Protoplasma DOI 10.1007/s00709-014-0739-4
ORIGINAL ARTICLE
Screening and identification of phytotoxic volatile compounds in medicinal plants and characterizations of a selected compound, eucarvone Yukari Sunohara & Yohei Baba & Shigeru Matsuyama & Kaori Fujimura & Hiroshi Matsumoto
Received: 11 September 2014 / Accepted: 23 November 2014 # Springer-Verlag Wien 2014
Abstract Screening and identification of phytotoxic volatile compounds were performed using 71 medicinal plant species to find new natural compounds, and the characterization of the promising compound was investigated to understand the mode of action. The volatile compounds from Asarum sieboldii Miq. showed the strongest inhibitory effect on the hypocotyl growth of lettuce seedlings (Lactuca sativa L.cv. Great Lakes 366), followed by those from Schizonepeta tenuifolia Briquet and Zanthoxylum piperitum (L.) DC.. Gas chromatography–mass spectrometry (GC/MS) identified four volatile compounds, αpinene (2,6,6-trimethylbicyclo[3.1.1]hept-2-ene), β-pinene (6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane), 3-carene (3,7,7-trimethylbicyclo[4.1.0]hept-3-ene), and eucarvone (2,6,6-trimethy-2,4-cycloheptadien-1-one), from A. sieboldii, and three volatile compounds, limonene (1-methyl-4-(1methylethenyl)-cyclohexene), menthone (5-methyl-2-(propan2-yl)cyclohexan-1-one), and pulegone (5-methyl-2-propan-2ylidenecyclohexan-1-one), from S. tenuifolia. Among these volatile compounds, eucarvone, menthone, and pulegone exhibited strong inhibitory effects on both the root and shoot growth of lettuce seedlings. Eucarvone-induced growth inhibition was species-selective. Cell death, the generation of reactive oxygen species (ROS), and lipid peroxidation were induced in susceptible finger millet seedlings by eucarvone treatment, whereas this compound (≤158 μM) did not cause the increase
of lipid peroxidation and ROS production in tolerant maize. The results of the present study show that eucarvone can have strong phytotoxic activity, which may be due to ROS overproduction and subsequent oxidative damage in finger millet seedlings. Keywords Allelochemical . Asarum sieboldii Miq . Eucarvone . Lipid peroxidation . Phytotoxicity . Reactive oxygen species (ROS) Abbreviations DHE Dihydroethidium DMSO Dimethyl sulfoxide DW Distilled water FDA Fluorescein diacetate FID Flame ionization detector GC Gas chromatography GC-MS Gas chromatography–mass spectrometry GR50 The dose required to cause a 50 % reduction in plant growth OH· Hydroxyl radical PI Propidium iodide ROS Reactive oxygen species SOD Superoxide dismutase
Handling Editor: Peter Nick Yukari Sunohara and Yohei Baba contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00709-014-0739-4) contains supplementary material, which is available to authorized users. Y. Sunohara (*) : Y. Baba : S. Matsuyama : K. Fujimura : H. Matsumoto Graduate school of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan e-mail: [email protected]
Introduction Plant-derived secondary metabolites play important roles in chemical defense mechanisms against multiple enemies including herbivores, pathogens, and other plants (Langenheim 1994). On the other hand, secondary metabolites are also useful sources of lead compounds for novel herbicide development (Duke et al. 2000a). In most cases, phytotoxic action mechanisms of natural products are considered as being
Y. Sunohara et al.
different from those of synthetic herbicides (Dayan et al. 1999, 2000; Duke et al. 1996). Plant terpenoids are one of major chemical class of plantderived secondary metabolites. Their functions are very diverse and have been reported as allelochemicals, pheromones, phytoalexins, signal molecules, and membrane constituents (Haig 2008). Plant terpenoids and terpenes are the main volatile constituents of essential oils in aromatic plants and have been reported as the active compounds for allelopathic activities in several plant species (Alsaadawi et al. 1985; Muller et al. 1964). Monoterpenes are hydrocarbons containing 10 carbon atoms, which consist of two isoprene units. Previous studies have showed that various volatile monoterpenes have inhibitory effects on seed germination and seedling growth (Alsaadawi et al. 1985; Muller and Muller 1964; Nishida et al. 2005; Romagni et al. 2000; Singh et al. 2002, 2006, 2009; Vaughn and Spencer 1993; Zunino and Zygadlo 2004). The monoterpenes 1,8-cineole (1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane) and 1,4-cineole {1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane}, components of essential oils from Eucalyptus spp. and Artemisia spp. (Halligan 1975), were previously reported to inhibit seedling growth of Echinochloa crusgalli and Cassia obtusifolia (Romagni et al. 2000). The 2-benzyl ether-substituted analog of 1,4-cineole is the commercial herbicide cinmethylin {exo-(±)-1-methyl-4-(1-methylethyl)-2-[(2methylphenyl) methoxy]-7-oxabicyclo[2.2.1]heptane}, and the benzyl ether side chain acts to reduce its volatility and to enhance formation of the more active compound (Duke et al. 2000b). D-Limonen, a monoterpene extended from orange peels, has also been used as an commercial herbicide (Fitzhenry 2005). Thus, natural volatile compounds containing terpenoids could be sources of lead compounds for environmentally safer novel herbicides. However, very little have been used as lead structures for herbicides, even though an enormous number of terpenoid structures were known (Vaughn and Spencer 1993), and the phytotoxic action mechanisms of terpenoids are not yet fully understood in plants. Medicinal plants contain a relatively large number of secondary metabolites, and allelopathic activities have been frequently found in those plants (Fujii et al. 1990). Several medicinal plant species belonging to the families Annonaceae, Apocynaceae, Euphorbiaceae, Guttiferae, Leguminosae, Myrtaceae, and Rubiaceae were reported to have strong allelopathic activity and inhibit seedling growth of lettuce (Fujii et al. 2003). In addition, previous studies have showed that several medicinal plant species also have phytotoxic effects on seed germination and seedling growth of several plant species including noxious weeds, and medicinal plants have been considered to have the potential to be used for weed management in crop fields (Han et al. 2008; Islam and Kato-Noguchi 2013; Sodaeizadeh et al. 2009). Therefore, medicinal plants could be useful sources for novel natural herbicide development.
This study was aimed to find new natural phytotoxic volatile compounds with applicability for environmentally safer novel herbicides from various medicinal plants and investigate physiological responses to the promising compound in order to understand the mode of action of the compound.
Material and methods Screening of medicinal plants for allelopathic activity of volatile substances Dried samples of 71 medicinal plants (Table 1) were purchased from a local supplier (Yanagido Yakkyoku, Ehime, Japan). These samples were stocked in an incubator at 25 °C and were then ground finely with the Japanese traditional grinder “Yagen” immediately prior to the experiment. The bioassay for volatile allelopathy was conducted by the dish pack method, which was developed by Fujii et al. (2005) and Sekine et al. (2007). A ground sample (1 g) was put into one of the six wells, 0-mm-distance well (source well), in a six-well multidish (Nunc, external dimensions: 128×86 mm, 35-mmdiameter wells; Fig. S1), and filter paper was then placed into each of the five other wells. A total of 0.7 ml of distilled water (DW) was then added to each of the five wells, and five pregerminated seeds of lettuce (Lactuca sativa L.cv. Great Lakes 366), which had been incubated for 1 day in the dark at 25 °C until the radicle length became approximately 1–2 mm, were placed onto each filter paper. The multidish was covered, and the side was sealed with adhesive tape to prevent the loss of compounds due to volatilization. A control multidish was prepared in the same manner, except that the source well contained no medicinal plant sample. The degrees of vapor diffusion and phytotoxic activity were estimated by the relationship between the degree of growth inhibition and its distance from the source well. The root and shoot lengths were measured after the dishes were kept for 3 days in the dark at 25 °C. Determination of volatile compounds from Asarum sieboldii Miq. and Schizonepeta tenuifolia Briquet. The volatile compounds from A. sieboldii and S. tenuifolia were analyzed using the headspace method. One gram of finely ground dried roots and spikes from A. sieboldii and S. tenuifolia, respectively, was placed into a glass vial (5 ml). Glass vials were tightly sealed with a rubber plug and an aluminum stopper using a crimper, and incubated at 25 °C in the dark for 1 and 3 days. A headspace vapor sample (0.5 ml) was withdrawn from the glass vial through the rubber plug with a gas-tight syringe and was then analyzed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS). GC-MS analyses were performed using a
Characterization of phytotoxic compound eucarvone Table 1
Effects of volatile substances from 71 medicinal plant species on the seedling growth of lettuce Growtha (% of control)
Species name Family name
Scientific name
Used part
Aristolochiaceae Lamiaceae Rutaceae Lamiaceae Lauraceae Orobanchaceae Apiaceae Lamiaceae Gentianaceae Pyrolaceae Pinaceae Apocynaceae Fabaceae Polygalaceae
Asarum sieboldii Miq. Schizonepeta tenuifolia Briquet Zanthoxylum piperitum (L.) DC. Mentha × piperita L. Laurus nobilis L. Cistanche salsa (CA Mey) G.Beck. Angelica keiskei Koidzumi Melissa officinalis L. Swertia japonica Makino Pyrola japonica Klenze. Pinus densiflora Sieb. et Zucc. Apocynum venetum L. Styphonolobium japonicum (L.) Schott Polygala tenuifolia Willd.
