2116
J. Agric. Food Chem. 1999, 47, 2116-2119
Phytotoxins from the Leaves of Laggera decurrens
L. Van Puyvelde,* f J. Bosselaers,* C. Stevens,1 N. De Kimpe,1 J. Van Gestel,* and
P. Van Damme§
Departments of Organic Chemistry and Plant Production, Faculty of Agricultural and Applied Biological
Sciences, University of Gent, Coupure Links 653, B-9000 Gent, Belgium, and Plant Protection Research,
Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, Belgium
Upon biological screening of a series of African medicinal plants, substantial phytotoxic activity
was found in the leaves of Laggera decurrens (Vahl.) Hepper & Wood (Asteraceae), using a Lemna
minor bioassay. Bioassay-guided fractionation of the leaves led to the isolation of two physiologically
active compounds: 3-hydroxythymoquinone and 5-acetoxy-2-hydroxythymol, causing death of Lemna
minorin the 25—100 ftM range. Symptoms were a rapidly developing chlorosis, followed by necrosis
of fronds. The compounds also inhibited growth and germination of the grass weed Agrostis capillaris
down to 250 fiM. The mode of action of both compounds could not be elucidated, but they do not
appear to be photosystem II inhibitors.
Keywords: Laggera decurrens; Asteraceae: 3-hydroxythymoquinone;
phytotoxicity
INTRODUCTION
Laggera decurrens (Vahl.) Hepper & Wood (Asteraceae), formerly known as Blumea decurrens (Vahl.)
Merxm. or B. gariepina DC., is a halfshrub found in
southern Africa (Anderberg, 1991) and is well-known
for its use in traditional medicine. In Namibia, an
extract of the leaves or the roots is drunk to relieve
stomach pains and is also used against acne (Van den
Eynden and Van Damme, 1993). Some attention has
been devoted to the constituents of L. decurrens. /3-isocomene, silphinene, modhephene, thymol, its acetate,
thymoquinone, and the corresponding acetoxy derivative
2-acetoxythymoquinone as well as the phenolic compounds 5-acetoxy-2-hydroxythyrnol, 2-acetoxy-5-hydroxythymol, 5-acetoxy-2-hydroxythymol acetate, and a diol,
7/3,12-dihydroxyhimachal-2-ene (Bohlmann et al., 1985).
Upon biological screening of a series of African medicinal plants, phytotoxic activity was found in the leaves
of L. decurrens using a Lemna minor bioassay. This
paper describes the bioassay-guided isolation of the
active principles and their phytotoxic properties.
MATERIALS AND METHODS
Plant Material. Leaves of L. decurrens were collected in
the Namib desert in Namibia in February 1992. The plant was
identified by one of the authors (P.V.D.), and a voucher
specimen (VdE 2.4 e) was deposited at the National Herbarium
of Namibia in Windhoek. The specimen was air-dried and
ground to a powder.
Extraction and Isolation. Powdered leaves (1000 g) of L.
decurrens were successively extracted until exhaustion with
hexane, CHC13> EtOAc, MeOH, and H2O and concentrated
under reduced pressure. The hexane and the CHCls extracts,
which showed phytotoxic activity in the L. minor bioassay,
were further fractionated.
* Author to whom correspondence should be addressed
(telephone 32 9 264 59 59; fax 32 9 264 62 43; e-mail
luc.vanpuyvelde@rug.ac.be).
f
Department of Organic Chemistry.
*s Plant Protection Research.
Department of Plant Production.
5-acetoxy-2-hydroxythymol;
The CHC13 fraction was extracted with MeOH/H2O (1:1).
The CHCls phase was evaporated under reduced pressure to
give a CHC13-1 extract (31.04 g; 3.1%) and the MeOH removed
from the MeOH/H2O extract. The H2O phase was extracted
with CHCls to give a CHCl3-2 extract (1.5 g; 0.15%) and with
EtOAc (3.0 g; 0.3%). The phytotoxic CHC13-1 extract was
chromatographed on silica gel (glash LC) (Merck, 230—400
mesh, 700 g) and eluted with hexane, hexane/EtOAc, and
EtOAc/MeOH mixtures. A total of 160 fractions (100 mL each)
were collected and combined into 16 fractions on the basis of
similar TLC profiles. TLC analyses were carried out on TLC
plates (Merck, silica gel 60 F254) using UV and 10% H2SO4 (10
min at 105 °C) as developing agents. The phytotoxic fraction
3 (900 mg), eluted with hexane/EtOAc (7:3) (3700-4000 mL),
was rechromatographed on silica gel (flash LC) and eluted with
hexane/EtOAC (9:1) (1000 mL) and EtOAc (1000 mL). A total
of 60 fractions (50 mL each) were collected and combined into
6 fractions. Fraction 2 (0.2 g; 0.02%), eluted with hexane/
EtOAc (9:1) (600-800 mL), was finally purified on preparative
TLC (Merck TLC plates, silica gel 60F254+366, 2 mm thickness).
