Dehydrozaluzanin C: A potent plant growth regulator with potential use as a natural herbicide template

Article abstract of DOI:10.1016/S0031-9422(00)00070-4

The natural product dehydrozaluzanin C (former DHZ) is a sesquiterpene lactone obtained from different weeds of the Compositae family. Its potential as a plant growth regulator has been evaluated by using a phytotoxic allelopathic bioassay, where the commercial herbicide Logran is used as internal reference. The evaluation is made based on their effects on germination and growth over several dicotyledon and monocotyledon species. The activity was tested in the range of 1000-0.001 μM. In almost all cases, DHZ was more active than the internal reference at 1000 μM, and its activity fell below the level of the internal reference at 100 μM. These results confirm DHZ as a potent plant growth regulator and good candidate for the development of new herbicide models. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(00)00070-4

Phytochemistry 54 (2000) 165±171
www.elsevier.com/locate/phytochem
Dehydrozaluzanin C: a potent plant growth regulator with
potential use as a natural herbicide template
p
Francisco A. Macõas*, Juan C.G. Galindo, Jose M.G. Molinillo, Diego Castellano
 Â
Departamento de QuõÂmica OrgaÂnica, Facultad de Ciencias, Universidad de CaÂdiz, c/ RepuÂblica Saharaui s/n. Apdo. 40, 11510-Puerto Real, CaÂdiz,
Spain
Received 12 July 1999; received in revised form 14 February 2000
Abstract
The natural product dehydrozaluzanin C (former DHZ) is a sesquiterpene lactone obtained from di€erent weeds of the
Compositae family. Its potential as a plant growth regulator has been evaluated by using a phytotoxic allelopathic bioassay,
1
where the commercial herbicide Logran is used as internal reference. The evaluation is made based on their e€ects on
germination and growth over several dicotyledon and monocotyledon species. The activity was tested in the range of 1000±0.001
mM. In almost all cases, DHZ was more active than the internal reference at 1000 mM, and its activity fell below the level of the
internal reference at 100 mM. These results con®rm DHZ as a potent plant growth regulator and a good candidate for the
development of new herbicide models. 7 2000 Elsevier Science Ltd. All rights reserved.
1
Keywords: Sesquiterpene lactones; Guaianolides; Dehydrozaluzanin C; Logran ; Plant growth regulator; Phytotoxicity; Natural herbicides
1
. Introduction
actions among plants could be another useful
approach for the development of new classes of envir-
onmentally soft herbicides (Duke and Abbas, 1995).
Allelopathic compounds are, in fact, potential sources
of phytotoxins that could lead to design of new che-
motypes of herbicides. Surprisingly, their potential is
still relatively untapped compared to the microbial
compounds (Hay, 1999).
Modern agricultural techniques heavily rely on the
use of pesticides and herbicides to get control of pests
and weeds. However, the economic and environmental
costs of such procedures are rapidly increasing, and
sometimes, these compounds become ine€ective due to
the appearance of resistance and cross-resistance
phenomena. Natural phytotoxins constitute a large
reservoir of underdeveloped compounds with high
agricultural potential, and which might target receptor
sites not yet exploited by synthetic herbicides (Duke
and Abbas, 1995; Duke et al., 1997). Nowadays, mi-
crobial compounds have rendered many active natural
products that should be used as new herbicides (Ama-
gasa et al., 1994). The study of the chemical inter-
Sesquiterpene lactones constitute a numerous group
of compounds with several biological activities, includ-
ing the allelopathic ones (Picman and Picman, 1984;
Macõas et al., 1993, 1996). Dehydrozaluzanin C (3, for-
Â
mer DHZ) is a sesquiterpene lactone with a guaiano-
lide skeleton that has been isolated from di€erent
members of the Compositae family (Bohlman and Le
Van, 1977; Bohlman et al., 1978, 1980, 1980a, 1980b,
1
982; Bohlman and Zdero, 1982). It has also been
reported to inhibit root growth (Asakawa and Take-
moto, 1979; Kalsi et al., 1984), and recently, there was
a ®rst study relative to their mode of action (Galindo
et al., 1999), however, no further work has been
reported. We are actually conducting a systematic
p
Part 10 in the Series ``Natural products as allelochemicals'', for
Part 9, see Macõ
Corresponding author. Tel.: +34-956-016370; fax: +34-956-
16288.
E-mail address: famacias@uca.es (F.A. Macõ
Â
as et al. (1999).
*
0
Â
as).
0031-9422/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00070-4