Root Spike Fruit Leaf Leaf Shoot and root Root Leaf Shoot and root Shoot and root Leaf Leaf Flower bud Root
33.3±0.4 34.6±1.5 45.8±1.7 49.2±2.4 49.8±1.2 69.5±8.3 71.8±5.4 73.4±1.3 75.1±3.2 75.4±3.2 77.2±1.3 77.4±6.3 77.7±0.7 77.8±4.3
56.9±2.5 77.1±0.8 66.7±4.9 92.9±1.6 62.3±2.2 19.0±3.0 55.3±4.7 73.0±0.7 86.3±2.1 29.1±0.4 100.0±5.6 56.2±8.2 103.6±9.1 70.5±6.3
Fabaceae Myrtaceae Ebenaceae Lauraceae Asteraceae Moraceae Fagaceae Commelinaceae Lamiaceae Rubiaceae Fabaceae Lamiaceae Cupressaceae Fumariaceae Saururaceae Apiaceae Lauraceae
Aspalathus linearis (N.L.Burm.) R.Dahlgr. Psidium guajava L. Diospyros kaki Thunb. Lindera strychnifolia (Sieb. et Zucc.) F. Villar Tagetes spp. Ficus carica L. Quercus salicina Blume Commelina communis L. Glechoma hederacea L. var. grandis Kudo Uncaria gambir Roxburgh Astragalus membranaceus Bunge Perilla frutescens Brittion var. crispa Decne Platycladus orientalis (L.f.) Franco Corydalis ambigua Cham. et Schltdl. Houttuynia cordata Thunb. Angelica acutiloba Kitagawa Lindera umbellata Thunb.
Leaf Leaf Leaf Root Flower Leaf Ramulus and leaf Shoot and root Shoot and root Ramulus and leaf Root Leaf Leaf Tuber Shoot Root Bark
78.9±1.7 82.1±3.3 83.0±2.1 83.5±5.4 84.0±2.0 84.1±5.8 84.4±1.6 84.9±8.5 85.0±0.9 85.0±5.1 85.4±4.0 85.9±2.6 86.0±4.7 86.2±2.7 86.6±2.2 86.8±1.2 87.6±2.5
89.0±2.6 84.7±2.4 78.7±2.3 76.8±6.1 82.7±2.2 81.2±13.5 97.1±1.7 72.3±4.1 100.0±5.4 89.5±1.5 83.2±1.8 108.6±4.2 91.4±5.6 104.1±6.4 95.5±1.8 79.9±13.0 95.7±2.0
Asteraceae
Echinacea angustifolia L. Moench.
Root
88.6±2.4
95.2±5.7
Lamiaceae Saxifragaceae
Leonurus japonicus Houtt. Hydrangea macrophylla Seringe var. thunbergii Makino Aloe arborescens Mill. Taxus cuspidata Sieb. & Zucc. Ajuga decumbens Thunb. Arctostaphylos uva-ursi (L.) spreng. Morus alba L. Hypericum erectum Thunb. Chamaecrista nomame (Siebold) H.Ohashi Lycopus lucidus Turcz. Epimedium grandiflorum Morr. var. thunbergianum (Miq.) Nakai Orthosiphon aristatus (Blume) Miq. Thujopsis dolabrata (Thunb. Ex L.f.) Sieb. & Zucc. Eleutherococcus senticosus Harms
Shoot and root Leaf
88.7±2.3 89.2±3.4
73.6±2.1 72.1±3.1
Leaf Leaf Shoot and root Leaf Leaf Shoot and root Shoot and root Shoot and root Shoot
89.3±0.4 89.5±1.0 89.9±2.6 89.9±2.0 90.3±0.9 90.3±3.8 90.8±1.0 90.9±2.5 92.0±0.7
89.0±3.7 107.7±3.0 66.4±4.6 100.4±7.5 104.9±3.6 106.0±5.5 98.8±4.9 86.4±0.7 97.6±1.7
Leaf Leaf Root
92.0±3.0 92.4±2.6 92.5±0.9
93.9±3.4 99.2±5.4 96.2±2.5
Aloaceae Taxaceae Lamiaceae Ericaceae Moraceae Clusiaceae Caesalpiniaceae Lamiaceae Berberidaceae Lamiaceae Cupressaceae Araliaceae
Hypocotyl
Root
Y. Sunohara et al. Table 1 (continued) Growtha (% of control)
Species name Family name
Scientific name
Used part
Liliaceae Gesneriaceae Lamiaceae Actinidiaceae Fabaceae Poaceae Ginkgoaceae Lamiaceae Eucommiaceae Lamiaceae Simaroubaceae Asteraceae Fabaceae Liliaceae
Polygonatum falcatum A. Gray Conandron ramondioides Sieb. et Zucc. Isodon japonicus (Burm.f.)H.Hara Actinidia polygama (Sieb. et Zucc.) Planch. ex Maxim. Lathyrus palustris L. subsp. pilosus (Cham.) Hult. Sasa veitchii Carr. Ginkgo biloba L. Elsholtzia ciliata (Thunb.) Hyl. Eucommia ulmoides Oliv. Scutellaria baicalensis Georgi. Picrasma quassioides Benn. Cirsium japonicum Fisch. ex DC. Desmodium styracifolium (Osb.) Merr. Polygonatum odoratum (Mill.) Druce var. pluriflorum (Miq.) Ohwi Scutellaria barbata D. DON. Chaenomeles sinensis (Thouin.) Koehne Phragmites australis (Cav.) Trin. ex Steud. Stevia rebaudiana L. Akebia quinata (Houtt)Decne Gymnema sylvestre R. Br. Coptis japonica (Thunb.)Makino Clematis chinensis Osbeck Lycium chinense Mill. Zanthoxylum schinifolium Siebold & Zucc. Saxifraga stolonifera Curtis
Rhizome Shoot and root Shoot Insect gall of fruit Shoot and root Leaf Leaf Shoot and root Leaf Root Heart wood Root Leaf Rhizome
Lamiaceae Rosaceae Poaceae Asteraceae Lardizabalaceae Asclepiadaceae Ranunculaceae Ranunculaceae Solanaceae Rutaceae Saxifragaceae a
Shoot and root Fruit Root Leaf stem Leaf Rhizome Root leaf Leaf and fruit Leaf
Hypocotyl
Root
93.0±3.2 93.1±1.5 93.6±0.9 93.9±2.8 93.9±4.0 93.9±2.0 94.1±5.3 94.2±3.7 94.5±1.6 94.7±2.5 95.3±1.2 95.5±2.0 96.3±3.2 96.6±2.3
88.2±5.7 82.5±7.6 87.6±0.5 68.4±3.0 94.5±1.9 104.4±4.4 92.4±3.7 95.7±3.2 122.0±2.5 97.9±3.8 101.3±3.9 94.7±7.3 97.1±2.2 81.3±10.6
97.2±1.2 98.5±1.6 99.2±2.9 99.8±3.5 100.0±0.7 100.2±1.3 100.5±2.5 101.0±2.1 101.3±3.0 102.7±3.3 104.3±3.3
106.4±9.5 104.5±8.1 90.3±1.5 93.4±4.4 94.1±5.6 115.0±4.9 103.8±1.3 106.7±7.3 102.7±2.9 106.4±4.5 98.9±6.1
Growth was determined by measuring the hypocotyl and root length of lettuce seedlings in 41-mm wells
gas chromatograph (Agilent Technology 6890N) connected online to a JEOL mass spectrometer (JEOL-MS-600H), equipped with an HP-5MS (Agilent Technology) fused-silica capillary column (25 m×0.25 mm i.d., 0.25 μm film thickness) in the electron impact mode (70 eV). The gas chromatographic conditions were as follows: the injection port was set at 250 °C; the oven temperature programme was 40 °C (1 min held), then increased to 250 °C at 10 °C/min, and held for 3 min at the final temperature; carrier gas, He (1 ml/min). A headspace vapor sample (0.5 ml) was injected manually in the splitless mode (sampling time: 1.0 min). GC-MS data of the volatile compounds from A. sieboldii and S. tenuifolia were analyzed using TSS 2000 software (Shrader Analytical and Consulting Laboratories, Inc.). A preliminary identification was performed by the NIST mass library database (National Institute of Standards and Technology, Gaithersburg, MD) and was further confirmed by comparisons of retention times and mass spectra with those of the standard compounds.
