Article

pubs.acs.org/jnp

Phytotoxic and Nematicidal Components of Lavandula luisieri

Luis F. Julio,† Alejandro F. Barrero,*,‡ M. Mar Herrador del Pino,‡ Jesús F. Arteaga,§ Jesús Burillo,⊥
Maria Fe Andres,† Carmen E. Díaz,∥ and Azucena González-Coloma*,†
†
Instituto de Ciencias Agrarias, Consejo Superior de Investigaciones Científicas, Serrano 115-bis, 28006 Madrid, Spain
‡
Departamento de Química Orgánica, Instituto de Biotecnología, Universidad de Granada, Campus de Fuente Nueva, s/n, 18071
Granada, Spain
§
CIQSO, Center for Research in Sustainable Chemistry and Department of Chemical Engineering, Physical Chemistry, and Organic
Chemistry, Facultad de Ciencias Experimentales, Universidad de Huelva, Campus el Carmen, 21071 Huelva, Spain
⊥
Departamento de Ciencia, Tecnología y Universidad, Centro de Investigación y Tecnología Agroalimentaria de Aragón, Gobierno de
Aragón, Avenida Montañana, 930, Zaragoza, Spain
∥
Instituto de Productos Naturales y Agrobiología, Consejo Superior de Investigaciones Científicas, Avenida Astrofísico F. Sánchez, 3,
38206 La Laguna, Tenerife, Spain
*
S Supporting Information

ABSTRACT: Several preparations were obtained from the aerial parts of predomesticated Lavandula luisieri, including the
essential oil and ethanolic, hexane, and ethyl acetate extractives. Additionally, pilot plant vapor pressure extraction was carried out
at a pressure range of 0.5−1.0 bar to give a vapor pressure oil and an aqueous residue. A chemical study of the hexane extract led
to the isolation of six necrodane derivatives (1, 2, and 4−7), with four of these (1, 2, 5, and 7) being new, as well as camphor, a
cadinane sesquiterpene (9), tormentic acid, and ursolic acid. The EtOAc and EtOH extracts contained a mixture of phenolic
compounds with rosmarinic acid being the major component. Workup of the aqueous residue resulted in the isolation of the
necrodane 3 and (1R*,2S*,4R*)-p-menth-5-ene-1,2,8-triol (8), both new natural compounds. The structures of the new
compounds were established based on their spectroscopic data. The phytotoxic and nematicidal activities of these compounds
were evaluated.

Lavandula luisieri (Rozeira) Riv.-Mart (Lamiaceae)1 is a small
aromatic shrub endemic to the Iberian Peninsula. Previous
studies have shown that L. luisieri essential oil contains 1,8-
cineole, lavandulol, linalool, and their acetates, in addition to a
series of compounds with a 1,2,2,3,4-pentamethylcyclopentane
(necrodane) structure.2−4 L. luisieri essential oil has also proven
to have antifungal, antibacterial, and antioxidant effects.4−8 This
volatile oil inhibits β-secretase (BACE-1) and thus represents a
promising therapeutic alternative for Alzheimer’s disease,9 due
to its content of 2,3,4,4,-tetramethyl-5-methylenecyclopent-2-
enone.10
The chemotype distribution and resultant bioactivity of the
essential oils of samples of L. luisieri exhibit wide variations in
the Iberian Peninsula. The major components found were
camphor, 1,8-cineole, and 2,3,4,4-tetramethyl-5-methylene-2-
© XXXX American Chemical Society and
American Society of Pharmacognosy
cyclopenten-1-one, for central and southern populations,11 and
trans-α-necrodyl acetate for western samples. A preliminary
experimental cultivation of L. luisieri yielded essential oils with
insect antifeedant effects stronger than those of wild-grown
plants.12−14 Additionally, CO2 supercritical extracts of L. luisieri
showed an increased concentration of necrodane-type ketones
and stronger insect antifeedant effects than hydrodistilled and
ethanolic extracts.15 On the basis of these results, we have
initiated the domestication of L. luisieri to obtain a viable variety
for natural-product-based biopesticide production.
In this contribution, compounds with a necrodane skeleton
and other secondary metabolites from the vapor pressure
Received: June 29, 2015
A DOI: 10.1021/acs.jnatprod.5b00501
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Article

