663 0.46 ? 207 95 260 85 73
13␣-Angeloyloxylupanine 2748 11.25 346 246 134 112 55 231
13␣-Tigloyloxylupanine 2762 0.24 8.01 346 246 134 207 231 55 246
n.i. 42 2830 1.35 ? 207 97 264 57 281 84 55
4␤-Tigloyloxylupanine 2864 0.34 0.37 346 134 148 246 55 207
a Retention indices.
 J.K. Przybylak et al. / Industrial Crops and Products 21 (2005) 1–7 5

Table 2
The percentage content of main alkaloids in relation with the total alkaloid content
Lupinus sp. Total alkaloid Major alkaloids (percentage of total alkaloid content)
content (%)
L. rotundiflorus 3.5 Lupanine (62.2), ␣-isolupanine (18.6), 3␤-hydroxylupanine (7.7), aphyllidine (1.5), n.i. 15 (1.4),
n.i. 20 (1.2) and other 15 minor (<1%) alkaloids
L. montanus 2.6 13-Hydroxylupanine (24.6), lupanine (18.7), sparteine (16.8), tetrahydrorhombifoline (13.6),
13␣-angeloyloxylupanine (11.6), 13␣-tigloyloxylupanine (8.0), n.i. 4 (1.6), 3␤-hydroxylupanine
(1,1) and other five minor (<1%) alkaloids
L. exaltatus 2.7 Lupanine (47.3), epiaphylline (11.9), ␣-isolupanine (10.6), n.i. 20 (7.8), 3␤-hydroxylupanine
(7.1), aphylline (5.4), n.i. 12 (3.7), 11,12-dehydrolupanine (1.1), 5,6-dehydro-␣-isolupanine (1.2)
and other nine minor (<1%) alkaloids
L. mexicanus 2.8 Lupanine (82.8), 3␤-hydroxylupanine (8.1), multiflorine (2.6), aphylline (2.5), epiaphylline (1.7)
and other seven minor (<1%) alkaloids
L. elegans 2.6 n.i. 32 (51.6), n.i. 20 (26), n.i. 29 (21.4), aphylline (1.0) and traces of lupanine
L. madrensis 2.0 Multiflorine (29.5), n.i. 35 (21.8), 13-hydroxylupanine (11), n.i. 34 (7.4), n.i. 20 (7),
3␤-hydroxylupanine (6.7), lupanine (5.5), angustifoline (5.1), 3␤,13␣-dihydroxylupanine (5,96),
n.i. 43 (1.4) and other three minor (<1%) alkaloids

of extraction employed by us and Wink. From our post-precipitation supernatant are alkaloids, minerals
investigation it follows that the main alkaloids of L. (ash) and some unidentified compounds (Table 3).
elegans are n.i. 32 (51.6%), next n.i. 20 (26%) and n.i. Table 4 data illustrate the effect of ALP on the
29 (21.4%). growth and yield of paprika. As follows, the use of
The greatest numbers of alkaloids occurred in ALP in all doses (80,320 and 1600 mg d.w./pot) was
L. rotundiflorus (3.5%). From among the tricyclic caused an increase in the paprika fruit yield. The
quinolizidine alkaloids tetrahydrorhombifoline oc- yield’s increase as a result of the application of the
curs in considerable amounts in L. montanus. In this doses 320 and 1600 mg/pot was statistically signifi-
species angustifoline as a minor alkaloid has also been cant. The increase in the paprika fruit yield was in
recorded. Angustifoline is one of the major alkaloids correlation with the applied doses of ALP. ALP was
of L. madrensis. also found to have beneficial effect on the growth of
Wink et al. (1995), having studied 36 species of paprika, leading to higher vegetative mass of plants.
Northern America lupines, found that they do not ac- The dry vegetative masses of paprika obtained after
cumulate multiflorine in considerable amounts. L. ma- application of all doses of ALP were statistically sig-
drensis, which we investigated, wanders away from nificantly higher in comparison to the control sample.
these observations, since its main alkaloid is exactly The beneficial effect of lupin extracts on the growth
multiflorine. and yield of various cultivated plants is well know
The exact data concerning the percent content of (Kant and Hijazi, 1987, 1991; Cwojdziński et al.,
main alkaloids in relation to the total alkaloid con- 1989; Gulewicz et al., 1997). Up to now, the studies
tent in the lupins species examined are presented in
Table 2. Table 3
The chemical composition of alkaloid lupin preparation
3.2. The effect of ALP on the growth and Compounds % in dry weight
yield of paprika Carbohydrates 7.04
Non volatile organic acid 9.85
The extract of alkaloids-rich lupin seeds consists Protein (N × 6.25) 1.40
mainly of sugars (about 50%), sucrose and raffinose Free amino acids, peptides 12.98
family oligosaccharides (Gulewicz, 1991). By precip- Alkaloids 20.96
Ash 19.98
itation thereof with ethanol, 7% of them remain still in Others 27.79
solution. Predominant components of the L.exaltatus
6 J.K. Przybylak et al. / Industrial Crops and Products 21 (2005) 1–7

Table 4
Effect of Lupinus exaltatus extract on yied of fruits and vegetative part of Capsicum annuum
No. Doses of Yield of fresh mass of Yield of dry mass of
lupin extract fruits (in g /1 plant) vegetative part
in mg d.w./pot (in g/1 plant)
1 0 98.44 ± 6.79∗ a∗∗ 22.62 ± 2.35∗ A∗∗
2 80 119.32 ± 23.57 a 26.87 ± 0.91 B
3 320 159.27 ± 25.61 b 28.63 ± 4.23 B
4 1600 213.98 ± 44.88 c 32.74 ± 3.61 C
∗Standard deviation.
∗∗ Letters a, b and c (for fresh mass of fruits) and A, B and C (for dry mass of vegetative part) following entries indicate statistical
significance at P < 0.05.

were carried out on extracts obtained during debit- Acknowledgements

tering process of cultivated species of alkaloid-rich
lupin seeds. These extracts contain a considerable This work is a result of the bilateral scientific co-
amount (about 50%) of sugars, mainly raffinose operation joint project between Poland and Mexico.
family oligosaccharides (RFOs) which are potential These studies were supported by the State Committee
prebiotiocs—an ingredient of healthy diet. Planning for Scientific Research (grant KBN 3P06T05023) and
the utilization of these extract as yield increasing CONACYT.
agents in plant cultivation, these sugars can be easily
removed by alcohol precipitation (Gulewicz et al.,
2000). Up to now, the so modified extract (after sug- References
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