Plant growth inhibitory activity of volatile compounds from A. sieboldii and S. tenuifolia Authentic samples of α-pinene (2,6,6trimethylbicyclo[3.1.1]hept-2-ene) and (-)-β-pinene (1S,5S)-(6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane) were purchased from Wako Pure Chemicals (Osaka, Japan), and (+)3-carene ((1S,6R)-3,7,7- trimethylbicyclo[4.1.0]hept-3-ene), eucarvone (2,6,6-trimethy-2,4-cycloheptadien-1-one), and (±)limonene (1-methyl-4-(1-methylethenyl)-cyclohexene) were obtained from Tokyo Chemical Industry (Tokyo, Japan). (+)Pulegone ((5R)-5-methyl-2-(propan-2-ylidene) cyclohexan-1one) and menthone (5-methyl-2-(propan-2-yl)cyclohexan-1-one) were purchased from Sigma-Aldrich Co. (St. Louis, USA) and Nacalai Tesque (Kyoto, Japan), respectively. Five pre-germinated seeds, which had been incubated for 1– 3 days in the dark at 25 °C until the radicle length became approximately 2–3 mm, were placed onto a filter paper
Characterization of phytotoxic compound eucarvone
impregnated with DW (0.7 ml) in a 30-ml glass vial (33 mm diameter). Each authentic sample was added to hexane (100 %) to give concentrations of 0, 750, 7,500, 37,500, 75,000, and 150,000 ppm (v/v). Ten microliters of the authentic sample solution was spotted onto a piece of filter paper (1 cm×4 cm), and the solvent hexane was allowed to volatilize in the air for 10 sec. The authentic sample-spotted filter paper was then hanged from the rubber plug of the 30-ml glass vial, and the glass vial was tightly sealed as above. The concentrations of authentic samples in the airspace within the glass vial were calculated assuming that the spotted compounds volatilize completely without any loss due to adsorption or leakage. The final concentration of each authentic sample in the 30-ml glass vial was converted from parts per million to mole per liter and shown in each figures. The root and shoot lengths were measured after 3 days in controlled conditions (25 °C, in the dark). The concentrations of GR10 (the dose required to cause a 10 % reduction in plant growth), GR20, and GR50 were calculated using the equations shown in Fig. 1. In addition, to investigate the effect of eucarvone on the seedling growth of several plant species, 11 plant species were used. The 11 plant species tested were as follows: maize (Zea mays L. cv. Honey Bantam), cucumber (Cucumis sativus L. cv. Shimoshirazu Jibai), lettuce, velvetleaf (Abutilon avicennae Geartn.), tomato (Solanum lycopersicum L. cv. Kantaro), rice (Oryza sativa L. cv. Nipponbare), wheat (Triticum aestivum L. cv. Norin 61), slender amaranth (Amaranthus viridis L.), finger millet (Eleusine coracana (L.) Gaertn.), goosegrass (Eleusine indica (L.) Gaertn.), and green foxtail (Setaria viridis (L.) Beauv.). The GR50 concentrations of shoot and root elongation were calculated using the equations shown in Fig. 2. Determination of cell viability Root cell viability was evaluated by staining with fluorescein diacetate (FDA) and propidium iodide (PI) (Pan et al. 2001; Sunohara and Matsumoto 2008). Root segments (1.0 cm length from the tip) were excised from the 3-day-old seedlings of finger millet and maize treated with 0, 53, or 158 μM eucarvone for 3 h. The roots were stained with a mixture solution of 13 μg/ml FDA (Aldrich Chemical Co., Milwaukee, USA) and 0.5 μg/ml PI, containing 5 % dimethyl sulfoxide (DMSO) for 10 min, and were then washed with distilled water. Fluorescence images of the stained roots were observed using a fluorescence microscope (Nikon E600 with a B-2A filter [excitation 450–490 nm, emission >520 nm]; Nikon Corp., Tokyo, Japan). Root cell viability was also evaluated by Evans blue staining (Sunohara and Matsumoto 2008; Tamás et al. 2004). Root segments (1.0 cm length from the tip) were taken from the eucarvone-treated seedlings as above. The root segments were stained with a 0.25 % (w/v) aqueous solution of Evans blue (Sigma Chemical Co., St. Louis, USA) for 1 h at room temperature. The stained roots were then washed with distilled water for 15 min. Evans blue, which was taken into the root segments,
was then extracted using 500 μl of N,N-dimethylformamide solution (100 %) without grinding. After the root tips were allowed to soak in the N,N-dimethylformamide solution overnight 30 °C in the dark, the absorbance of the released Evans blue was spectrophotometrically measured at 600 nm. Detection of ROS production Production of O2− was estimated using the redox-sensitive probe, dihydroethidium (DHE), in accordance with the procedures of Yamamoto et al. (2002) with minor modification (Sunohara and Matsumoto 2008; Sunohara et al. 2011). Root segments (1.0 cm length from the tip) were taken from the eucarvone-treated seedlings as above and then stained with 10 μM DHE solution containing 1 % acetone in 100 μM CaCl2, by gently shaking for 30 min at room temperature in the dark. After washing the root segments with distilled water to remove residual dye, the roots were observed using a fluorescence microscope (Nikon E600 with a B-2A filter [excitation 450–490 nm, emission ≥520 nm]; Nikon, Tokyo, Japan). The fluorescence of oxyethidium derived from DHE oxidation by O2− was observed using the microscope. Lipid peroxidation The histochemical detection of lipid peroxidation was performed using the procedures described by Pompella et al. (1987) and Yamamoto et al. (2001) with minor modification. Root segments (1.0 cm length from the tip) were taken from the eucarvonetreated seedlings as above. The roots were stained with Schiff’s reagent which detects aldehydes that originate from lipid peroxides for 20 min. After the reaction, roots were rinsed with a sulfide solution (0.5 % (w/v) K2S2O5 in 0.05 M HCl). The stained roots were kept in the sulfide solution to retain the staining color. Roots stained with Schiff’s reagent were observed under a light microscope (Nikon E600, Nikon Corp., Tokyo, Japan). Statistical analyses All results were represented as the means±S.E. of at least three replicates, and all experiments were repeated at least twice. Relationships were considered to be significant when P 320 µM
20
20 00
50
100
150
200
250
300
00
350
50
100
Concentration (μM)
(c) 3-Carene
Growth (% of control)
Growth (% of control)
100 80 60
y(●) = 108.71e-0.0009x, R² = 0.8823 GR10 = 209.9 µM, GR20 > 318 µM
40
y(○) = 116.51e-0.0010x, R² = 0.7861 GR10 = 258.2 µM, GR20 > 318 µM
20 0
50
100
150 200 250 Concentration (μM)
300
100 80 60
y(●) = 104.62e-0.0006x, R² = 0.7195 GR10 = 250.9 µM, GR20 > 308 µM
40
0
50
100
150 200 Concentration (μM)
250
300
350
(g) Pulegone 120 100
y(●) = 92.599e-0.01x, R² = 0.935 GR20 = 14.1 µM, GR50 = 59.3 µM
80
y(○) = 97.695e-0.013x, R² = 0.9291 GR20 = 16.0 µM, GR50 = 59.3 µM
60 40 20 0
50
100
150 200 Concentration (μM)
250
300
y(○) = 126.28e-0.012x, R² = 0.979 GR20 = 37.3 µM, GR50 = 76.6 µM
60 40
0
50
100
150 200 Concentration (μM)
250
300
350
(f) Menthone y(●) = 124.34e-0.011x, R² = 0.9532 GR20 = 42.0 µM, GR50 = 86.8 µM
100 80
y(○) = 120.34e-0.012x, R² = 0.9586 GR20 = 38.0 µM, GR50 = 78.5 µM
60 40 20
y(○) = 100.55e-0.0008x, R² = 0.6246 GR10 = 138.6 µM, GR20 > 308 µM
20
80
120 Growth (% of control)
Growth (% of control)
350
y(●) = 94.481e-0.011x, R² = 0.9826 GR20 = 15.8 µM, GR50 = 60.6 µM
100
0
350
120
Growth (% of control)
300
20
(e) Limonene
140
0
250
120
120
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200
(d) Eucarvone
140
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150
Concentration (μM)
350
0
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150 200 Concentration (μM)
250
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350
Characterization of phytotoxic compound eucarvone
Fig.