essential oil and organic (ethanol, ethyl acetate, and hexane)
and aqueous extracts of experimentally cultivated L. luisieri
specimens are reported. Additionally, their biocidal effects
(phytotoxic and nematicidal effects) have been tested. The
necrodane derivatives isolated, 1−3, 5, and 7, and compound 8
are new natural products.
triterpene acids tormentic acid17 and ursolic acid.17,18 The
EtOAc and EtOH extracts contained a mixture of phenols, with
rosmarinic acid19 being the major component.
The molecular formula of 1 was determined as C10H16O2 by
HREIMS ([M]+, m/z 168.1147). This together with the NMR
data (Table 1) suggested a monoterpene with three degrees of
unsaturation, possessing a cyclopentane structure with a gem-
dimethyl group and two methyl groups on a double bond. The
IR spectrum showed two absorption bands at νmax 3406 and
1686 cm−1 due to a hydroxy group and an α,β-unsaturated
ketone, respectively. The hydroxy group corresponded to a
primary alcohol, as deduced from NMR data. The 13C NMR
spectrum confirmed the presence of a tetrasubstituted double
bond conjugated with a ketone group. These data were in
agreement with a necrodane skeleton,2 and therefore, the
structure of 1 was assigned as 5-(hydroxymethyl)-2,3,4,4-
tetramethylcyclopent-2-enone.
Compound 2 showed a molecular formula of C12H18O3 as
established by HREIMS ([M]+, m/z 210.1254). Its IR spectrum
showed absorption bands due to an acetate group (1734 cm−1)
and an α,β-unsaturated ketone (1668 cm−1). The NMR data
(Table 1) indicated the presence of a necrodane structure
closely related to that of 1, with the main differences being the

■ RESULTS AND DISCUSSION

Tables S1 and S2 (Supporting Information) show the
phytotoxic, nematicidal, and insect antifeedant effects of the
different L. luisieri extracts. Workup of the aqueous and hexane
extract and vapor pressure essential oil led to the isolation of six
necrodane derivatives (1−7). Five of these (1−3, 5, and 7) are
new, whereas 6 (characterized as the methyl ester 6a) and 4
were previously reported.2 Additionally, camphor and 10-
hydroxy-4(5)-cadinen-3-one (9)16 were also identified. When
the hexane extract was cooled to room temperature, an
insoluble fraction was separated from the solution. The study of
this insoluble material led to the isolation of the known
strong deshielding of the H-6 signals (Δδ = δ(2) − δ(1) = 0.70
and 0.41 ppm) and the presence of signals corresponding to an
acetyl group. These data allowed 2 to be identified as 2,2,3,4-
tetramethyl-5-oxocyclopent-3-en-1-ylmethyl acetate.
Compound 3 showed a molecular formula of C10H16O3
established by HRESITOFMS ([M + Na]+, m/z 207.0991).
The NMR data (Table 1) indicated the presence of a
necrodane structure closely related to 1, with the main
differences being the strong deshielding of the C-5 signal
(Δδ = δ(3) − δ(1) = 20.4 ppm) and the absence of the proton
H-5, substituted by a hydroxy group instead. The presence of a
hydroxy group at C-5 was confirmed by the HMBC correlation
of the corresponding carbon signal with the H-6 hydroxy-

