Screening for Plant Volatile Emissions with Allelopathic Activity and the Identification of L-Fenchone and 1,8-Cineole from Star Anise (Illicium verum) Leaves

One hundred and thirty-nine medicinal plant species were screened for their allelopathic activity through volatile emissions using Lactuca sativa as a test plant. Volatile emissions from the leaves of star anise (Illicium verum) showed the highest inhibition (100%) on the radicle and hypocotyl growth. Using headspace gas collection and gas chromatography-mass spectrometry (GC-MS), seven major volatile compounds from the leaves of star anise, including α-pinene, β-pinene, camphene, 1,8-cineole, D-limonene, camphor, and L-fenchone were detected. To determine volatile compounds that may contribute to the inhibitory activity of star anise, the allelopathic potential of individual volatiles from star anise was evaluated using the cotton swab bioassay. The EC50 was calculated for each of the seven identified compounds. L-fenchone showed the strongest growth inhibitory activity (EC50 is 1.0 ng/cm3 for radicle and hypocotyl growth of lettuce), followed by 1,8-cineole, and camphene. This is the first report that L-fenchone could be an important volatile allelochemical from the leaves of star anise. From the actual concentration of each volatile compound in headspace and EC50 value, we concluded that the four volatile compounds, including L-fenchone, 1,8-cineole, β-pinene, and camphene are the most important contributors to the volatile allelopathy of star anise.


Introduction
Allelopathy refers to any direct or indirect harmful or beneficial effect by an organism (mostly plants) on another species through the production of bioactive compounds that are released into the environment [1]. Besides, the importance of allelopathic interaction between plants in nature, screening, and identification of natural compounds with high allelopathic activity is one direction in the search for new natural herbicides that could augment current weed control approaches. Several natural

Screening of Allelopathic Activity
Allelopathic activity of volatile emissions from 139 plant species (Appendix A) using lettuce as a test plant was evaluated using the Dish pack method [13,14]. The top 30 plants with the highest allelopathic activity are presented in Table 1. Generally, 59% and 50% of the screened plants inhibited hypocotyl and radicle growth of lettuce seedlings respectively at different degrees (Figures 1 and 2). Other plant species demonstrated either a lack of inhibitory activity or exhibited stimulatory activity up to 38.9% (Epimedium sagittatum) and 95.0% (Pimenta racemosa) for lettuce radicle and hypocotyl, respectively. The highest radicle and hypocotyl inhibition (100%) were observed for the volatile constituents of Illicium verum or star anise leaves. I. verum (Illiciaceae) is an aromatic evergreen tree distributed in North America, the West Indies, and Eastern Asia, and is known for the use of its fruits in traditional Chinese medicine and the food industry due to unique secondary metabolites, such as terpenoids, phenylpropanoids, lignans, and benzoquinones [15,16]. Both the leaves and fruits have a strong aroma with a distinctive licorice taste [17]. Star anise is known for its insecticidal activity [18], antifungal [19], and antimicrobial [20] properties. However, there is no information about the allelopathic properties of star anise through volatile emissions. Therefore, this plant was chosen as a candidate species for further identification of volatile compounds.    Several other plants with high plant growth inhibitory activity were also identified in this study. For example, the volatiles from Crateva religiosa or sacred garlic pear, suppressed the hypocotyl growth by 86.2%, followed by Shorea robusta, Artabotrys uncinatus, Sinomenium acutum, Dendrobium sp. (with inhibitory activity ranging from 34.4 to 30.3%). Regarding the inhibitory effect on radicle growth, Argemone mexicana or Mexican poppy suppressed 73.5% of lettuce radicle growth. Twentyfour other plant species showed radicle inhibition from 10.5% (Zingiber officinale, ginger) to 26.3% (Derris malaccensis). Generally, there was no significant correlation between radicle and hypocotyl growth, which can be due to differences in the mode of action of volatile compounds and their availability for lettuce seedlings. Although this study focused on the screening of plants with high plant growth inhibitory activity, several species showed a stimulatory effect, especially on hypocotyl growth. Some of these species include, but not limited to, Pimenta racemose (95% of stimulatory) from the myrtle family and Citrus hystrix (70.8%) or kaffir lime. Several other plants with high plant growth inhibitory activity were also identified in this study. For example, the volatiles from Crateva religiosa or sacred garlic pear, suppressed the hypocotyl growth by 86.2%, followed by Shorea robusta, Artabotrys uncinatus, Sinomenium acutum, Dendrobium sp. (with inhibitory activity ranging from 34.4 to 30.3%). Regarding the inhibitory effect on radicle growth, Argemone mexicana or Mexican poppy suppressed 73.5% of lettuce radicle growth. Twenty-four other plant species showed radicle inhibition from 10.5% (Zingiber officinale, ginger) to 26.3% (Derris malaccensis). Generally, there was no significant correlation between radicle and hypocotyl growth, which can be due to differences in the mode of action of volatile compounds and their availability for lettuce seedlings. Although this study focused on the screening of plants with high plant growth inhibitory activity, several species showed a stimulatory effect, especially on hypocotyl growth. Some of these species include, but not limited to, Pimenta racemose (95% of stimulatory) from the myrtle family and Citrus hystrix (70.8%) or kaffir lime.
While the data presented in Table 1 were obtained from wells that were located 41 mm from the plant source, the distance from the source of volatiles had a significant effect on the observed inhibitory activity. In this regard, Figure 3 shows that the growth inhibitory effect of volatiles from star anise decreased as a function of a distance from a well for both radicle and hypocotyl.