1 Effects of the volatile compounds from A. sieboldii and S. tenuifolia on the shoot (filled circle) and root (empty circle) growth of lettuce seedlings at the germination stage 3 days after treatment. Mean control values (cm) and standard errors were as follows: a α-pinene: 1.30 ±0.09 (shoot), 1.64±0.13 (root); b β-pinene: 1.39±0.02 (shoot), 2.22± 0.18 (root); c 3-carene: 1.13 ± 0.10 (shoot), 1.67 ± 0.24 (root); d eucarvone: 1.23±0.03 (shoot), 2.14±0.22 (root); e limonene: 1.22±0.04 (shoot), 2.12±0.21 (root); f menthone: 1.23±0.06 (shoot), 2.00±0.02 (root); and g pulegone: 1.23±0.03 (shoot), 2.18±0.22 (root)
A total of 71 species (41 families) including Lamiaceae (13 species), Fabaceae (6 species), and Asteraceae (4 species) were tested. Among the species, the rate of inhibition of hypocotyl elongation between 80 and 100 % in the nearest (41 mm) well of the multidish (cf. Fig. S1) was observed in 0 species, between 60 and 79 % in 2 species, between 40 and 59 % in 3 species, between 20 and 39 % in 10 species, and 308, 38.0, and 16.0 μM for α-pinene, β-pinene, 3-carene, eucarvone, limonene, menthone, and pulegone, respectively. In shoot growth, the GR20 values were approximately 70.6, 85.9, >318, 15.8, >308, 42.0, and 14.1 μM for α-pinene, β-pinene, 3-carene, eucarvone, limonene, menthone, and pulegone, respectively. The order of the strength of the seven volatile compounds as inhibitors of lettuce shoot and root growth based on the GR20 values was estimated as pulegone > eucarvone > menthone > α-pinene > β-pinene > limonene ≒ 3-carene. These results support the previous reports by Vaughn and Spencer (1993, 1996), in which several monoterpenoids having oxygencontaining functional groups had strong phytotoxic effects on seed germination (cf. Fig. S4), and that by Vokou et al. (2003), in which ketones were the most phytotoxic, followed by aldehydes, ethers, alcohols, and phenols, with acetates and hydrocarbons being the least phytotoxic in lettuce seedlings in 47 monoterpenoids of different chemical groups. Mentone and pulegone are known to be components in peppermint essential oil (Maffei and Sacco 1987), and mentone has been reported to induce sprout growth suppression in potato tubers (Coleman et al. 2001). Pulegone has been identified as one of the most strong allelochemicals, was shown to be nearly four times more toxic than HCN on the seed germination of radish
Y. Sunohara et al.
Characterization of phytotoxic compound eucarvone
Fig. 2
Effects of eucarvone on the shoot (filled circle) and root (empty circle) growth of 11 plant species at the germination stage 3 days after treatment. The mean control values (cm) and standard errors were as follows: a maize: 2.42±0.18 (shoot), 5.83±0.22 (root); b cucumber: 1.21 ±0.27 (shoot), 4.69±0.71 (root); c lettuce: 1.23±0.03 (shoot), 2.14±0.22 (root); d velvetleaf: 2.06±0.52 (shoot), 3.94±0.17 (root); e tomato: 2.87± 0.10 (shoot), 5.54±0.38 (root); f rice: 1.48±0.21 (shoot), 5.03±0.25 (root); g wheat: 4.01 ± 0.16 (shoot), 6.03 ± 0.71 (root); h slender amaranth: 1.36±0.05 (shoot), 1.58±0.16 (root); i finger millet: 1.16± 0.07 (shoot), 1.55±0.07 (root); j goosegrass: 1.70±0.07 (shoot), 1.97± 0.07 (root); and k green foxtail: 3.67±0.09 (shoot), 2.25±0.06 (root)
(Asplund 1968), and was also shown to possess antimicrobial (Oumzil et al. 2002) and insecticidal activities (Park et al. 2006). Eucarvone has been reported to exhibit strong insecticidal activity (Kim and Park 2008). However, to the best of our knowledge, this is the first study to show that eucarvone can have potent phytotoxic activity, and the strength of which is almost equal to that of pulegone. In addition, nothing has been done to explore phytotoxic mechanisms of eucarvone. Therefore, we focused on eucarvone and further examined its phytotoxic activity. Effect of eucarvone on the seedling growth of several plant species at the germination stage The phytotoxic activities of eucarvone on initial growth were investigated using six crop species, maize, cucumber, lettuce, tomato, rice, and wheat, and five weed species, velvetleaf, slender amaranth, finger millet, goosegrass, and green foxtail. Eucarvone-induced growth inhibition was species-selective, while seedling growth was suppressed by eucarvone in a concentration-dependent manner in most of the species tested (Fig. 2). Among the 11 plant species, maize was the most tolerant, and green foxtail, goosegrass, and finger millet were relatively susceptible. The GR50 values of shoot growth determined 3 days after treatment in maize, green foxtail, goosegrass, and finger millet were estimated to be approximately >316, 21.2, 25.7, and 27.0 μM, respectively (Fig. 2). Regarding root growth, the GR50 values in maize, green foxtail, goosegrass, and finger millet were approximately >316, 20.9, 7.3, and 24.3 μM, respectively. Maize and finger millet were selected as examples of tolerant and susceptible species, respectively, for further experiments to characterize the phytotoxic action of eucarvone. Effect of eucarvone on cell viability The viability of root cells was examined by a double-staining method using FDA and PI. PI enters membrane-damaged cells, but not membrane-intact cells; then, it can be detected by its red fluorescence (Umebayashi et al. 2003). FDA easily enters living cells and then hydrolized by esterase, producing fluorescein, which can be detected by its green fluorescence
(Umebayashi et al. 2003). Hence, red and green fluorescences were used to assess dead and living cells, respectively. In 53 μM eucarvone treatment, red fluorescence was observed in the upper part of root cap of finger millet 3 h after treatment, and in 158 μM eucarvone, red fluorescence was observed in a whole part of the root tip (Fig. 3a). In maize root tips, a slight difference among control and the eucarvone (53 and 158 μM) treatments was observed in the fluorescences. Faint red fluorescence was observed in the small part of control root tip, and the faint red areas were slightly increased in 53 and 158 μM eucarvone-treated root tips (Fig. 3b). These results suggest that eucarvone (53–158 μM) strongly induce cell death in especially finger millet root tips 3 h after treatment. Cell viability in finger millet and maize roots were also determined by Evans blue staining to quantify the rates of dead cells. Evans blue is a non-permeating dye that enter membrane-injured cells and is unable to enter membraneintact cells. Hence, Evans blue staining has been used to assess cellular integrity (Gaff and Okong'o-Ogola 1971). In finger millet, 158 μM eucarvone significantly enhanced Evans blue uptake in root tip cells from 3 h after treatment. At 53 μM eucarvone, Evans blue uptake was not enhanced 24 h after treatment in finger millet root cells (Fig. 4a), while red fluorescence showing cell death was observed in the root tip 3 h after 53 μM eucarvone treatment (Fig. 3a). The inconsistency of these results may be due to the detection sensitivity between the two staining methods. In maize roots, eucarvone (
ORIGINAL ARTICLE
Screening and identification of phytotoxic volatile compounds in medicinal plants and characterizations of a selected compound, eucarvone Yukari Sunohara & Yohei Baba & Shigeru Matsuyama & Kaori Fujimura & Hiroshi Matsumoto
Received: 11 September 2014 / Accepted: 23 November 2014 # Springer-Verlag Wien 2014
Abstract Screening and identification of phytotoxic volatile compounds were performed using 71 medicinal plant species to find new natural compounds, and the characterization of the promising compound was investigated to understand the mode of action. The volatile compounds from Asarum sieboldii Miq. showed the strongest inhibitory effect on the hypocotyl growth of lettuce seedlings (Lactuca sativa L.cv. Great Lakes 366), followed by those from Schizonepeta tenuifolia Briquet and Zanthoxylum piperitum (L.) DC.. Gas chromatography–mass spectrometry (GC/MS) identified four volatile compounds, αpinene (2,6,6-trimethylbicyclo[3.1.1]hept-2-ene), β-pinene (6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane), 3-carene (3,7,7-trimethylbicyclo[4.1.0]hept-3-ene), and eucarvone (2,6,6-trimethy-2,4-cycloheptadien-1-one), from A. sieboldii, and three volatile compounds, limonene (1-methyl-4-(1methylethenyl)-cyclohexene), menthone (5-methyl-2-(propan2-yl)cyclohexan-1-one), and pulegone (5-methyl-2-propan-2ylidenecyclohexan-1-one), from S. tenuifolia. Among these volatile compounds, eucarvone, menthone, and pulegone exhibited strong inhibitory effects on both the root and shoot growth of lettuce seedlings. Eucarvone-induced growth inhibition was species-selective. Cell death, the generation of reactive oxygen species (ROS), and lipid peroxidation were induced in susceptible finger millet seedlings by eucarvone treatment, whereas this compound (≤158 μM) did not cause the increase