Table 1. NMR Spectroscopic Data (500 MHz, CDCl3) for Compounds 1−3, 5, 6a, and 7

1 2 3 5 6a 7
δH (J in δH (J in δH (J in δH (J in
position δC, type δH (J in Hz) δC, type δH (J in Hz) δC, type Hz) δC, type Hz) δC, type Hz) δC, type Hz)
1 213.6, C 205.7, C 209.2, C 196.0, C 145, CH 7.12 s 160.8, C
2 136.4, C 134.0, C 131.3, C 137.6, C 130.3, C 119.7, C
3 180.3, C 176.1, C 176.8, C 173.9, C 156.5, C 156.1, C
4 46.4, C 44.4, C 47.4, C 44.4, C 53.5, C 44.5, C
5 59.8, CH 2.30, dd 55.7, CH 2.47, dd 80.2, C 152.1, C 142.9, C 172.2, C
(5.7, 8.9) (4.2, 9.2)
6 63.2, CH2 3.77, dd 62.3, CH2 4.18, dd 66.2, CH2 3.57, m 114.4, 5.34, s 163.9, C 15.3, CH3 1.97, d
(5.7, (9.2, CH2 (0.95)
10.5) 11.9)
3.83, dd 4.53, dd 3.64, m 6.04, s
(8.9, (4.2,
10.5) 11.9)
7 10.5, CH3 1.66, s 8.3, CH3 1.67, s 8.0, CH3 1.72, s 56.1, CH2 4.42, s 12.2, CH3 1.86, d 13.2, CH3 1.98, d
(0.95) (0.95)
8 14.5, CH3 1.94, s 11.9, CH3 1.95, s 12.4, CH3 1.98, s 11.3, CH3 2.02, s 9.9, CH3 1.79, d 25.6, CH3 1.49, s
(0.95)
9 25.0, CH3 1.20, s 22.8, CH3 1.23, s 19.7, CH3 1.106, s 25.4, CH3 1.25, s 21.4, CH3 1.16, s 25.6, CH3 1.49, s
10 29.2, CH3 1.02, s 27.0, CH3 1.09, s 24.8, CH3 1.113, s 25.4, CH3 1.25, s 21.4, CH3 1.16, s
OH 3.30, br s
COCH3 21.1, CH3 2.05, s
COCH3 171.0, C
OCH3 50.7, CH3 3.74, s

B DOI: 10.1021/acs.jnatprod.5b00501
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Article

methylene protons and the methyl singlets at C-9 and C-10. Scheme 1. Possible Biosynthetic Pathway of Necrodanes in
These data allowed compound 3 to be assigned as 5-hydroxy-5- L. lusieri
(hydroxymethyl)-2,3,4,4-tetramethylcyclopent-2-en-1-one.
The molecular formula of 5 was determined as C10H14O2 by
HREIMS ([M]+, m/z 166.0991). Its IR spectrum showed
absorption bands characteristic of a hydroxy (3427 cm−1) and a
carbonyl group (1684 cm−1). The hydroxy group corresponded
to a primary allyl alcohol, as shown by the NMR spectroscopic
data (Table 1). These values together with other signals
observed in the NMR spectra showed that 5 is a hydroxylated
derivative of 4. A careful study of the HMBC and NOESY
correlations (Figure 1) allowed the spatial arrangement of these

Figure 1. HMBC and NOESY correlations of compound 5.