1
66
F.A. MacõÂas et al. / Phytochemistry 54 (2000) 165±171
Fig. 1. Semi-synthesis of dehydrozaluzanin C (3) from dehydrocostuslactone (1).
evaluation of the phytotoxic activity of sesquiterpene
lactones looking for the structural requirements needed
Thus, discussion will focus on the activities at 100 and
1000 mM.
for the activity (Macõ
Â
as et al., 1992, 1999). Based on
The e€ect of DHZ (1000 mM) on germination is
higher than that obtained with the same concentration
these previous results, we have selected DHZ as one of
the most promising candidate for further studies.
In the present paper, biological data of the phyto-
toxic activity of DHZ on eight di€erent plant species is
presented, regarding three main macroscopic par-
ameters: germination, and root and shoot elongation.
1
The commercial herbicide Logran (59.4% terbume-
trine and 0.6% triasulfuron), previously selected as in-
Â
ternal standard for this type of bioassay (Macõas et al.,
000), is used, aiming at two di€erent objectives: (a) to
2
validate the results with a well-known active com-
pound or formulation and (b) to establish the relative
levels of activity of the natural product with respect to
commercial herbicides.
2. Results and discussion
Treatment of dehydrocostuslactone (1) with SeO2
and terc-butyl-hydroperoxide, as previously described
Kalsi et al., 1984; Macõas et al., 1992), a€orded a
(
Â
major product isozaluzanin C (2), which was easily
oxidized to DHZ (3) using a chloroformic solution of
pyridinium chlorochromate (PCC) (Fig. 1).
The e€ects produced by DHZ and the internal refer-
1
ence (Logran ) on the germination, and root and
shoot length of di€erent monocotyledon and dicotyle-
don species are presented in Tables 1 and 2 and Figs.
2
1
and 3. The tested concentrations ranged between
000 and 0.001 mM. Plant species were selected as
representatives of main monocotyledon and dicotyle-
don weeds and important commercial crops (Macõas et
al., 2000).
Â
2.1. Dicotyledon species
The species assayed were lettuce (Lactuca sativa cv.
nigra and cv. roman), tomato (Lycopersicon esculentum
L. cv. Tres Cantos), carrot (Daucus carota L. cv.
Coral) and cress (Lepidium sativum L.). From Table 1
and Fig. 2, no relevant activities below 10 mM were
1
Fig. 2. Selected e€ects of DHZ and Logran on germination and
growth development. 1: Lactuca sativa cv. nigra; 2: Lactuca sativa
cv. roman; 3: Lycopersicon esculentum L.; 4: Daucus carota L.; 5:
Lepidium sativum L.
1
observed for Logran , or for DHZ in almost all cases.

F.A. MacõÂas et al. / Phytochemistry 54 (2000) 165±171
167

1
68
F.A. MacõÂas et al. / Phytochemistry 54 (2000) 165±171
1
1
of Logran (Fig. 2). Moreover, DHZ is active on
species like carrot and cress where the internal stan-
dard shows no activity. On the other hand, 100 mM
treatments of the herbicide maintain the levels of ac-
tivity for both species of lettuce around 50%, while
DHZ (100 mM) has no activity.
to Logran at 100 and 1000 mM, the activity of the
herbicide being lower than that showed by DHZ (100
mM).
Among the dicotyledon species tested, cress was
most sensitive to DHZ, while all the other species were
not sensitive to DHZ at 100 mM, cress growth (root
and shoot parameters) is still strongly inhibited.
Tomato root length is the other parameter inhibited
by DHZ at this concentration.
Root growth is strongly inhibited by DHZ (1000
mM) in all the tested species (Fig. 2). Lettuce and
tomato are similarly inhibited by both treatments;
cress is more sensitive to Logran but the herbicide is
still less active than DHZ at this concentration; carrot
1
2.2. Monocotyledon species
remains insensitive to herbicide. Treatment with DHZ
1
(
tains the activity in lettuce, tomato and cress, while
100 mM) again changes the situation: Logran main-
The species assayed were onion (Allium cepa L. cv.
Valenciana), wheat (Triticum aestivum L. cv. Cortex),
barley (Hordeum vulgare L. cv. Wellam) and maize
(Zea mays L. cv. Oropesa). The situation is similar to
that observed for dicotyledon species (Fig. 3): DHZ
1
DHZ inhibitory e€ects are lower than Logran levels
in lettuce and tomato, cress is equally inhibited by
DHZ and the internal standard. It is also remarkable
that though the level of inhibition obtained in tomato
fall below that of the herbicide, it is still signi®cant to
reach at the 40% value.
A similar situation is observed for the shoot length
parameter (Fig. 2). The main di€erence is the lower
sensitivity of tomato to DHZ at 1000 mM, and of cress
1
has a higher level of activity than Logran at 1000
mM, but its activity is lowered at 100 mM below the
levels of the herbicide.
1
Regarding germination, Logran showed no rel-
evant activities on onion, or maize, while wheat shows
minor e€ects; barley is strongly a€ected both by Log-
Table 2
a
Germination and growth e€ects of DHZ (3) and Logran on di€erent monocotyledoneous plant species
Concentration DHZ (3) Logran
Allium cepa Triticum aestivum Hordeum vulgare Zea mays Allium cepa Triticum aestivum Hordeum vulgare Zea mays
Germination
�
�
�
�
�
�
�
3
4
5
6
7
8
9
10
10
10
10
10
10
10
M
M
M
M
M
M
M
� 36
� 3
� 6
1
� 63
� 1
3
� 96
� 11
� 5
� 16
� 4
1
� 18
� 4
0
� 36
� 14
� 58
� 30
� 9
� 82
� 58
� 20
� 2
� 15
15
� 12
11
13
� 7
0
� 11
� 7
� 4
� 4
� 17
� 16
29
22
� 14
� 2
3
� 9
� 8
� 11
18
� 40
� 9
0
� 16
� 38
� 2
5
� 14
� 15
Root length
�
�
�
�
�
�
�
3
4
5
6
7
8
9
10
10
10
10
10
10
10
M
M
M
M
M
M
M
� 90
� 19
� 12
9
� 82
� 4
� 1
2
� 40
� 13
� 25
� 8
� 50
12
� 80
� 72
� 66
� 63
� 49
� 41
� 25
� 28
� 31
� 24
� 5
� 3
� 5
2
� 54
� 46
4
� 81
� 60
� 37
0
19
8
45
41
42
2
14
� 2
8
� 33
� 16
9
17
31
� 12
� 17
43
40
0
� 1
34
Shoot length
�
�
�
�
�
�
�
3
4
5
6
7
8
9
10
10
10
10
10
10
10
M
M
M
M
M
M
M
� 79
� 10
� 12
� 5
3
� 55
� 4
2
� 15
� 7
� 19
� 9
� 5
� 7
8
� 66
� 66
� 61
� 59
� 44
� 42
� 21
� 26
� 25
� 26
0
� 55
� 20
� 13
� 1
� 71
� 15
7
� 14
� 3
� 7
0
� 9
16
� 12
� 13
� 13
� 6
0
� 2
� 6
� 1
� 6
� 4
� 16
� 16
� 4
17
20
9
� 3
a
Values are presented as percentage di€erences from the control (e.g., 16% means 116% compared with the control). Values are signi®cantly
di€erent from the control with P > 0.05 for the Welch's test. a: P < 0.01; b: 0.01 < P < 0.05.