Four elutions with hexane/EtOAc (9:1) afforded 3-hydroxythymoquinone (105 mg; 0.01%) (1).
The hexane fraction was extracted with MeOH/H2O (9:1).
The hexane phase was evaporated under reduced pressure
(25.6 g, 2.5%) and the MeOH removed from the MeOH/H2O
extract. The H2O phase was extracted with CHCls to give a
CHCls extract (15.0 g, 1.5%). This CHCls extract was chromatographed on silica gel (flash LC) and eluted with hexane,
hexane/EtOAC, and EtOAc/MeOH mixtures. A total of 200
fractions (50 mL each) were collected and combined into 13
fractions on the basis of similar TLC profiles. The active
fraction 9 (1500 mg), eluted with hexane/EtOAc (60:40) (45005000 mL), was rechromatographed on silica gel and eluted
with hexane, hexane/EtOAC, and EtOAc/MeOH mixtures. A
total of 180 fractions (50 mL each) were collected and combined
into 16 fractions. Fraction 8-10 (804 mg, 0.08%), eluted with
hexane/EtOAc (70:30) (4200-5000 mL), afforded 5-acetoxy-2hydroxythymol (216 mg; 0.02%) (2).
3-Hydroxythymoquinone (1): orange crystals (CHCls/hexane); mp 165.1-165.4 °C; IR (KBr) v^ = 3280 (br, OH), 1610
(C=O) cm-'; 'H NMR (270 MHz, CDC13) d 1.24 (3H, d, J =
6.9 Hz, CHMe2), 2.05 (3H, d, J= 1.6 Hz, Me), 3.18 (IH, sept,
J= 6.9 Hz, CHMe2), 6.47 (IH, q, J= 1.6 Hz, CH), 6.98 (IH,
s, OH);I3C NMR (68 MHz, CDC13) <3 14.72 (Me), 19.84 (Me2),
10.1021/jf980029a CCC: $18.00 ® 1999 American Chemical Society
Published on Web 04/14/1999
Phytotoxins from L. decurrens Leaves
J. Agric. Food Chem., Vol. 47, No. 5, 1999
20
10
5
Concentration (mg/1)
2.5
1.25
0.63
0.31
2117
Control
(0.5%
DMSO)
atrazin
'.«.
juglone
thymoquinone
Compound 1
Compound 2
Figure 1. Activity of compounds 1 and 2 in the L. minor bioassay (representative experiment).
24.08 (CH), 125.50 (CCHMe2), 135.83 (CH), 140.52 (CMe),
150.76 (C-OH), 184.51 (C=O), 187.35 (O=O); EI-MS (70 eV),
mlz (rel int %) 180 [M+] (100), 165 [M+ - Me] (28), 152 (23),
147 (25), 137 (30), 109 (21), 83 (30), 69 (28), 55 (26).
.OH
OH
1
AcO
2
5-Acetoxy-2-hydroxythymol flight brown crystals (CHCls/
hexane); mp 113.9-114.5 °C; IR (KBr) vmax = 3450 (OH), 1725
(C=O) cm-'; 'H NMR (270 MHz, CDCls) 6 1.29 (6H, d, J =
7.3 Hz, Me2), 1.94 (3H, s, MeC=C), 2.31 (3H, s, MeC=O), 3.09
(IH, sept, J= 7.3 Hz, CHMe2), 5.65 (IH, s, OH), 5.76 (IH, s,
OH), 6.17 (IH, s, CH arom); I3C NMR (68 MHz, CDCls) 6 15.23
(Me), 20.65 (Me2), 21.08 (Me), 25.89 (CHMe2), 114.43 (CH
arom), 122.67 (C-Me), 123.50 (C-Me2), 135.53 (C-OH),
141.65 (C-OAc), 144.32 (C-OH), 171.76 (C=O); EI-MS (70
eV), m/z (rel int %) 224 [M+] (33), 182 [M - ketene]+ (86), 167
[182 - Me]+ (100).