substituents on the ring to be determined unambiguously, and

therefore 5 was identified as 2-(hydroxymethyl)-3,4,4-trimeth-
yl-5-methylenecyclopent-2-enone. Table 2. NMR Spectroscopic Data (500 MHz, CDCl3) for
The molecular formula of 7 was determined as C9H13O3 by Compound 8
HREIMS ([M + H]+, m/z 169.0861). This formula suggested a
monoterpene with four degrees of unsaturation. Its IR position δC, type δH (J in Hz) COSY HMBCa NOESY
spectrum showed three major absorption bands at νmax 1783, 1 69.8, C
1737, and 1052 cm−1, due to the presence of a CO−O−CO 2 73.3, CH 3.86, dd (6.0, H-3 1, 4, 6, 8 H-7
group. The IR and NMR data (Table 1) allowed the structure 2.8)
of 2H-pyran-2,6(3H)-dione to be established for 7 with a gem- 3 27.4, α 1.95, dd (6.0, 1, 2, 4, 5 H-9, H-
CH2 2.8) 10
dimethyl group and two methyl groups on a double bond.
β 1.86, dd H-7, H-9,
These data were confirmed by 2D NMR studies (HSQC and (13,9, 6.9) H-10
HMBC) and permitted the assignment of 7 as 3,3,4,5- 4 42.7, CH 2.35, m H-3 3, 5, 6, 7 H-2, H-9,
tetramethyl-2H-pyran-2,6(3H)-dione, a new nor-necrodane H-10
compound. 5 129.5, 5.93, dd (10.4, H-6, H-4 1, 3, 4 H-9, H-
The necrodane carbon skeleton (1,2,2,3,4-pentamethylcyclo- CH 2.8) 10
pentane) of 1−7 indicates that their biosynthesis did not 6 133.5, 5.75, ddd (10.4, H-5 2, 4, 8 H-7
CH 2.5, 0.9)
involve prenylation between the monoterpene precursors, 7 24.1, 1.34, s 1, 2, 6
DMAPP and IPP. The involvement of DMAPP and isoprene CH3
(derived from the removal of HOPP from IPP) in their 8 72.7, C
biosynthesis has been proposed.20 Enzymes with acid centers 9 28.3, 1.29, s 4, 7, 10
(prenyltransferase and/or cyclase) could selectively protonate CH3
the disubstituted double bond of the isoprene, leading to 10 26.9, 1.24, s 4, 7, 9
CH3
prenylation−cyclization−deprotonation that would form nec- a
rodols. These necrodols could be the precursors of other HMBC correlations, optimized for 6 Hz, are from proton(s) stated to
necrodanes found in L. luisieri through oxidation, eliminations, the indicated carbon.
and acetylation (Scheme 1).
Workup of the neutralized-lyophilized aqueous residue
resulted in the isolation of compound 8. The molecular
formula of 8 was determined as C10H17O3 by HREIMS ([M]+,
m/z 185.1181), indicating two degrees of unsaturation. The 1D
and 2D NMR data (Table 2) were consistent with a p-
menthane skeleton with three hydroxy groups at the C-1, C-2,
Figure 2. Significant NOESY correlations of compound 8.
and C-8 positions and one double bond between the C-5 and
C-6 positions.21 The observed NOE effects of H-2β with CH3-7
and H-4 in the NOESY spectrum indicated a syn relationship
between the hydroxyisopropyl group at the C-4 position and sativa. Necrodanes 1, 2, 6, 6a, and 7, cadinane 9, and rosmarinic
hydroxy groups at the C-1 and C-2 positions (Figure 2). These acid inhibited the germination of L. sativa between 24 and 72 h
data allowed compound 8 to be proposed as (1R*,2S*,4R*)-p- (up to 168 h for 6a), but only 7 reduced its root growth at all
menth-5-ene-1,2,8-triol. The absolute configuration at the doses tested. Compounds 1, 2, 5, 6, and 7 inhibited both the
stereogenic centers has not been confirmed. germination and growth of L. perenne. Cadinane 9 showed
Table 3 shows the results of the phytotoxic effects of the pure similar effects. The new nor-necrodane 7 was the only
compounds. Overall, L. perenne was more sensitive than L. compound with phytotoxic effects against both plant species.
C DOI: 10.1021/acs.jnatprod.5b00501
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Article