of lipid peroxidation and ROS production in tolerant maize. The results of the present study show that eucarvone can have strong phytotoxic activity, which may be due to ROS overproduction and subsequent oxidative damage in finger millet seedlings. Keywords Allelochemical . Asarum sieboldii Miq . Eucarvone . Lipid peroxidation . Phytotoxicity . Reactive oxygen species (ROS) Abbreviations DHE Dihydroethidium DMSO Dimethyl sulfoxide DW Distilled water FDA Fluorescein diacetate FID Flame ionization detector GC Gas chromatography GC-MS Gas chromatography–mass spectrometry GR50 The dose required to cause a 50 % reduction in plant growth OH· Hydroxyl radical PI Propidium iodide ROS Reactive oxygen species SOD Superoxide dismutase
Handling Editor: Peter Nick Yukari Sunohara and Yohei Baba contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00709-014-0739-4) contains supplementary material, which is available to authorized users. Y. Sunohara (*) : Y. Baba : S. Matsuyama : K. Fujimura : H. Matsumoto Graduate school of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan e-mail: [email protected]
Introduction Plant-derived secondary metabolites play important roles in chemical defense mechanisms against multiple enemies including herbivores, pathogens, and other plants (Langenheim 1994). On the other hand, secondary metabolites are also useful sources of lead compounds for novel herbicide development (Duke et al. 2000a). In most cases, phytotoxic action mechanisms of natural products are considered as being
Y. Sunohara et al.
different from those of synthetic herbicides (Dayan et al. 1999, 2000; Duke et al. 1996). Plant terpenoids are one of major chemical class of plantderived secondary metabolites. Their functions are very diverse and have been reported as allelochemicals, pheromones, phytoalexins, signal molecules, and membrane constituents (Haig 2008). Plant terpenoids and terpenes are the main volatile constituents of essential oils in aromatic plants and have been reported as the active compounds for allelopathic activities in several plant species (Alsaadawi et al. 1985; Muller et al. 1964). Monoterpenes are hydrocarbons containing 10 carbon atoms, which consist of two isoprene units. Previous studies have showed that various volatile monoterpenes have inhibitory effects on seed germination and seedling growth (Alsaadawi et al. 1985; Muller and Muller 1964; Nishida et al. 2005; Romagni et al. 2000; Singh et al. 2002, 2006, 2009; Vaughn and Spencer 1993; Zunino and Zygadlo 2004). The monoterpenes 1,8-cineole (1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane) and 1,4-cineole {1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane}, components of essential oils from Eucalyptus spp. and Artemisia spp. (Halligan 1975), were previously reported to inhibit seedling growth of Echinochloa crusgalli and Cassia obtusifolia (Romagni et al. 2000). The 2-benzyl ether-substituted analog of 1,4-cineole is the commercial herbicide cinmethylin {exo-(±)-1-methyl-4-(1-methylethyl)-2-[(2methylphenyl) methoxy]-7-oxabicyclo[2.2.1]heptane}, and the benzyl ether side chain acts to reduce its volatility and to enhance formation of the more active compound (Duke et al. 2000b). D-Limonen, a monoterpene extended from orange peels, has also been used as an commercial herbicide (Fitzhenry 2005). Thus, natural volatile compounds containing terpenoids could be sources of lead compounds for environmentally safer novel herbicides. However, very little have been used as lead structures for herbicides, even though an enormous number of terpenoid structures were known (Vaughn and Spencer 1993), and the phytotoxic action mechanisms of terpenoids are not yet fully understood in plants. Medicinal plants contain a relatively large number of secondary metabolites, and allelopathic activities have been frequently found in those plants (Fujii et al. 1990). Several medicinal plant species belonging to the families Annonaceae, Apocynaceae, Euphorbiaceae, Guttiferae, Leguminosae, Myrtaceae, and Rubiaceae were reported to have strong allelopathic activity and inhibit seedling growth of lettuce (Fujii et al. 2003). In addition, previous studies have showed that several medicinal plant species also have phytotoxic effects on seed germination and seedling growth of several plant species including noxious weeds, and medicinal plants have been considered to have the potential to be used for weed management in crop fields (Han et al. 2008; Islam and Kato-Noguchi 2013; Sodaeizadeh et al. 2009). Therefore, medicinal plants could be useful sources for novel natural herbicide development.
This study was aimed to find new natural phytotoxic volatile compounds with applicability for environmentally safer novel herbicides from various medicinal plants and investigate physiological responses to the promising compound in order to understand the mode of action of the compound.
Material and methods Screening of medicinal plants for allelopathic activity of volatile substances Dried samples of 71 medicinal plants (Table 1) were purchased from a local supplier (Yanagido Yakkyoku, Ehime, Japan). These samples were stocked in an incubator at 25 °C and were then ground finely with the Japanese traditional grinder “Yagen” immediately prior to the experiment. The bioassay for volatile allelopathy was conducted by the dish pack method, which was developed by Fujii et al. (2005) and Sekine et al. (2007). A ground sample (1 g) was put into one of the six wells, 0-mm-distance well (source well), in a six-well multidish (Nunc, external dimensions: 128×86 mm, 35-mmdiameter wells; Fig. S1), and filter paper was then placed into each of the five other wells. A total of 0.7 ml of distilled water (DW) was then added to each of the five wells, and five pregerminated seeds of lettuce (Lactuca sativa L.cv. Great Lakes 366), which had been incubated for 1 day in the dark at 25 °C until the radicle length became approximately 1–2 mm, were placed onto each filter paper. The multidish was covered, and the side was sealed with adhesive tape to prevent the loss of compounds due to volatilization. A control multidish was prepared in the same manner, except that the source well contained no medicinal plant sample. The degrees of vapor diffusion and phytotoxic activity were estimated by the relationship between the degree of growth inhibition and its distance from the source well. The root and shoot lengths were measured after the dishes were kept for 3 days in the dark at 25 °C. Determination of volatile compounds from Asarum sieboldii Miq. and Schizonepeta tenuifolia Briquet. The volatile compounds from A. sieboldii and S. tenuifolia were analyzed using the headspace method. One gram of finely ground dried roots and spikes from A. sieboldii and S. tenuifolia, respectively, was placed into a glass vial (5 ml). Glass vials were tightly sealed with a rubber plug and an aluminum stopper using a crimper, and incubated at 25 °C in the dark for 1 and 3 days. A headspace vapor sample (0.5 ml) was withdrawn from the glass vial through the rubber plug with a gas-tight syringe and was then analyzed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS). GC-MS analyses were performed using a
Characterization of phytotoxic compound eucarvone Table 1
Effects of volatile substances from 71 medicinal plant species on the seedling growth of lettuce Growtha (% of control)
Species name Family name
Scientific name
Used part
Aristolochiaceae Lamiaceae Rutaceae Lamiaceae Lauraceae Orobanchaceae Apiaceae Lamiaceae Gentianaceae Pyrolaceae Pinaceae Apocynaceae Fabaceae Polygalaceae
Asarum sieboldii Miq. Schizonepeta tenuifolia Briquet Zanthoxylum piperitum (L.) DC. Mentha × piperita L. Laurus nobilis L. Cistanche salsa (CA Mey) G.Beck. Angelica keiskei Koidzumi Melissa officinalis L. Swertia japonica Makino Pyrola japonica Klenze. Pinus densiflora Sieb. et Zucc. Apocynum venetum L. Styphonolobium japonicum (L.) Schott Polygala tenuifolia Willd.