Biological Assays. Fractionation was guided using a L.
minor bioassay: an appropriate amount of the test fractions,
dissolved in acetone, was added to 4 mL of Pirson and Seidel
medium (Pirson and Seidal, 1950) in the wells of square 25well multiwell plates. The maximum 0nal acetone concentration in the medium was 1%. Each well was subsequently
inoculated with one axenically cultured L. minor L. plant in
the four-leaf stage. The plates were incubated in the laboratory
at 27 °C under continuous cool white fluorescent illumination
(90 [imoVm2 s photosynthetically active radiation). The percentage of phytotoxicity (necrosis and growth inhibition), as
compared to untreated controls, was evaluated visually after
2 weeks by estimating the well surface area covered by green,
healthy leaf tissue. Acetone (1%) was used as a negative
control. Juglone (5-hydroxy-l,4-naphthoquinone, 97% pure,
Janssen Chimica, Geel, Belgium), thymoquinone (2-isopropyl5-methyl-l,4-benzoquinone, Sigma), and atrazine [2-chloro-4(ethylamino)-6-(isopropylamino)-6-triazine, Janssen Chimica]
were used as reference compounds. To obtain a quantitative
measure of compound activity, the experiment was repeated
in quadruplicate in standard 24-well multiwell plates, with
purified samples of compounds 1 and 2 and thymoquinone and
juglone as reference compounds, by determining fresh weight
of the Lemna plants in each well after 2 weeks of incubation.
For further study of the phytotoxic properties of compounds
1 and 2, selected agar-grown weed species were used. Compounds 1 and 2 were dissolved at 0.2 M in DMSO and added
in appropriate amounts to liquid Hoagland's mineral nutrient
medium containing 0.8% of agar (Hoagland and Arnon, 1950).
The maximum final DMSO concentration in the medium was
0.5%. The medium was allowed to solidify in standard 24-well
multiwell plates, and the wells were subsequently seeded with
Arabidopsis thaliana (L.) Heynh., SolanumnigrumL., Agrostis
capillaris L., or Poa annua L. Wells were then incubated in a
Weiss climate room with a 16 h day at 22 °C and an 8 h night
at 20 °C. Light intensity was 8 ^mol/m2 s photosynthetically
active radiation during the first 5 days (germination) and 90
^mol/m2 s photosynthetically active radiation thereafter. Evaluation was performed 1 week after sowing by visually scoring
plant injury and death using a linear, 11-stage rating scale
as follows: 0 = no growth, 1 = 1-10%, 2 = 11-20%, 3 = 2130%, 4 = 31-40%, 5 = 41-50%, 6 = 51-60%, 7 = 61-70%, 8
= 71-80%, 9 = 81-90%, 10 = 91-100% healthy green leaf
tissue as compared to controls. Experiments were repeated
four times. DMSO (0.5%) was used as a negative control.
Juglone and thymoquinone were used as reference compounds.
To evaluate effects on germination, compounds 1 and 2 were
dissolved at 0.2 M in DMSO and added in appropriate amounts
to deionized water. Six layers of filter paper (Schleicher and
Schuell 5892 ashless "white ribbon") were placed on the bottom
of the wells of standard multiwell plates, and 0.25 mL of
deionized water containing the test compounds was added to
them. Each well was subsequently seeded with 20 A. capillaris
seeds. The multiwell plates were then incubated in a Weiss
climate room, as described above. Evaluation was performed
after 2 weeks by counting the number of germinated seeds in
each well and calculating percentage germination. Experiments were repeated four times. DMSO (0.5%) was used as a
negative control. Juglone and thymoquinone were used as
reference compounds.
2118 J. Agric. Food Chem., Vol. 47, No. 5, 1999
Fluorescence transients from microwell-grown A. capillaris
plants were recorded using a Hansatech modulated fluorescence measurement system (Ogren and Baker, 1985), by
positioning single 16 mm diameter wells under the hole in the
instrument's leaf clip. Atrazine was used as a reference
compound.
RESULTS AND DISCUSSION
Bioassay-guided fractionation of the hexane and
chloroform extracts of the leaves of L. decurrens led to
the isolation of two phytotoxic compounds, 1 and 2.
The structure of 3-hydroxythymoquinone (1) was
deduced from the spectrometric data. The !H NMR
revealed the presence of a methyl, an isopropyl, a
hydroxy group (also visible in the IR spectrum), and an
aromatic proton. Because of the allylic coupling of the
methyl group with the olefinic proton, the methyl group
is located at the ortho position of the olefinic proton.
The 13C NMR data showed two carbonyl signals together
with three quaternary carbons (125—150 ppm) and a
CH signal at 135 ppm, characteristic of a quinone
moiety. The mass spectral analysis (molecular weight
= 180) also confirmed the presence of the substituents
mentioned above. 2D-HETCOR and COSY measurements were also in agreement with the structure of
3-hydroxythymoquinone, which was synthesized (JozephNathan et al., 1987) and isolated before from Antiphiona
pinnatisecta (Zdero and Bohlmann, 1989). These chemical findings allow one to accept the relative positions of
the methyl and isopropyl groups in 3-hydroxythymoquinone.