Table 3. Phytotoxic Effects of Compounds 1−9

Lactuca sativa Lolium perenne
germinationa growtha germinationa growtha
compound mg/mL 24 h 144 h root 72 h 168 h root leaf
1 0.20 84.0 ± 5.0 100.0 ± 0.0 94.3 ± 7.2 36.7 ± 3.6b 67.6 ± 9.4 74.7 ± 9.5b 63.6 ± 9.3b
0.10 50.0 ± 27.0b 93.5 ± 13.6 77.2 ± 13.3b 76.9 ± 16.5b
0.05 68.8 ± 15.4b 96.8 ± 7.2 89.3 ± 11.7 94.1 ± 17.6
2 0.20 66.0 ± 7.0b 100.0 ± 0.0 88.9 ± 7.6 20.0 ± 6.7b 73.0 ± 8.3b 73.0 ± 12.6b 56.2 ± 12.7b
0.10 36.7 ± 8.5b 59.5 ± 3.5b 88.7 ± 13.8 74.9 ± 14.0b
0.05 75.0 ± 23.4b 83.9 ± 7.0 80.3 ± 11.3 82.5 ± 18.4
4 0.20 87.0 ± 9.0 100.0 ± 0.0 124.6 ± 9.6 75.0 ± 23.4b 106.5 ± 7.1 99.1 ± 12.4 104.2 ± 18.1
5 0.20 117.0 ± 9.0 108.1 ± 8.8 123.2 ± 7.9 68.8 ± 15.4b 100.0 ± 7.0 52.9 ± 7.3b 110.4 ± 14.7
6 0.20 24.0 ± 9.0b 100.0 ± 0.0 108.4 ± 8.2 50.0 ± 11.8b 85.3 ± 10.4 47.1 ± 6.8b 68.3 ± 10.9b
0.10 82.7 ± 16.9 100.0 ± 10.2 86.5 ± 10.1 66.6 ± 10.9b
0.05 62.5 ± 20.6b 96.8 ± 8.9 106.9 ± 12.7 112.8 ± 20.2
6a 0.20 0.0 ± 0.0b 67.5 ± 6.3b 97.8 ± 15.1 128.6 ± 11.0 100.0 ± 7.3 129.4 ± 18.4 123.0 ± 15.8
7 0.20 0.0 ± 0.0b 92.5 ± 2.5 61.6 ± 3.9b 33.3 ± 4.1b 83.8 ± 5.6 2.5 ± 2.8b 59.5 ± 7.5b
0.10 5.0 ± 2.9b 95.0 ± 2.9 74.4 ± 5.2b 46.7 ± 12.9b 81.1 ± 5.8 28.9 ± 4.9b 63.6 ± 10.2b
0.05 0.0 ± 0.0b 100.0 ± 6.1 56.9 ± 6.0b 87.5 ± 28.1 103.2 ± 8.2 86.5 ± 12.0 112.5 ± 20.5
8 0.20 87.5 ± 5.1 92.5 ± 4.8 77.1 ± 3.7b 81.3 ± 17.7 106.5 ± 10.3 99.1 ± 12.6 107.9 ± 18.1
9 0.20 3.3 ± 3.3b 100.0 ± 0.0 151.9 ± 13.8 29.6 ± 6.1b 43.6 ± 8.8 55.2 ± 13.5b 51.6 ± 16.6b
0.10 9.4 ± 6.0b 100.0 ± 6.1 144.8 ± 16.2 76.5 ± 15.5b 100.0 ± 5.8 90.6 ± 12.3 88.1 ± 14.2
carvonec 0.20 27.3 ± 13.5b 100.0 ± 0.0 95.3 ± 5.4 88.2 ± 15.0 97.4 ± 3.9 92.2 ± 11.5 100.7 ± 13.6
a
% Control. bp < 0.05, Mann−Whitney U-test. cPositive control.

Additionally, compound 7 was nematicidal (LD50 and LD90
values of 0.24 and 0.52 μg/μL with 95% confidence limits of
0.23−0.26 and 0.49−0.56, respectively), and 6 showed a
moderate effect (53.9 ± 5.1% J2 mortality at 0.5 μg/μL), while
3 and 8, isolated from the aqueous residue, were not active.
Necrodane-type compounds have been reported as moderate
insect antifeedants.15 However, there are no reports on their
phytotoxic or nematicidal activity. These compounds are
structurally related to cyclopentenone oxylipins that inhibit
seed germination in Arabidopsis thaliana.22
Additionally, γ-pyrones, γ-pyridones, and pyrandiones have
been recognized as efficient−moderate photosystem II
inhibitors.23,24 In this work, the pyrandione-related compound
7 showed a strong phytotoxic effect that could be related to
photosystem II inhibition. Additionally, cadinane-type sesqui-
terpenes showed antigermination activity against lettuce and
radish seeds25,26 and insecticidal and ixodicidal effects.25,27
blocks), containing four 10 m rows with 104 plants per row (49.92
m2) at a distance of 1.20 × 0.40 m (0.48 m2/plant). The experimental
field was established in March 2008 with plants produced from seeds
collected in June 2007 from a wild population located in Pueblo
Nuevo del Bullaque (Ciudad Real, Spain; latitude: 39°27′41″ N,
longitude: 4°24′34″ W, altitude: 733 m) and germinated in a
commercial nursery. Flowering aerial parts of the wild and cultivated
plants collected in June 2009 were dried in the shade at room
temperature and ground for extraction.
Extraction and Isolation. Hydrodistillation (essential oil) was
performed using a Clevenger-type apparatus (0.8% yield) according to
the method recommended by the European Pharmacopoeia (http://
www.edqm.eu/en/Homepage-628.html). Pilot plant vapor pressure
extraction (vapor pressure oil) (0.2% yield) was carried out in a
stainless steel distillation plant equipped with a 100 kg distillation
chamber, a 500 L vessel, and a pressure range of 0.5−1.0 bar. The
water collected after the essential oil was decanted (1.16 L) was
filtered to give an acidic water residue (aqueous residue, 4.5 mg/mL of
organic extract, pH 3.2). Then, 155 mL of this aqueous residue were

