Phytotoxic and Nematicidal Components of Lavandula luisieri

Jan 22, 2016 - ABSTRACT: Several preparations were obtained from the aerial parts of predomesticated Lavandula luisieri, including the essential oil a...
1 downloads 0 Views 1MB Size
Download PDF
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. 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

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 1,8cineole, 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-2enone.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

Received: June 29, 2015

A

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

Journal of Natural Products

Article

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 gemdimethyl 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)-2,3,4,4tetramethylcyclopent-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 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 2,2,3,4tetramethyl-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-

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.

■

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 10hydroxy-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

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

δC, type

1 2 3 4 5

213.6, C 136.4, C 180.3, C 46.4, C 59.8, CH

6

63.2, CH2

δH (J in Hz)

2 δC, type 205.7, C 134.0, C 176.1, C 44.4, C 55.7, CH

7

10.5, CH3

2.30, dd (5.7, 8.9) 3.77, dd (5.7, 10.5) 3.83, dd (8.9, 10.5) 1.66, s

8

14.5, CH3

1.94, s

11.9, CH3

9 10 OH COCH3 COCH3 OCH3

25.0, CH3 29.2, CH3

1.20, s 1.02, s 3.30, br s

δH (J in Hz)

8.3, CH3

2.47, dd (4.2, 9.2) 4.18, dd (9.2, 11.9) 4.53, dd (4.2, 11.9) 1.67, s

62.3, CH2

3 δC, type

5 δH (J in Hz)

209.2, C 131.3, C 176.8, C 47.4, C 80.2, C 66.2, CH2

δC, type

6a δH (J in Hz)

196.0, C 137.6, C 173.9, C 44.4, C 152.1, C 3.57, m

114.4, CH2

3.64, m

δC, type 145, CH 130.3, C 156.5, C 53.5, C 142.9, C

5.34, s

7 δH (J in Hz) 7.12 s

163.9, C

δH (J in Hz)

160.8, C 119.7, C 156.1, C 44.5, C 172.2, C 15.3, CH3

1.97, d (0.95)

13.2, CH3 25.6, CH3

1.98, d (0.95) 1.49, s

25.6, CH3

1.49, s

6.04, s

8.0, CH3

1.72, s

56.1, CH2

4.42, s

12.2, CH3

1.95, s

12.4, CH3

1.98, s

11.3, CH3

2.02, s

9.9, CH3

22.8, CH3 27.0, CH3

1.23, s 1.09, s

19.7, CH3 24.8, CH3

1.106, s 1.113, s

25.4, CH3 25.4, CH3

1.25, s 1.25, s

21.4, CH3 21.4, CH3

1.86, d (0.95) 1.79, d (0.95) 1.16, s 1.16, s

21.1, CH3 171.0, C

2.05, s 50.7, CH3

3.74, s

B

δC, type

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. These data allowed compound 3 to be assigned as 5-hydroxy-5(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

Scheme 1. Possible Biosynthetic Pathway of Necrodanes in L. lusieri

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-trimethyl-5-methylenecyclopent-2-enone. The molecular formula of 7 was determined as C9H13O3 by HREIMS ([M + H]+, m/z 169.0861). This formula suggested a monoterpene with four degrees of unsaturation. Its IR spectrum showed three major absorption bands at νmax 1783, 1737, and 1052 cm−1, due to the presence of a CO−O−CO group. The IR and NMR data (Table 1) allowed the structure of 2H-pyran-2,6(3H)-dione to be established for 7 with a gemdimethyl group and two methyl groups on a double bond. These data were confirmed by 2D NMR studies (HSQC and HMBC) and permitted the assignment of 7 as 3,3,4,5tetramethyl-2H-pyran-2,6(3H)-dione, a new nor-necrodane compound. The necrodane carbon skeleton (1,2,2,3,4-pentamethylcyclopentane) of 1−7 indicates that their biosynthesis did not involve prenylation between the monoterpene precursors, DMAPP and IPP. The involvement of DMAPP and isoprene (derived from the removal of HOPP from IPP) in their biosynthesis has been proposed.20 Enzymes with acid centers (prenyltransferase and/or cyclase) could selectively protonate the disubstituted double bond of the isoprene, leading to prenylation−cyclization−deprotonation that would form necrodols. These necrodols could be the precursors of other necrodanes found in L. luisieri through oxidation, eliminations, 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 pmenthane skeleton with three hydroxy groups at the C-1, C-2, 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 hydroxy groups at the C-1 and C-2 positions (Figure 2). These data allowed compound 8 to be proposed as (1R*,2S*,4R*)-pmenth-5-ene-1,2,8-triol. The absolute configuration at the stereogenic centers has not been confirmed. Table 3 shows the results of the phytotoxic effects of the pure compounds. Overall, L. perenne was more sensitive than L.