Evaluation of EC50 of Volatiles from Star Anise Volatiles
Inhibitory activity of the authentic volatile compounds varied from D-limenone (EC50 is 105.7

Evaluation of EC 50 of Volatiles from Star Anise Volatiles
Inhibitory activity of the authentic volatile compounds varied from D-limenone (EC 50 is 105.7 ng/cm 3 and 24 5 ng/cm 3 for hypocotyl and radicle, respectively, less the inhibitor) to L-fenchone (EC 50 is 1.0 ng/cm 3 for radicle and hypocotyl, most potent inhibitory activity). Similar to this study, volatile terpenes, including camphor, 1,8-cineole, α-pinene, and β-pinene, were identified from the invasive perennial weed mugwort (Artemisia vulgaris), and their potential role in mugwort establishment and proliferation in introduced habitats was suggested to be as a result of their phytotoxicity [26]. The determination of EC 50 in the headspace (Table 2) of the seven compounds showed that L-fenchone was the most potent plant growth inhibitor (EC 50 is 1.0 ng/cm 3 for both radicle and hypocotyl), followed by 1,8-cineole and camphene. Kaur et al. [27] demonstrated that the volatiles from the essential oil of Eucalyptus tereticornis, including α-pinene (32.5%) and 1,8-cineole (22.4%), significantly suppressed early seedling growth and seedling vigour of Amaranthus viridis. 1,8-Cineole is known to be a potent plant growth regulator and can inhibit mitosis, which leads to growth abnormities, inhibits respiration of isolated mitochondria, and aspartate synthase [28]. In fennel seeds, L-fenchone is well known to be present in sufficient amounts, but L-fenchone was never reported as potent plant growth inhibitors. This is the first report that L-fenchone could be an important volatile allelochemical from the leaves of the star anise. From the actual concentration of each volatile compound in the headspace and EC 50 value, we concluded that four volatile compounds, 1,8-cineole, β-pinene, camphene, and L-fenchone ( Figure 4) were the most important contribution for plant growth inhibitory activity in the headspace of star anise. determination of EC50 of radicle and hypocotyl growth of lettuce seedlings by 1-decyne in the vapor phase and was found at the concentration of 0.5 ng/mL [29] and by safranal-1.2 µg/L (ppb) [30].
Additionally, previous results also demonstrated that octyl acetate, a major volatile from H. sosnowskyi fruits, had lower EC50 for radicle and hypocotyl growth (64 and 57 ng/cm 3 , respectively), than the predominant octanal (EC50 is 20 and 9 ng/cm 3 respectively), however, octanal was suggested to be the major contributor to its allelopathic activity based on total activity estimation [14].

Plant Material
Plant materials (leaves) were collected from 139 plant species in the Botanical Garden of Showa Pharmaceutical University, Tokyo (Japan), from May to June 2013. All samples were dried in an oven The cotton swab method, following GC-MS analysis was previously successfully applied for the determination of EC 50 of radicle and hypocotyl growth of lettuce seedlings by 1-decyne in the vapor phase and was found at the concentration of 0.5 ng/mL [29] and by safranal-1.2 µg/L (ppb) [30].
Additionally, previous results also demonstrated that octyl acetate, a major volatile from H. sosnowskyi fruits, had lower EC 50 for radicle and hypocotyl growth (64 and 57 ng/cm 3 , respectively), than the predominant octanal (EC 50 is 20 and 9 ng/cm 3 respectively), however, octanal was suggested to be the major contributor to its allelopathic activity based on total activity estimation [14].

Plant Material
Plant materials (leaves) were collected from 139 plant species in the Botanical Garden of Showa Pharmaceutical University, Tokyo (Japan), from May to June 2013. All samples were dried in an oven at 60 • C for 4 h and then stored in paper bags placed in plastic bags in a refrigerator (4 • C) before their use.