■
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured with a PerkinElmer model 343 polarimeter. IR spectra
were recorded on a PerkinElmer 1600 FT spectrometer. NMR
experiments were carried out on a Bruker AMX2 500 MHz or a Varian
Direct-Drive 500 NMR spectrometer. Chemical shifts were calculated
using the solvent as internal standard (CDCl3, at δH 7.26 and δC 77.0).
High-resolution mass spectra were recorded using a Micromass
Autospec instrument at 70 eV. Column chromatography (CC) was
performed on silica gel 40−70 μm (Merck). Precoated silica gel 60
F 254 (Merck) plates were used for TLC. Preparative flash
chromatography was carried out on a column 5 cm in diameter with
a height of 22 and 2.5 cm diameter silica cartridges (40−70 μm) in a
Jones Flash Chromatography apparatus. Semipreparative HPLC was
performed on a Shimadzu LC-20AD HPLC with an ACE 5 SIL
column (250 mm × 10 mm, 5 μm particle size).
Plant Material. Lavandula luisieri plants were cultivated in an
experimental field located in Comarca del Campo de Cariñena,
Aguarón, Zaragoza, Spain (16 m, 41°19′13.33″ N; 1°19′53.9″ W). The
experimental design consisted of three random blocks (2 m between
extracted with dichloromethane (150 mL × 3) to give an organic
fraction (230 mg, 0.15% yield). Next, 50 mL of aqueous residue was
neutralized at pH 6.62 with 2 N NaOH and lyophilized to give a dry
residue (36.7 mg, 0.073% yield). The organic extractions (hexane,
EtOAc, and EtOH) were carried out in a Soxhlet for 12 h (131 g, 1.2%,
0.76%, and 12.5% yield, respectively). The insoluble material from the
hexane extraction (1.60 g) was filtered, and the solution obtained was
washed with a 2 N NaOH solution. The aqueous layer was acidified
with 2 N HCl at pH 2 and extracted with tert-butyl methyl ether. Both
organic layers were washed with brine, dried with anhydrous Na2SO4,
filtered, and concentrated under a vacuum to afford 687 and 724 mg of
a neutral and an acid fraction, respectively.
The soluble hexane extract (8.4 g) was fractionated by column
chromatography over silica gel using hexane/tert-butyl methyl ether
mixtures of increasing polarity, affording camphor (714 mg),28,29 4
(103 mg), 2 (148 mg), 1 (86 mg), and 9 (360 mg).16 Additional
workup of the hexane extract (3.1 g) by flash silica gel column
chromatography eluted with a hexane/EtOAc gradient (0−30%
EtOAc) at 50 mL/min gave eight fractions. Compound 9 (110 mg)
was isolated from fraction 7. Further chromatography of fraction 5 on
a 20 g silica gel prepacked flash cartridge (ExtraBond Flash OT SI, 20
g, 70 mL, 26.8 × 154 mm, Scharlau), eluted with hexane/EtOAc, gave