Root Spike Fruit Leaf Leaf Shoot and root Root Leaf Shoot and root Shoot and root Leaf Leaf Flower bud Root
33.3±0.4 34.6±1.5 45.8±1.7 49.2±2.4 49.8±1.2 69.5±8.3 71.8±5.4 73.4±1.3 75.1±3.2 75.4±3.2 77.2±1.3 77.4±6.3 77.7±0.7 77.8±4.3
56.9±2.5 77.1±0.8 66.7±4.9 92.9±1.6 62.3±2.2 19.0±3.0 55.3±4.7 73.0±0.7 86.3±2.1 29.1±0.4 100.0±5.6 56.2±8.2 103.6±9.1 70.5±6.3
Fabaceae Myrtaceae Ebenaceae Lauraceae Asteraceae Moraceae Fagaceae Commelinaceae Lamiaceae Rubiaceae Fabaceae Lamiaceae Cupressaceae Fumariaceae Saururaceae Apiaceae Lauraceae
Aspalathus linearis (N.L.Burm.) R.Dahlgr. Psidium guajava L. Diospyros kaki Thunb. Lindera strychnifolia (Sieb. et Zucc.) F. Villar Tagetes spp. Ficus carica L. Quercus salicina Blume Commelina communis L. Glechoma hederacea L. var. grandis Kudo Uncaria gambir Roxburgh Astragalus membranaceus Bunge Perilla frutescens Brittion var. crispa Decne Platycladus orientalis (L.f.) Franco Corydalis ambigua Cham. et Schltdl. Houttuynia cordata Thunb. Angelica acutiloba Kitagawa Lindera umbellata Thunb.
Leaf Leaf Leaf Root Flower Leaf Ramulus and leaf Shoot and root Shoot and root Ramulus and leaf Root Leaf Leaf Tuber Shoot Root Bark
78.9±1.7 82.1±3.3 83.0±2.1 83.5±5.4 84.0±2.0 84.1±5.8 84.4±1.6 84.9±8.5 85.0±0.9 85.0±5.1 85.4±4.0 85.9±2.6 86.0±4.7 86.2±2.7 86.6±2.2 86.8±1.2 87.6±2.5
89.0±2.6 84.7±2.4 78.7±2.3 76.8±6.1 82.7±2.2 81.2±13.5 97.1±1.7 72.3±4.1 100.0±5.4 89.5±1.5 83.2±1.8 108.6±4.2 91.4±5.6 104.1±6.4 95.5±1.8 79.9±13.0 95.7±2.0
Asteraceae
Echinacea angustifolia L. Moench.
Root
88.6±2.4
95.2±5.7
Lamiaceae Saxifragaceae
Leonurus japonicus Houtt. Hydrangea macrophylla Seringe var. thunbergii Makino Aloe arborescens Mill. Taxus cuspidata Sieb. & Zucc. Ajuga decumbens Thunb. Arctostaphylos uva-ursi (L.) spreng. Morus alba L. Hypericum erectum Thunb. Chamaecrista nomame (Siebold) H.Ohashi Lycopus lucidus Turcz. Epimedium grandiflorum Morr. var. thunbergianum (Miq.) Nakai Orthosiphon aristatus (Blume) Miq. Thujopsis dolabrata (Thunb. Ex L.f.) Sieb. & Zucc. Eleutherococcus senticosus Harms
Shoot and root Leaf
88.7±2.3 89.2±3.4
73.6±2.1 72.1±3.1
Leaf Leaf Shoot and root Leaf Leaf Shoot and root Shoot and root Shoot and root Shoot
89.3±0.4 89.5±1.0 89.9±2.6 89.9±2.0 90.3±0.9 90.3±3.8 90.8±1.0 90.9±2.5 92.0±0.7
89.0±3.7 107.7±3.0 66.4±4.6 100.4±7.5 104.9±3.6 106.0±5.5 98.8±4.9 86.4±0.7 97.6±1.7
Leaf Leaf Root
92.0±3.0 92.4±2.6 92.5±0.9
93.9±3.4 99.2±5.4 96.2±2.5
Aloaceae Taxaceae Lamiaceae Ericaceae Moraceae Clusiaceae Caesalpiniaceae Lamiaceae Berberidaceae Lamiaceae Cupressaceae Araliaceae
Hypocotyl
Root
Y. Sunohara et al. Table 1 (continued) Growtha (% of control)
Species name Family name
Scientific name
Used part
Liliaceae Gesneriaceae Lamiaceae Actinidiaceae Fabaceae Poaceae Ginkgoaceae Lamiaceae Eucommiaceae Lamiaceae Simaroubaceae Asteraceae Fabaceae Liliaceae
Polygonatum falcatum A. Gray Conandron ramondioides Sieb. et Zucc. Isodon japonicus (Burm.f.)H.Hara Actinidia polygama (Sieb. et Zucc.) Planch. ex Maxim. Lathyrus palustris L. subsp. pilosus (Cham.) Hult. Sasa veitchii Carr. Ginkgo biloba L. Elsholtzia ciliata (Thunb.) Hyl. Eucommia ulmoides Oliv. Scutellaria baicalensis Georgi. Picrasma quassioides Benn. Cirsium japonicum Fisch. ex DC. Desmodium styracifolium (Osb.) Merr. Polygonatum odoratum (Mill.) Druce var. pluriflorum (Miq.) Ohwi Scutellaria barbata D. DON. Chaenomeles sinensis (Thouin.) Koehne Phragmites australis (Cav.) Trin. ex Steud. Stevia rebaudiana L. Akebia quinata (Houtt)Decne Gymnema sylvestre R. Br. Coptis japonica (Thunb.)Makino Clematis chinensis Osbeck Lycium chinense Mill. Zanthoxylum schinifolium Siebold & Zucc. Saxifraga stolonifera Curtis
Rhizome Shoot and root Shoot Insect gall of fruit Shoot and root Leaf Leaf Shoot and root Leaf Root Heart wood Root Leaf Rhizome
Lamiaceae Rosaceae Poaceae Asteraceae Lardizabalaceae Asclepiadaceae Ranunculaceae Ranunculaceae Solanaceae Rutaceae Saxifragaceae a
Shoot and root Fruit Root Leaf stem Leaf Rhizome Root leaf Leaf and fruit Leaf
Hypocotyl
Root
93.0±3.2 93.1±1.5 93.6±0.9 93.9±2.8 93.9±4.0 93.9±2.0 94.1±5.3 94.2±3.7 94.5±1.6 94.7±2.5 95.3±1.2 95.5±2.0 96.3±3.2 96.6±2.3
88.2±5.7 82.5±7.6 87.6±0.5 68.4±3.0 94.5±1.9 104.4±4.4 92.4±3.7 95.7±3.2 122.0±2.5 97.9±3.8 101.3±3.9 94.7±7.3 97.1±2.2 81.3±10.6
97.2±1.2 98.5±1.6 99.2±2.9 99.8±3.5 100.0±0.7 100.2±1.3 100.5±2.5 101.0±2.1 101.3±3.0 102.7±3.3 104.3±3.3
106.4±9.5 104.5±8.1 90.3±1.5 93.4±4.4 94.1±5.6 115.0±4.9 103.8±1.3 106.7±7.3 102.7±2.9 106.4±4.5 98.9±6.1
Growth was determined by measuring the hypocotyl and root length of lettuce seedlings in 41-mm wells
gas chromatograph (Agilent Technology 6890N) connected online to a JEOL mass spectrometer (JEOL-MS-600H), equipped with an HP-5MS (Agilent Technology) fused-silica capillary column (25 m×0.25 mm i.d., 0.25 μm film thickness) in the electron impact mode (70 eV). The gas chromatographic conditions were as follows: the injection port was set at 250 °C; the oven temperature programme was 40 °C (1 min held), then increased to 250 °C at 10 °C/min, and held for 3 min at the final temperature; carrier gas, He (1 ml/min). A headspace vapor sample (0.5 ml) was injected manually in the splitless mode (sampling time: 1.0 min). GC-MS data of the volatile compounds from A. sieboldii and S. tenuifolia were analyzed using TSS 2000 software (Shrader Analytical and Consulting Laboratories, Inc.). A preliminary identification was performed by the NIST mass library database (National Institute of Standards and Technology, Gaithersburg, MD) and was further confirmed by comparisons of retention times and mass spectra with those of the standard compounds.