The structure of 5-acetoxy-2-hydroxythymol (2) could
also be deduced from the spectroscopic data, although
more advanced NMR techniques had to be used to
distinguish between the different isomers. The substituents on the aromatic ring were easily found from the
'H NMR, that is, a methyl, an isopropyl, two hydroxy
groups, and an acetoxy group (confirmed by a carbonyl
absorption in the IR and a carbonyl signal in the 13C
NMR). The structure of the pentasubstituted aromatic
ring was also confirmed by the mass spectral analysis,
which showed a molecular ion at mlz22$. As mentioned
before, the relative position of the substituents on the
aromatic ring had to be determined by ECHO—INADEQUATE and NOESEL experiments. ECHO-INADEQUATE measurements revealed cross-peaks between
171.76 and 141.65 ppm and between 15.23 and 139.33
ppm, proving that the methyl group is located at the
ortho position of a hydroxy group and that the acetoxy
group is attached to the carbon atom resonating at
141.65 ppm. A NOESEL experiment with irradiation
at d 6.17 led to a considerable reduction of the complexity of the signals at 141.65 ppm (the signal becomes a
doublet), proving that the acetoxy group is located at
the ortho position of the aromatic proton. Combining
this information led to the conclusion that the active
compound was 5-acetoxy-2-hydroxythymol (2). The positioning of the substituents is in agreement with some
monoterpene analogues isolated from Relhania species
(Tsichritzis and Jakupovic, 1990), and compound 2 was
also isolated from Blumea alata (3) and Antiphiona
species (Zdero and Bohlmann, 1989) (no 13C spectral
data given). Compound 2 was obtained as a crystalline
product, although it has been reported as an oil by
Bohlmann and co-workers (Bohlmann et ail., 1985; Zdero
and Bohlmann, 1989).
Bioassay-guided fractionation of the extracts using L.
minorshowed that compounds 1 and 2 completely killed
Van PuyveJde et al.
Table 1. Effect of 1 and 2 on Fresh Weight Accumulation
of L. minor Plants As Compared to Juglone and
Thymoquinone*
test
compd
100
1
7.6* (4.3)
2
6.9* (1.8)
juglone 101.8(3.9)
thymo96.4(8.6)
quinone
concentration (x 10 6 M)
1.56
6.25
25
34.9* (9.3)
42.8(11.3)
98.2(6.4)
104.9(11.3)
0.39
60.7 (3.0) 66.0 (9.6) 67.2 (4.3)
72.5(13.4) 66.1(9.1) 99.3(22.1)
102.8(5.7) 117.0(23.2)100.8(9.3)
98.7(7.5) 96.0(4.6) 103.4(7.7)
a
Growth is expressed as milligrams of fresh weight per treatment after 2 weeks of incubation, mean of four replicates. Standard
deviation shown in parentheses. Control = 102.8 (13.5). Asterisks
indicate significant difference from control at 1% level using
Student's test.
Table 2. Effect of 1 and 2 on Agar-Grown A. capillaris
Plants As Compared to Juglone and Thymoquinone"
concentration (x 10 6 M)
test
compound
250
125
63
1000
500
0*(0)
10(0)
1
1*(1)
8(1)
4*(D
2
10(1)
0*(0)
8(1)
0*(0)
1*(D
juglone
8(2)
10(1)
0*(0)
2*(«
10(0)
10(0)
thymoquinone
4(2)
10(1)
9(1)
3
Plant injury is expressed as a score from a 0—10 rating scale,
mean of four replicates. Standard deviation shown in parentheses.
Control = 10 (1). Asterisks indicate significant difference from
control at 1% level using Student's test.
1*(1)
Table 3. Effect of 1 and 2 on Germination of A. capillaris
Seeds As Compared to Juglone and Thymoquinone"
concentration (x 10~6 M)
50
25
test compound
400
200
100
1
25
(
1
4
)
54(3)
63
(10)
58(3)
5* (4)
8* (6) 15(8) 54(8) 63 (12) 59 (19)
2.
juglone
1*(3) 15 (11) 31 (3) 44 (24) 51 (18)
thymoquinone 40(11) 43 (10) 58 (13) 54(5) 60 (4)
a
Percentage germination, 2 weeks after sowing, mean of four
replicates. Standard deviation shown in parentheses. Control =
64 (10). Asterisks indicate significant difference from control at
1% level using Student's test.