Table 2. NMR Spectroscopic Data (500 MHz, CDCl3) for Compound 8 position 1 2 3

4 5 6 7 8 9 10

δC, type

δH (J in Hz)

69.8, C 73.3, CH 3.86, dd (6.0, 2.8) 27.4, α 1.95, dd (6.0, CH2 2.8) β 1.86, dd (13,9, 6.9) 42.7, CH 2.35, m 129.5, CH 133.5, CH 24.1, CH3 72.7, C 28.3, CH3 26.9, CH3

5.93, dd (10.4, 2.8) 5.75, ddd (10.4, 2.5, 0.9) 1.34, s

HMBCa

COSY H-3

NOESY

1, 4, 6, 8

H-7

1, 2, 4, 5

H-9, H10 H-7, H-9, H-10 H-2, H-9, H-10 H-9, H10 H-7

H-3

3, 5, 6, 7

H-6, H-4

1, 3, 4

H-5

2, 4, 8 1, 2, 6

1.29, s

4, 7, 10

1.24, s

4, 7, 9

a

HMBC correlations, optimized for 6 Hz, are from proton(s) stated to the indicated carbon.

Figure 2. Significant NOESY correlations of compound 8.

sativa. Necrodanes 1, 2, 6, 6a, and 7, cadinane 9, and rosmarinic acid inhibited the germination of L. sativa between 24 and 72 h (up to 168 h for 6a), but only 7 reduced its root growth at all doses tested. Compounds 1, 2, 5, 6, and 7 inhibited both the germination and growth of L. perenne. Cadinane 9 showed similar effects. The new nor-necrodane 7 was the only 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 compound 1

2

4 5 6

6a 7

8 9 carvonec a

growtha

mg/mL

24 h

144 h

root

0.20 0.10 0.05 0.20 0.10 0.05 0.20 0.20 0.20 0.10 0.05 0.20 0.20 0.10 0.05 0.20 0.20 0.10 0.20

84.0 ± 5.0

100.0 ± 0.0

94.3 ± 7.2

66.0 ± 7.0b

100.0 ± 0.0

88.9 ± 7.6

87.0 ± 9.0 117.0 ± 9.0 24.0 ± 9.0b

100.0 ± 0.0 108.1 ± 8.8 100.0 ± 0.0

124.6 ± 9.6 123.2 ± 7.9 108.4 ± 8.2

67.5 ± 6.3b 92.5 ± 2.5 95.0 ± 2.9 100.0 ± 6.1 92.5 ± 4.8 100.0 ± 0.0 100.0 ± 6.1 100.0 ± 0.0

97.8 61.6 74.4 56.9 77.1 151.9 144.8 95.3

0.0 ± 0.0b 0.0 ± 0.0b 5.0 ± 2.9b 0.0 ± 0.0b 87.5 ± 5.1 3.3 ± 3.3b 9.4 ± 6.0b 27.3 ± 13.5b