Dish Pack Method
The dish pack method [13] was used to determine the allelopathic activity of naturally emitted volatile compounds of test plant materials ( Figure 5). Briefly, 2 g of dried material was placed in one of the 6-well multi-dish plastic plate (3.5 cm d., Nunc Company). The distances from the well where the sample was placed (source well) to the center of other wells were 41, 58, 82 and 92 mm (Figure 5a). In each of the other 5 wells, the filter paper was placed, and 0.7 mL of distilled water was added. Then, 7 seeds of Lactuca sativa, var. Great Lakes 366 (Takii seed Co., Japan) were placed on top. The plastic plates were sealed tightly and incubated for 3 days at 22 • C under dark conditions. A multi-dish plastic plate with a blank source well was used as the control treatment. The lengths of lettuce radicle and hypocotyl were measured, and the allelopathic activity was expressed as a percentage of radicle or hypocotyl inhibition at wells located 41 mm from the plant source.
volatile compounds of test plant materials ( Figure 5). Briefly, 2 g of dried material was placed in one of the 6-well multi-dish plastic plate (3.5 cm d., Nunc Company). The distances from the well where the sample was placed (source well) to the center of other wells were 41, 58, 82 and 92 mm (Figure 5a). In each of the other 5 wells, the filter paper was placed, and 0.7 mL of distilled water was added. Then, 7 seeds of Lactuca sativa, var. Great Lakes 366 (Takii seed Co., Japan) were placed on top. The plastic plates were sealed tightly and incubated for 3 days at 22 °C under dark conditions. A multidish plastic plate with a blank source well was used as the control treatment. The lengths of lettuce radicle and hypocotyl were measured, and the allelopathic activity was expressed as a percentage of radicle or hypocotyl inhibition at wells located 41 mm from the plant source.

Headspace Gas Chromatography-Mass Spectrometry
Plant material (1 g) was placed into a 20 mL sealed glass vial (GRACE, Japan) and incubated at 60 °C for 1 hour. Then, headspace gas (200 µL) was collected using a 1000 µL micro-syringe (MS-GAN100, Ito Corporation, Tokyo, Japan), and injected into a gas chromatography-mass spectrometry set-up (GC-MS-QP 2010 Plus system, Shimadzu, Japan) using an EQUITY-5 column (0.25 mm × 30 m × 0.25 µm, Supelco) [14]. Helium gas was used as a carrier with a total flow rate of 29 mL/min. The injection temperature was 200 °C with a column head pressure of 61.3 kPa. The oven temperature was increased at a rate of 10 °C/min to 200 °C from 60 °C and kept constant for 30 min at the end. Mass spectra were recorded at 70 eV and compared with an in-house mass spectral library (NIST and Wiley). The samples analyzed using the headspace GC-MS were the leaves of the Illicium verum and the volatile compounds, including α-pinene, β-pinene, camphene, 1,8-cineole, D-limonene, camphor, and L-fenchone.

Cotton Swab Method
The cotton swab method [14] was used to evaluate the plant growth inhibitory activity of the leaves of I. verum and authentic volatile compounds 1,8-cineole, beta-pinene, camphene, D-limonene, Figure 5. Testing the effect of leave volatiles from star anise on radicle and hypocotyl growth (%) of lettuce seedlings as a function of distance from plant material using the Dish Pack method (a,b), samples for GC-MS analysis (c), and cotton swab method (d).

Headspace Gas Chromatography-Mass Spectrometry
Plant material (1 g) was placed into a 20 mL sealed glass vial (GRACE, Japan) and incubated at 60 • C for 1 h. Then, headspace gas (200 µL) was collected using a 1000 µL micro-syringe (MS-GAN100, Ito Corporation, Tokyo, Japan), and injected into a gas chromatography-mass spectrometry set-up (GC-MS-QP 2010 Plus system, Shimadzu, Japan) using an EQUITY-5 column (0.25 mm × 30 m × 0.25 µm, Supelco) [14]. Helium gas was used as a carrier with a total flow rate of 29 mL/min. The injection temperature was 200 • C with a column head pressure of 61.3 kPa. The oven temperature was increased at a rate of 10 • C/min to 200 • C from 60 • C and kept constant for 30 min at the end. Mass spectra were recorded at 70 eV and compared with an in-house mass spectral library (NIST and Wiley). The samples analyzed using the headspace GC-MS were the leaves of the Illicium verum and the volatile compounds, including α-pinene, β-pinene, camphene, 1,8-cineole, D-limonene, camphor, and L-fenchone.

Cotton Swab Method
The cotton swab method [14] was used to evaluate the plant growth inhibitory activity of the leaves of I. verum and authentic volatile compounds 1,8-cineole, beta-pinene, camphene, D-limonene, α-pinene, camphor, and L-fenchone, which were identified by GC-MS analysis as major volatile compounds. Briefly, 10 mL of 0.75% agar solution was added to a 20 mL glass vial, and after agar solidification, 7 seeds of lettuce were placed into each vial. A half of double-tipped cotton [14] was vertically inserted into the agar, and an appropriate amount (0.1, 0.2, and 0.3 µL) of the authentic compound was added on the cotton swab. The concentrations of the compounds were 0.001, 0.01, 0.1, and 1 ppm. The glass vial was closed by a pressure cap and incubated for 3 days at 22 • C. The length of the radicle and hypocotyl of the lettuce seedlings were measured, and the inhibition of the radicle and hypocotyl of the lettuce seedlings was plotted against the applied amount of an authentic compound.