F.A. MacõÂas et al. / Phytochemistry 54 (2000) 165±171
169
1
ran and DHZ (1000 mM). In germination, the higher
1000 mM. A dose-response e€ect was obtained with
the herbicide for all the tested species.
sensitivity to the treatments was shown by barley, fol-
lowed by wheat and onion. Maize remained insensi-
tive.
No other phytotoxic carbonyl containing guaiano-
lides have been described yet, but some allelopathic
isoguaianolides with an additional carbonyl group
have been reported. Ambrosin and its 11,13-dihydro
derivative damsin (isolated from Parthenium hystero-
phorus ) have been reported to inhibit germination and
growth (Kanchan and Jayachandra, 1980); conferti-
¯orin (Ambrosia conferti¯ora ) (Fischer and Mabry,
1985) strongly inhibits the germination of Amaranthus
palmeri (49%, 100 mM) and A. retro¯exus (21%, 10
mM); and partenin (P. hysterophorus ) also exerts an in-
hibitory e€ect on the germination of A. palmeri (40%,
1 mM) and carrot (24%, 100 mM) (Fischer et al.,
1989). The e€ects obtained with DHZ are higher than
Wheat root length development was more a€ected
1
by DHZ than Logran , while onion root inhibition
exhibited the same levels for both treatments; barley
and maize had slightly lower activities with DHZ
(
1000 mM) than with the internal standard. However,
1
while the levels of activity of Logran were still im-
portant with the two following dilutions, DHZ (100
mM) showed no activity.
Shoot length is strongly inhibited by DHZ at 1000
mM in onion and wheat, while barley and maize are
not a€ected. Logran shows no activity with wheat,
while onion, barley and maize are strongly inhibited at
1
1
Fig. 3. Selected e€ects of DHZ and Logran on germination and growth development. 6: Allium cepa L.; 7: Triticum aestivum L.; 8: Hordeum
vulgare L.; 9: Zea mays L.