± ± ± ± ± ± ± ±

germinationa 72 h

15.1 3.9b 5.2b 6.0b 3.7b 13.8 16.2 5.4

36.7 50.0 68.8 20.0 36.7 75.0 75.0 68.8 50.0 82.7 62.5 128.6 33.3 46.7 87.5 81.3 29.6 76.5 88.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.6b 27.0b 15.4b 6.7b 8.5b 23.4b 23.4b 15.4b 11.8b 16.9 20.6b 11.0 4.1b 12.9b 28.1 17.7 6.1b 15.5b 15.0

growtha 168 h

root

leaf

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

74.7 ± 9.5b 77.2 ± 13.3b 89.3 ± 11.7 73.0 ± 12.6b 88.7 ± 13.8 80.3 ± 11.3 99.1 ± 12.4 52.9 ± 7.3b 47.1 ± 6.8b 86.5 ± 10.1 106.9 ± 12.7 129.4 ± 18.4 2.5 ± 2.8b 28.9 ± 4.9b 86.5 ± 12.0 99.1 ± 12.6 55.2 ± 13.5b 90.6 ± 12.3 92.2 ± 11.5

63.6 ± 9.3b 76.9 ± 16.5b 94.1 ± 17.6 56.2 ± 12.7b 74.9 ± 14.0b 82.5 ± 18.4 104.2 ± 18.1 110.4 ± 14.7 68.3 ± 10.9b 66.6 ± 10.9b 112.8 ± 20.2 123.0 ± 15.8 59.5 ± 7.5b 63.6 ± 10.2b 112.5 ± 20.5 107.9 ± 18.1 51.6 ± 16.6b 88.1 ± 14.2 100.7 ± 13.6

67.6 93.5 96.8 73.0 59.5 83.9 106.5 100.0 85.3 100.0 96.8 100.0 83.8 81.1 103.2 106.5 43.6 100.0 97.4

9.4 13.6 7.2 8.3b 3.5b 7.0 7.1 7.0 10.4 10.2 8.9 7.3 5.6 5.8 8.2 10.3 8.8 5.8 3.9

% Control. bp < 0.05, Mann−Whitney U-test. cPositive control. 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 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

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 sesquiterpenes showed antigermination activity against lettuce and radish seeds25,26 and insecticidal and ixodicidal effects.25,27

■

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 D

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

Journal of Natural Products

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).

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 chromatographed 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-2en-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-en1-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

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.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): [email protected]. Tel/Fax: +34958243318. *E-mail (A. Gonzál ez-Coloma): [email protected]. Tel: +34917452500. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work has been partially supported by grants CTQ201238219-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. E

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

Journal of Natural Products

■

Article

(34) Andres, M. F.; Gonzalez-Coloma, A.; Sanz, J.; Burillo, J.; Sainz, P. Phytochem. Rev. 2012, 11, 371−390.