Plant growth inhibitory activity of volatile compounds from A. sieboldii and S. tenuifolia Authentic samples of α-pinene (2,6,6trimethylbicyclo[3.1.1]hept-2-ene) and (-)-β-pinene (1S,5S)-(6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane) were purchased from Wako Pure Chemicals (Osaka, Japan), and (+)3-carene ((1S,6R)-3,7,7- trimethylbicyclo[4.1.0]hept-3-ene), eucarvone (2,6,6-trimethy-2,4-cycloheptadien-1-one), and (±)limonene (1-methyl-4-(1-methylethenyl)-cyclohexene) were obtained from Tokyo Chemical Industry (Tokyo, Japan). (+)Pulegone ((5R)-5-methyl-2-(propan-2-ylidene) cyclohexan-1one) and menthone (5-methyl-2-(propan-2-yl)cyclohexan-1-one) were purchased from Sigma-Aldrich Co. (St. Louis, USA) and Nacalai Tesque (Kyoto, Japan), respectively. Five pre-germinated seeds, which had been incubated for 1– 3 days in the dark at 25 °C until the radicle length became approximately 2–3 mm, were placed onto a filter paper
Characterization of phytotoxic compound eucarvone
impregnated with DW (0.7 ml) in a 30-ml glass vial (33 mm diameter). Each authentic sample was added to hexane (100 %) to give concentrations of 0, 750, 7,500, 37,500, 75,000, and 150,000 ppm (v/v). Ten microliters of the authentic sample solution was spotted onto a piece of filter paper (1 cm×4 cm), and the solvent hexane was allowed to volatilize in the air for 10 sec. The authentic sample-spotted filter paper was then hanged from the rubber plug of the 30-ml glass vial, and the glass vial was tightly sealed as above. The concentrations of authentic samples in the airspace within the glass vial were calculated assuming that the spotted compounds volatilize completely without any loss due to adsorption or leakage. The final concentration of each authentic sample in the 30-ml glass vial was converted from parts per million to mole per liter and shown in each figures. The root and shoot lengths were measured after 3 days in controlled conditions (25 °C, in the dark). The concentrations of GR10 (the dose required to cause a 10 % reduction in plant growth), GR20, and GR50 were calculated using the equations shown in Fig. 1. In addition, to investigate the effect of eucarvone on the seedling growth of several plant species, 11 plant species were used. The 11 plant species tested were as follows: maize (Zea mays L. cv. Honey Bantam), cucumber (Cucumis sativus L. cv. Shimoshirazu Jibai), lettuce, velvetleaf (Abutilon avicennae Geartn.), tomato (Solanum lycopersicum L. cv. Kantaro), rice (Oryza sativa L. cv. Nipponbare), wheat (Triticum aestivum L. cv. Norin 61), slender amaranth (Amaranthus viridis L.), finger millet (Eleusine coracana (L.) Gaertn.), goosegrass (Eleusine indica (L.) Gaertn.), and green foxtail (Setaria viridis (L.) Beauv.). The GR50 concentrations of shoot and root elongation were calculated using the equations shown in Fig. 2. Determination of cell viability Root cell viability was evaluated by staining with fluorescein diacetate (FDA) and propidium iodide (PI) (Pan et al. 2001; Sunohara and Matsumoto 2008). Root segments (1.0 cm length from the tip) were excised from the 3-day-old seedlings of finger millet and maize treated with 0, 53, or 158 μM eucarvone for 3 h. The roots were stained with a mixture solution of 13 μg/ml FDA (Aldrich Chemical Co., Milwaukee, USA) and 0.5 μg/ml PI, containing 5 % dimethyl sulfoxide (DMSO) for 10 min, and were then washed with distilled water. Fluorescence images of the stained roots were observed using a fluorescence microscope (Nikon E600 with a B-2A filter [excitation 450–490 nm, emission >520 nm]; Nikon Corp., Tokyo, Japan). Root cell viability was also evaluated by Evans blue staining (Sunohara and Matsumoto 2008; Tamás et al. 2004). Root segments (1.0 cm length from the tip) were taken from the eucarvone-treated seedlings as above. The root segments were stained with a 0.25 % (w/v) aqueous solution of Evans blue (Sigma Chemical Co., St. Louis, USA) for 1 h at room temperature. The stained roots were then washed with distilled water for 15 min. Evans blue, which was taken into the root segments,
was then extracted using 500 μl of N,N-dimethylformamide solution (100 %) without grinding. After the root tips were allowed to soak in the N,N-dimethylformamide solution overnight 30 °C in the dark, the absorbance of the released Evans blue was spectrophotometrically measured at 600 nm. Detection of ROS production Production of O2− was estimated using the redox-sensitive probe, dihydroethidium (DHE), in accordance with the procedures of Yamamoto et al. (2002) with minor modification (Sunohara and Matsumoto 2008; Sunohara et al. 2011). Root segments (1.0 cm length from the tip) were taken from the eucarvone-treated seedlings as above and then stained with 10 μM DHE solution containing 1 % acetone in 100 μM CaCl2, by gently shaking for 30 min at room temperature in the dark. After washing the root segments with distilled water to remove residual dye, the roots were observed using a fluorescence microscope (Nikon E600 with a B-2A filter [excitation 450–490 nm, emission ≥520 nm]; Nikon, Tokyo, Japan). The fluorescence of oxyethidium derived from DHE oxidation by O2− was observed using the microscope. Lipid peroxidation The histochemical detection of lipid peroxidation was performed using the procedures described by Pompella et al. (1987) and Yamamoto et al. (2001) with minor modification. Root segments (1.0 cm length from the tip) were taken from the eucarvonetreated seedlings as above. The roots were stained with Schiff’s reagent which detects aldehydes that originate from lipid peroxides for 20 min. After the reaction, roots were rinsed with a sulfide solution (0.5 % (w/v) K2S2O5 in 0.05 M HCl). The stained roots were kept in the sulfide solution to retain the staining color. Roots stained with Schiff’s reagent were observed under a light microscope (Nikon E600, Nikon Corp., Tokyo, Japan). Statistical analyses All results were represented as the means±S.E. of at least three replicates, and all experiments were repeated at least twice. Relationships were considered to be significant when P 320 µM
20
20 00
50
100
150
200
250
300
00
350
50
100
Concentration (μM)
(c) 3-Carene
Growth (% of control)
Growth (% of control)
100 80 60
y(●) = 108.71e-0.0009x, R² = 0.8823 GR10 = 209.9 µM, GR20 > 318 µM
40
y(○) = 116.51e-0.0010x, R² = 0.7861 GR10 = 258.2 µM, GR20 > 318 µM
20 0
50
100
150 200 250 Concentration (μM)
300
100 80 60
y(●) = 104.62e-0.0006x, R² = 0.7195 GR10 = 250.9 µM, GR20 > 308 µM
40
0
50
100
150 200 Concentration (μM)
250
300
350
(g) Pulegone 120 100
y(●) = 92.599e-0.01x, R² = 0.935 GR20 = 14.1 µM, GR50 = 59.3 µM
80
y(○) = 97.695e-0.013x, R² = 0.9291 GR20 = 16.0 µM, GR50 = 59.3 µM
60 40 20 0
50
100
150 200 Concentration (μM)
250
300
y(○) = 126.28e-0.012x, R² = 0.979 GR20 = 37.3 µM, GR50 = 76.6 µM
60 40
0
50
100
150 200 Concentration (μM)
250
300
350
(f) Menthone y(●) = 124.34e-0.011x, R² = 0.9532 GR20 = 42.0 µM, GR50 = 86.8 µM
100 80
y(○) = 120.34e-0.012x, R² = 0.9586 GR20 = 38.0 µM, GR50 = 78.5 µM
60 40 20
y(○) = 100.55e-0.0008x, R² = 0.6246 GR10 = 138.6 µM, GR20 > 308 µM
20
80
120 Growth (% of control)
Growth (% of control)
350
y(●) = 94.481e-0.011x, R² = 0.9826 GR20 = 15.8 µM, GR50 = 60.6 µM
100
0
350
120
Growth (% of control)
300
20
(e) Limonene
140
0
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120
120
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(d) Eucarvone
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Concentration (μM)
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0
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150 200 Concentration (μM)
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Characterization of phytotoxic compound eucarvone
Fig.