D DOI: 10.1021/acs.jnatprod.5b00501
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
compound 7 (5 mg). Fraction 8 was chromatographed similarly and
eluted with hexane/EtOAc to yield compound 5 (9 mg).
The acid-insoluble material (724 mg) was fractionated by column
chromatography over silica gel using hexane/tert-butyl methyl ether/
EtOAc mixtures of increasing polarity to yield six fractions. Fraction 6
(EtOAc, 154 mg) was methylated with TMSCHN230 to afford a crude
product, which was subjected to column chromatography over silica
gel to yield methyl ursolate (30 mg) and methyl tormentate (79
mg).17,18
The plant material extracted with hexane was further extracted with
EtOAc. This EtOAc extract (1 g) was purified by column
chromatography over silica gel using hexane/tert-butyl methyl ether
mixtures of increasing polarity to yield rosmarinic acid (30 mg).19
The essential oil (52 g) was added to a Na2CO3 solution (25 mg/
mL) and stirred for 2 h. The water layer was treated with 10 g of NaCl,
extracted with dichloromethane (3 × 200 mL), and acidified with
diluted HCl to pH 3. The acid extract was partitioned with
dichloromethane (3 × 200 mL), the resulting organic extract dried
over Na2SO4, and the solvent evaporated to give 480 mg of a
crystalline yellow solid, which was purified by flash chromatography to
give 200 mg of compound 6 (white solid) and 20 mg of compound 7.
The water-residue organic fraction (230 mg) was purified by
column chromatography over silica gel using hexane/EtOAc mixtures
of increasing polarity, affording compound 3 (3.5 mg). The
neutralized-lyophilized aqueous residue (110 mg) was chromato-
graphed on a silica gel column eluted with EtOAc to yield compound
8 (3 mg).
5-(Hydroxymethyl)-2,3,4,4-tetramethylcyclopent-2-enone (1):
colorless syrup; [α]D −9.7 (c 1.0, CH2Cl2); IR (film) νmax 3406,
2960, 2931, 2875, 1686, 1465, 1378, 1330, 1240, 1041 cm−1; 1H NMR
data (CDCl3, 500 MHz), see Table 1; 13C NMR data (CDCl3, 125
MHz), see Table 1; EIMS m/z 168 [M]+ (53), 138 (100), 135 (80),
123 (99), 109 (44), 107 (79), 81 (49), 79 (39), 67 (32), 41 (34);
HREIMS m/z 168.1147 [M]+ (calcd for C10H16O2, 168.1150).
(2,2,3,4-Tetramethyl-5-oxocyclopent-3-en-1-yl)methyl acetate
(2): colorless syrup; [α]D −7.9 (c 1.0, CH2Cl2); IR (film) νmax 2953,
2928, 2859, 1734, 1668, 1452, 1370, 1262, 1231, 1093, 1017 cm−1; 1H
NMR data (CDCl3, 500 MHz), see Table 1; 13C NMR data (CDCl3,
125 MHz), see Table 1; EIMS m/z 210 [M]+ (8), 151 (19), 150 (33),
136 (11), 135 (100), 123 (14), 107 (41), 91 (10), 79 (9), 43 (33), 41
(10); HREIMS m/z 210.1254 [M]+ (calcd for C12H18O3, 210.1256).
5-Hydroxy-5-(hydroxymethyl)-2,3,4,4-tetramethylcyclopent-2-
en-1-one (3): colorless syrup; [α]D −7.0 (c 0.24, CHCl3); 1H NMR
data (CDCl3, 500 MHz), see Table 1; 13C NMR data (CDCl3, 125
MHz), see Table 1; EIMS m/z 184 [M]+ (1), 154 (100), 151 (29),
136 (53), 125 (47), 123 (40), 121 (29), 107 (23), 81 (20), 55 (24), 43
(46); HRESITOFMS m/z 207.0991 [M + Na]+ (calcd for C10H16O3,
207.0997).
2-(Hydroxymethyl)-3,4,4-trimethyl-5-methylenecyclopent-2-en-
1-one (5): 1H NMR data (CDCl3, 500 MHz), see Table 1; 13C NMR
data (CDCl3, 125 MHz), see Table 1; EIMS m/z 166 [M]+ (76), 151
(100), 149 (21), 137 (30), 135 (29), 123 (54), 105 (26), 95 (35), 91
(27), 79 (25), 67 (37), 59 (16); HREIMS m/z 166.0991 [M]+ (calcd
for C10H14O2, 166.0994).
Methyl 3,4,5,5-tetramethylcyclopenta-1,3-diene-1-carboxylate
(6a): 1H NMR data (CDCl3, 500 MHz), see Table 1; 13C NMR
data (CDCl3, 125 MHz), see Table 1; EIMS m/z 180 [M]+ (13), 165
(47), 157 (33), 137 (39), 125 (59), 107 (38), 99 (61), 91 (74), 71
(82); HREIMS m/z 180.1144 [M]+ (calcd for C11H16O2, 180.1150).
3,3,4,5-Tetramethyl-2H-pyran-2,6(3H)-dione (7): IR (film) νmax
2925, 1783, 1737, 1052 cm−1; 1H NMR data (CDCl3, 500 MHz),
see Table 1; 13C NMR data (CDCl3, 125 MHz), see Table 1; EIMS