1
70
F.A. MacõÂas et al. / Phytochemistry 54 (2000) 165±171
those reported with the above mentioned compounds.
DHZ presented a 40% inhibition on the less sensitive
carrot, while conferti¯orin is not active and partenin is
slightly active. Thus, comparison with other similar
active structures reveals DHZ as a very active and
promising lead product to be developed.
dehydrozaluzanin C (53%) and 2,3-dehydro-14-oxo-
dehydrocostuslactone (18%).
3.3. Seed germination and growth bioassays
Seeds of L. sativa cv. nigra and cv. roman, L. escu-
lentum L. cv. Tres Cantos, D. carota L. cv. Coral, L.
sativum L., A. cepa L. cv. Valenciana, and Z. mays L.
It is also important to note the di€erent pro®les of
activity showed by DHZ and the internal reference,
DHZ presenting a wider spectrum of activity. Though
DHZ could not maintain the activity with the dilution
Â
cv. Oropesa were obtained from FITO, S.L. (Barce-
lona, Spain). Hordeum vulgare L. cv. Wellam and T.
aestivum L. cv. Cortex were obtained from Rancho La
Merced, Junta de Andalucõa, Jerez, Cadiz, Spain. All
 Â
undersized or damaged seeds were discarded, and the
assayed seeds of uniform size were selected. Bioassays
were carried out in plastic Petri dishes of 9 cm diam-
eter lined with Whatman No. 1 ®lter paper.
Germination and growth bioassays were as follows:
L. sativum, 25 seeds per dish, 5 ml test solution, 4 days
dark, 258C and four replicates of each concentration;
L. sativa cv. roman and cv. four seasons, L. esculen-
tum, and A. cepa, 25 seeds per dish, 5 ml test solution,
5 days dark, 258C and four replicates of each concen-
tration; D. carota, 25 seeds per dish, 5 ml test solution,
7 days dark, 258C and four replicates of each concen-
tration; T. aestivum, H. vulgare, and Z. mays, 10 seeds
per dish, 5 ml test solution, 5 days dark, 258C and 10
1
at the same level as Logran , DHZ presented activity
at 1000 mM in species where the herbicide showed no
activity (tomato, carrot, cress and wheat). It is also
noticeable that DHZ can a€ect some parameters when
1
Logran is inactive (e.g., carrot germination). This
could be indicative of a di€erent mode of action, but
due to the nature of this study where only macroscopic
parameters, such as germination and growth, are
measured, this cannot be assured and needs to be stu-
died.
Based on the results of these experiments, DHZ can
be considered as a potent growth regulator, specially
for dicotyledon species. Consequently, it can be pro-
posed as a lead compound for further studies in the
development of new herbicides, since it presents ac-
tivity levels which are comparable (or even higher)
with those of a commercial herbicide and has an
apparently di€erent mode of action. Future research
should aim to enhance its activity, to modify the mol-
ecule in order to maintain the activity with dilution,
and to determine its speci®c site of action and the pro-
cesses a€ected at the molecular level. Another basic
aspect is to evaluate the toxicological activity of DHZ,
which should be preliminary investigated.
replicates of each concentration (Macõas et al., 2000).
Â
Test solutions (1000 mM) were prepared using H O±
2
MES (2-[N-morpholino]ethanesulfonic acid, 10 mM),
and the other solutions were obtained by dilution. Par-
allel controls were performed. All pH values were
adjusted to 6.0 before bioassay with MES. Osmotic
pressure values were measured on a vapor pressure
osmometer (WESCOR 5500) and ranged between 30
and 38 mOsmolar.
Data is presented as percentage di€erences from
control in graphics and tables (Figs. 2 and 3 and
Tables 1 and 2). Thus, zero represents the control;
positive values represent stimulation of the studied
parameter and negative values represent inhibition.
3
. Methods and materials
3
(
.1. Selenium dioxide (SeO )±terc-butylhydroperoxide
2
TBHP) oxidation
533 mg of 1 was dissolved in chloroform (0.03 M)
and 60 mg of SeO and 2 ml of TBHP (1:0, 5:2) were
3.4. Statistical treatment
2
added while stirring. After 10 min, the reaction mix-
ture was ®ltered through silica gel and then puri®ed by
Column Chromatography (CC) (n-hexane:EtOAc, 6:4)
to yield 55% of isozaluzanin C (2), 5a-hydroxy-dehy-
drocostuslactone (28%) and 5a-hydroxy-isozaluzanin
C (13%).
Germination and root and shoot length values were
tested by the Welch's test; di€erences between test sol-
utions and controls were signi®cant (P = 0.01) (Tables
1 and 2).
Acknowledgements
3.2. Oxidation of isozaluzanin C
This research was supported by the Direccio
eral de Investigacion Cientõ®ca y Tecnica, Spain (DGI-
CYT; Project No. PB95-1231). We thank FITO, S.A.
and Rancho La Merced, Junta de Andalucõa, Spain
for providing seeds for the bioassays.
Â
n Gen-
2
60 mg of 2 was dissolved in 25 ml of chloroform
Â
Â
Â
and then stirred with 465 mg of PCC (1:2). After 1 h,
the reaction mixture was ®ltered through silica gel and
then puri®ed by CC (n-hexane:EtOAc, 7:3) to yield
Â

F.A. MacõÂas et al. / Phytochemistry 54 (2000) 165±171
171
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