1 Effects of the volatile compounds from A. sieboldii and S. tenuifolia on the shoot (filled circle) and root (empty circle) growth of lettuce seedlings at the germination stage 3 days after treatment. Mean control values (cm) and standard errors were as follows: a α-pinene: 1.30 ±0.09 (shoot), 1.64±0.13 (root); b β-pinene: 1.39±0.02 (shoot), 2.22± 0.18 (root); c 3-carene: 1.13 ± 0.10 (shoot), 1.67 ± 0.24 (root); d eucarvone: 1.23±0.03 (shoot), 2.14±0.22 (root); e limonene: 1.22±0.04 (shoot), 2.12±0.21 (root); f menthone: 1.23±0.06 (shoot), 2.00±0.02 (root); and g pulegone: 1.23±0.03 (shoot), 2.18±0.22 (root)
A total of 71 species (41 families) including Lamiaceae (13 species), Fabaceae (6 species), and Asteraceae (4 species) were tested. Among the species, the rate of inhibition of hypocotyl elongation between 80 and 100 % in the nearest (41 mm) well of the multidish (cf. Fig. S1) was observed in 0 species, between 60 and 79 % in 2 species, between 40 and 59 % in 3 species, between 20 and 39 % in 10 species, and 308, 38.0, and 16.0 μM for α-pinene, β-pinene, 3-carene, eucarvone, limonene, menthone, and pulegone, respectively. In shoot growth, the GR20 values were approximately 70.6, 85.9, >318, 15.8, >308, 42.0, and 14.1 μM for α-pinene, β-pinene, 3-carene, eucarvone, limonene, menthone, and pulegone, respectively. The order of the strength of the seven volatile compounds as inhibitors of lettuce shoot and root growth based on the GR20 values was estimated as pulegone > eucarvone > menthone > α-pinene > β-pinene > limonene ≒ 3-carene. These results support the previous reports by Vaughn and Spencer (1993, 1996), in which several monoterpenoids having oxygencontaining functional groups had strong phytotoxic effects on seed germination (cf. Fig. S4), and that by Vokou et al. (2003), in which ketones were the most phytotoxic, followed by aldehydes, ethers, alcohols, and phenols, with acetates and hydrocarbons being the least phytotoxic in lettuce seedlings in 47 monoterpenoids of different chemical groups. Mentone and pulegone are known to be components in peppermint essential oil (Maffei and Sacco 1987), and mentone has been reported to induce sprout growth suppression in potato tubers (Coleman et al. 2001). Pulegone has been identified as one of the most strong allelochemicals, was shown to be nearly four times more toxic than HCN on the seed germination of radish
Y. Sunohara et al.
Characterization of phytotoxic compound eucarvone
Fig. 2
Effects of eucarvone on the shoot (filled circle) and root (empty circle) growth of 11 plant species at the germination stage 3 days after treatment. The mean control values (cm) and standard errors were as follows: a maize: 2.42±0.18 (shoot), 5.83±0.22 (root); b cucumber: 1.21 ±0.27 (shoot), 4.69±0.71 (root); c lettuce: 1.23±0.03 (shoot), 2.14±0.22 (root); d velvetleaf: 2.06±0.52 (shoot), 3.94±0.17 (root); e tomato: 2.87± 0.10 (shoot), 5.54±0.38 (root); f rice: 1.48±0.21 (shoot), 5.03±0.25 (root); g wheat: 4.01 ± 0.16 (shoot), 6.03 ± 0.71 (root); h slender amaranth: 1.36±0.05 (shoot), 1.58±0.16 (root); i finger millet: 1.16± 0.07 (shoot), 1.55±0.07 (root); j goosegrass: 1.70±0.07 (shoot), 1.97± 0.07 (root); and k green foxtail: 3.67±0.09 (shoot), 2.25±0.06 (root)
(Asplund 1968), and was also shown to possess antimicrobial (Oumzil et al. 2002) and insecticidal activities (Park et al. 2006). Eucarvone has been reported to exhibit strong insecticidal activity (Kim and Park 2008). However, to the best of our knowledge, this is the first study to show that eucarvone can have potent phytotoxic activity, and the strength of which is almost equal to that of pulegone. In addition, nothing has been done to explore phytotoxic mechanisms of eucarvone. Therefore, we focused on eucarvone and further examined its phytotoxic activity. Effect of eucarvone on the seedling growth of several plant species at the germination stage The phytotoxic activities of eucarvone on initial growth were investigated using six crop species, maize, cucumber, lettuce, tomato, rice, and wheat, and five weed species, velvetleaf, slender amaranth, finger millet, goosegrass, and green foxtail. Eucarvone-induced growth inhibition was species-selective, while seedling growth was suppressed by eucarvone in a concentration-dependent manner in most of the species tested (Fig. 2). Among the 11 plant species, maize was the most tolerant, and green foxtail, goosegrass, and finger millet were relatively susceptible. The GR50 values of shoot growth determined 3 days after treatment in maize, green foxtail, goosegrass, and finger millet were estimated to be approximately >316, 21.2, 25.7, and 27.0 μM, respectively (Fig. 2). Regarding root growth, the GR50 values in maize, green foxtail, goosegrass, and finger millet were approximately >316, 20.9, 7.3, and 24.3 μM, respectively. Maize and finger millet were selected as examples of tolerant and susceptible species, respectively, for further experiments to characterize the phytotoxic action of eucarvone. Effect of eucarvone on cell viability The viability of root cells was examined by a double-staining method using FDA and PI. PI enters membrane-damaged cells, but not membrane-intact cells; then, it can be detected by its red fluorescence (Umebayashi et al. 2003). FDA easily enters living cells and then hydrolized by esterase, producing fluorescein, which can be detected by its green fluorescence
(Umebayashi et al. 2003). Hence, red and green fluorescences were used to assess dead and living cells, respectively. In 53 μM eucarvone treatment, red fluorescence was observed in the upper part of root cap of finger millet 3 h after treatment, and in 158 μM eucarvone, red fluorescence was observed in a whole part of the root tip (Fig. 3a). In maize root tips, a slight difference among control and the eucarvone (53 and 158 μM) treatments was observed in the fluorescences. Faint red fluorescence was observed in the small part of control root tip, and the faint red areas were slightly increased in 53 and 158 μM eucarvone-treated root tips (Fig. 3b). These results suggest that eucarvone (53–158 μM) strongly induce cell death in especially finger millet root tips 3 h after treatment. Cell viability in finger millet and maize roots were also determined by Evans blue staining to quantify the rates of dead cells. Evans blue is a non-permeating dye that enter membrane-injured cells and is unable to enter membraneintact cells. Hence, Evans blue staining has been used to assess cellular integrity (Gaff and Okong'o-Ogola 1971). In finger millet, 158 μM eucarvone significantly enhanced Evans blue uptake in root tip cells from 3 h after treatment. At 53 μM eucarvone, Evans blue uptake was not enhanced 24 h after treatment in finger millet root cells (Fig. 4a), while red fluorescence showing cell death was observed in the root tip 3 h after 53 μM eucarvone treatment (Fig. 3a). The inconsistency of these results may be due to the detection sensitivity between the two staining methods. In maize roots, eucarvone (