m/z 169 [M + H]+ (11), 124 (66), 123 (20), 109 (60), 96 (9), 81
(100), 79 (12), 53 (12); HREIMS m/z 169.0861 [M + H]+ (calcd for
C9H13O3, 169.0865).
5-(2-Hydroxypropan-2-yl)-2-methylcyclohex-3-ene-1.2-diol (8):
colorless syrup; [α]D −15.7 (c 0.28, CHCl3); 1H NMR data
(CDCl3, 500 MHz), see Table 2; 13C NMR data (CDCl3, 125
MHz), see Table 2; EIMS m/z 185 [M − H]+ (1), 168 (2), 153 (4),
135 (4), 110 (94), 109 (55), 107 (11), 95 (77), 91 (12), 81 (12), 67
E DOI: 10.1021/acs.jnatprod.5b00501
Article
(20), 59 (100); HREIMS m/z 185.1181 [M − H]+ (calcd for
C10H17O3, 185.1178).
Insect Bioassays. Spodoptera littoralis and Myzus persicae colonies
were reared on an artificial diet and bell pepper (Capsicum annuum)
plants, respectively, and maintained at 22 ± 1 °C and >70% relative
humidity, with a photoperiod of 16:8 h (L:D) in a growth chamber.
Bioassays were conducted with newly emerged S. littoralis L6 larvae or
10 M. persicae adults as described previously.31 The organic extracts
and pure compounds were tested at initial concentrations of 100 and
50 μg/cm2, respectively.
Phytotoxic Activity. The experiments were conducted with
Lactuca sativa cv. Teresa (Fito, España) and Lolium perenne seeds
(100 seeds/test) in 12-well microplates as described previously.32 The
organic extracts and pure compounds were tested at initial
concentrations of 0.4 and 0.2 mg/mL (final concentration in the
well), respectively, and diluted serially (1:2 dilutions), if needed. The
aqueous extract was tested without dilution (100%) and then was
diluted serially. Germination was monitored for 6 (L. sativa) or 7 days
(L. perenne), and the root length measured at the end of the
experiment (25 plants were selected randomly for each experiment,
digitalized, and measured with the application ImageJ, http//rsb.info.
nih.gov./ij/). A nonparametric analysis of variance (ANOVA) was
performed on radical length data. Carvone (5 μg/μL) was included as
a positive control.33
Nematode Bioassays. A Meloydogine javanica population
maintained on Solanum lycopersicum plants (var. “Marmande”) in
pot cultures at 25 ± 1 °C and >70% relative humidity was used.
Second-stage juveniles (J2) hatched within a 24 h period from egg
masses handpicked from infected tomato roots were used. The
experiments were carried out in 96-well microplates (Becton,
Dickinson), as described previously.33 The organic extracts and pure
compounds were tested at initial concentrations of 1.0 and 0.5 mg/mL
(final concentration in the well) respectively, and diluted serially if
necessary. The aqueous extract was tested without dilution (100%)
and then was diluted serially. The number of dead juveniles was
recorded after 72 h. All treatments were replicated four times. The
data were determined as percent mortality corrected according to
Scheider−Orelli’s formula. Effective lethal doses (LC50 and LC90) were
calculated for the active pure compounds by probit analysis (five serial
dilutions, 0.5−0.01 mg/mL).
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00501.
1
H NMR, 13 C NMR, and HREIMS spectra of
compounds 1−8. Tables S1 and S2 with biological
effects (phytotoxic, antifeedant, and nematicidal) of L.
luisieri extracts (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail (A. F. Barrero): afbarre@ugr.es. Tel/Fax:
+34958243318.
*E-mail (A. Gonzál ez-Coloma): azu@ica.csic.es. Tel:
+34917452500.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work has been partially supported by grants CTQ2012-
38219-C03-01 (Spain), Junta de Andaluciá (Excellence Project
P08-FQM-3596) Spain, and JAE-CSIC (predoctoral fellowship
to L.F.J.). We thank R. Muñoz and F. de la Peña for their
technical assistance.
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products

■
Article

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F DOI: 10.1021/acs.jnatprod.5b00501
J. Nat. Prod. XXXX, XXX, XXX−XXX