the indicator organism down to 2.5 mg/L (14 ^M) and 5
mg/L (22 fiM), respectively, as can be seen in a photograph of a representative experiment (Figure 1). The
related compound thymoquinone was inactive at the
concentrations tested (0.3—20 mg/L), and the allelopathic quinone compound juglone, which was tested for
comparison, caused complete necrosis at 20 mg/L (115
fjM) only. In a separate experiment, the effects of
compounds 1 and 2 and thymoquinone and juglone on
L. minor fresh weight accumulation were compared at
five doses, ranging from 100 to 0.39^M. As can be seen
in Table 1, only compounds 1 and 2 significantly
inhibited fresh weight accumulation in the test plants
within this dose range, at concentrations down to 25
and 100 ^M, respectively. The higher doses of 1, 2, or
juglone caused a rapidly (within 48 h) developing
chlorosis in L. minor plants, followed by complete or
partial necrosis. The symptomology of plant injury was
markedly different from what could be observed with a
photosystem II inhibiting herbicidal compound such as
atrazine, which caused, down to 2.5 mg/L (12 fiM), a
slowly developing necrosis that took > 1 week to show
its full effect.
The phytotoxic properties of compounds 1 and 2 were
further studied on agar-grown seedlings of the weeds
A. thaliana, S. nigrum, A. capillaris, and P. annua.
Compounds 1 and 2 were mainly active on the grasses
Phytotoxins from L. decurrens Leaves
juglone
(0.25 mM)
thymoquinone
(0.25 mM)
J. Agric. Food Chem., Vol. 47, No. 5, 1999
compound 1
(0.25 mM)
compound 2
(0.25 mM)
check
(0.5% DMSO)
2119
atrazin
(0.025 mM)
Figure 2. Fluorescence induction curves of treated agar-grown A. capillaris plants.
and weak on the two dicotyledonous plants (data not
shown). This suggests a narrower spectrum than that
of juglone, which completely inhibited growth of all four
weeds down to 500 [iM. Subsequently, an experiment
on agar-grown A. capillaris, comparing compounds 1
and 2 with thymoquinone and juglone, was performed.
The results are summarized in Table 2.
Compounds 1 and 2 and juglone significantly inhibited growth of A. capillaris down to 250 ^M in this
experiment, whereas thymoquinone was inactive. The
affected plants were stunted, but no obvious chlorosis
could be observed, in contrast to the effects on L. minor.
Because the phytotoxicity observed appeared to be a
combination of growth retardation and germination
inhibition, a germination experiment with A. capillaris
seeds was also performed. The results are summarized
in Table 3. As it turned out, compounds 1 and 2 and
juglone inhibited A. capillaris germination, but results
were only significant at 400 fiM.
Interaction with photosystem II and inhibition of
photosynthetic light reactions have been described for
various types of quinones (Pfister et al., 1981; Renger
et al., 1988; Nimbal et al., 1996; Gonzales et al., 1997;
Rimando et al., 1998). Moreover, inhibition of the
photosynthesis by the well-known allelopathic quinone
compound juglone has been reported (Hejl et al., 1993),
although the main mode of action of this compound
appears to be inhibition of respiration (Perry et al., 1967;
Koeppe, 1972). To check for photosynthetic electron
transport inhibition as a possible mode of action for
compounds 1 and 2, fluorescence transients from microwell-grown A. capillaris plants were recorded using
a Hansatech modulated fluorescence measurement system (Ogren and Baker, 1985). The measurements
revealed normal fluorescence decay curves in plants
treated with 1, 2, thymoquinone, and juglone, as compared to the typically flat response curve in atrazinetreated plants. This rules out photosystem II inhibition
as a possible mode of action for compounds 1 and 2
(Figure 2).
To conclude, the L. minor bioassay proved to be useful
in guiding the isolation of the two phytotoxic compounds
3-hydroxythymoquinone (1) and 5-acetoxy-2-hydroxythymol (2) from the leaves of L. decurrens. The phytotoxic properties of compounds 1 and 2 have not been
reported before. The compounds are more potent than
the well-known allelopathic compound juglone on L.
minor but significantly weaker on the dicotyledonous
weeds tested. For the grass species A capillaris, on the
other hand, the inhibitory effect of compound 2 is
comparable to that of juglone, whereas compound 1 is
slightly weaker. Compounds 1 and 2 and juglone cause
growth retardation as well as germination inhibition in
A. capillaris. Results from variable fluorescence measurements rule out photosystem II as a possible mode
of action. The nonhydroxylated analogue thymoquinone
was nearly inactive in all assays performed.
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Received for review January 13, 1998. Revised manuscript
received January 29, 1999. Accepted February 6, 1999.
JF980029A
Jan Bosselaers