REFERENCES

(1) Rivas-Martínez, S. Lazaroa 1979, 1, 110. (2) Baldovini, N.; Lavoine-Hanneguelle, S.; Ferrando, G.; Dusart, G.; Lizzani-Cuvelier, L. Phytochemistry 2005, 66, 1651−1655. (3) García-Vallejo, M. I.; García-Vallejo, M. C.; Sanz, J.; Bernabé, M.; Velasco-Negueruela, A. Phytochemistry 1994, 36, 43−45. (4) Lavoine-Hanneguelle, S.; Casabianca, H. J. Essent. Oil Res. 2004, 16, 445−448. (5) Baptista, R.; Madureira, A. M.; Jorge, R.; Adao, R.; Duarte, A.; Duarte, N.; Lopes, M. M.; Teixeira, G. Evid.-Based Complement. Altern. Med. 2015, 2015, 570−521. (6) Matos, F.; Miguel, M. G.; Duarte, J.; Venâncio, F.; Moiteiro, C.; Correia, A. I. D.; Figueiredo, A. C.; Barroso, J. G.; Pedro, L. G. J. Essent. Oil Res. 2009, 21, 327−336. (7) Roller, S.; Ernest, N.; Buckle, J. J. Altern. Complement. Med. 2009, 15, 275−279. (8) Zuzarte, M.; Goncalves, M. J.; Cruz, M. T.; Cavaleiro, C.; Canhoto, J.; Vaz, S.; Pinto, E.; Salgueiro, L. Food Chem. 2012, 135, 1505−1510. (9) Videira, R.; Castanheira, P.; Grãos, M.; Salgueiro, L.; Faro, C.; Cavaleiro, C. Flavour Fragrance J. 2013, 28, 380−388. (10) Videira, R.; Castanheira, P.; Graos, M.; Resende, R.; Salgueiro, L.; Faro, C.; Cavaleiro, C. J. Nat. Prod. 2014, 77, 1275−1279. (11) Sanz, J.; Soria, A. C.; García-Vallejo, M. C. J. Chromatogr. 2004, 1024, 139−146. (12) González-Coloma, A.; Delgado, F.; Rodilla, J. M.; Silva, L.; Sanz, J.; Burillo, J. Biochem. Syst. Ecol. 2011, 39, 1−8. (13) González-Coloma, A.; Martín-Benito, D.; Mohamed, N.; GarcíaVallejo, M. C.; Soria, A. C. Biochem. Syst. Ecol. 2006, 34, 609−616. (14) González-Coloma, A.; López-Balboa, C.; Santana, O.; Reina, M.; Fraga, B. M. Phytochem. Rev. 2011, 10, 245−260. (15) Julio, L. F.; Martín, L.; Muñoz, R.; Mainar, A. M.; Urieta, J. S.; Sanz, J.; Burillo, J.; González-Coloma, A. Ind. Crops Prod. 2014, 58, 25−30. (16) Stærk, D.; Skole, B.; Jørgensen, F. S.; Budnik, B. A.; Ekpe, P.; Jaroszewski, J. W. J. Nat. Prod. 2004, 67, 799−805. (17) Numata, A.; Yang, P.; Takahashi, C.; Fujiki, R.; Nabae, M.; Fujita, E. Chem. Pharm. Bull. 1989, 37, 648−651. (18) Seo, S.; Tomita, Y.; Tori, K. J. Am. Chem. Soc. 1981, 103, 2075− 2080. (19) Kuhnt, M.; Rimpler, H.; Heinrich, M. Phytochemistry 1994, 36, 485−489. (20) Figadere, B. A.; McElfresh, J. S.; Borchardt, D.; Daane, K. M.; Bentley, W.; Millar, J. G. Tetrahedron Lett. 2007, 48, 8434−8437. (21) Naik, R. H.; Joshi, G. O.; Kulkarni, G. H. Indian J. Chem. 1986, 25B, 306−307. (22) Dave, A.; Graham, I. A. Front. Plant Sci. 2012, 3, 42. (23) Asami, T.; Yoshida, S.; Takahashi, N. Agric. Biol. Chem. 1986, 50, 469−474. (24) Oettmeier, W.; Jäger, J.; Masson, K. Biochim. Biophys. Acta, Bioenerg. 2006, 1757, 727−729. (25) Buchanan, G. O.; Williams, L. A.; Reese, P. B. Phytochemistry 2000, 54, 39−45. (26) Nawamaki, K.; Kuroyanagi, M. Phytochemistry 1996, 43, 1175− 1182. (27) Porter, R.; Reese, P.; Williams, L.; Williams, D. Phytochemistry 1995, 40, 735−738. (28) Barrero, A. F.; Herrador, M. M.; Arteaga, P.; Quílez, J.; Akssira, M.; Mellouki, F.; Akkad, S. J. J. Essent. Oil Res. 2005, 17, 166−168. (29) Demarco, P. V.; Doddrell, D.; Wenkert, E. J. Chem. Soc. D 1969, 1418−1420. (30) Hashimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm. Bull. 1981, 29, 1475−1478. (31) Burgueno-Tapia, E.; Castillo, L.; Gonzalez-Coloma, A.; JosephNathan, P. J. Chem. Ecol. 2008, 34, 766−771. (32) Martín, L.; Julio, L. F.; Burillo, J.; Sanz, J.; Mainar, A. M.; González-Coloma, A. Ind. Crops Prod. 2011, 34, 1615−1621. (33) De Martino, L.; Mancini, E.; Rolim de Almeida, L. F.; De Feo, V. Molecules 2010, 15, 6630−6637. F

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