Eco-Engineering, 18（2）, 77-81, 2006

Original Paper

Sundiversifolide from Exudates of Mexican Sunflower ( (Hemsl.) A. Gray) Achenes

Takako Kato*, Masamichi Yamashita**, Koji Hasegawa* and Kaori Tomita-Yokotani*

* Doctoral Program in Life Sciences and Bioengineering, University of Tsukuba Tsukuba, Ibaraki 305-8572, Japan ** Institute of Space and Astronautical Science /JAXA Yoshinodai, Sagamihara, Kanagawa 229-8510, Japan

(Received December 20, 2005; Accepted March 22, 2006)

ABSTRACT The purpose of this study was to confirm the existence of sundiversifolide, 4,15-dinor-3-hydroxy-1(5)-xanthene- 12,8-olide, as an allelopathic substance in two species of . This substance could be used to control an ecosystem by its allelopathic function. Sundiversifolide has species-specific allelopathic properties in germinating seeds (achenes) of the sunflower, L.cv. Taiyo. However, its distribution among other species of plants has not yet been elucidated. The allelopathic properties and the identification of their substances were investigated in the exudates from the achenes of the Mexican sunflower (Tithonia diversifolia (Hemsl.) A. Gray) and Leucanthemum paludosum cv. North Pole. In the exudates from the Mexican sunflower achens, the existence of sundiversifolide was identified by LC- ESI+/MS. In the exudates from the L. paludosum, sundiversifolide was not detected. The low polar fraction of the exudates from the achenes of the Mexican sunflower has an allelopathic function as does the sunflower . Allelopathic properties of achenes of Mexican sunflower was also examined. The ethylacetate-soluble fraction of the exudates inhibited the growth of cat’s-eyes (Veronica persica Poiret) seedlings and the conidial germination of useful fungi, i.e. Neurospora crassa. These allelopathic functions including sundiversifolide could be applied to regulate the growth in agricultural eco-systems.

Key words : Allelopathy, Growth regulator, Helianthus annuus L., Tithonia diversifolia, Sundiversifolide

substances. Khalid et al. (2002) reviewed that allelopathic 1. Introduction compounds can be used as natural herbicides and other Many chemicals have been identified as allelopathic pesticides; they are less disruptive of the global eco-system than substances in living and fallen leaves (Rice, 1984). There is are the synthetic agrochemicals. Sundiversifolide, 4,15-dinor-3- considerable information available regarding the allelopathy hydroxy-1(5)-xanthene-12,8-olide (Fig. 1 (A)), was isolated and phenomenon itself and potential allelochemicals. If allelopathic identified from the exudate of seeds (achenes) of the Taiyo substances could be identified, they can be used as a plant sunflower as a new plant species-specific allelopathic substance growth regulator. Since allelopathic substances are (Ohno et al., 2001). This substance was also isolated from the fundamentally natural compounds in plants, the use of these exudates of Russia sunflower after that (Kato et al., 2006). substances in agriculture might produce less damage against the However, the distribution in other species of plants has not yet natural eco-system compared to the use of artificial synthetic been reported. The stereoisomer of Sundiversifolide (Fig. 1 (B))

© The Society of Eco-Engineering （33）77 was also studied. Once we understand the allelopathy phenomenon and its mechanism, we might apply such knowledge in agricultural eco- systems, and provide an alternative to now widely applied synthesized chemicals. We are sure that this study would significantly contribute to this goal.

2. Materials and Methods 2.1 Plant and microbe materials The Mexican sunflower (Tithonia diversifolia (Hemsl.) A. Gray), sunflower (Helianthus annuus L. cv. Russia) achenes and Leucanthemum paludosum (Poir.) Bonnet et Barratte were used Fig. 1 Chemical structure of sundiversifolide (A) and diversifolide (B). as plant materials for the detection of the allelopathic activity or sundiversifolide. In the exudates form H. annuus L. cv. Russia has already been isolated and identified in the extract from the achenes, sundiversifolide has already isolated as cv. Taiyo (Kato root of the Mexican sunflower as a new substance, i.e., et al., 2006). A wild-type strain of Neurospora crassa 74A diversifolide (Kuo et al., 1999). The Mexican sunflower might obtained from the Fungal Genetics Stock Center at the have the ability to produce sundiversifolide, because it produces University of Kansas Medical Center, Kansas City, KS, was a stereoisomer of sundiversifolide. The sunflower and Mexican used as the test fungus. sunflower are in the same Helianthinae in family 2.2 Extraction of sundiversifolide (Konarev et al. 2002). These plants are native species around Mexican sunflower (Tithonia diversifolia (Hemsl.) A. Gray) North America. Furthermore, Konarev et al. (2002) have achenes (1450 achenes, 3 g) were sterilized in a 0.05% (w/v) reported that H. annuus and T. diversifolia had similar highly sodium hypochlorite solution for 10 minutes, soaked 10 minutes alkaline inhibitor of trypsin components. in running tap water and rinsed with three times distilled- The leaf extract from the Mexican sunflower has many deionized water. These achenes were placed in a 1 L biological activities, such as anti-inflammatory and analgesic Erlenmeyer flask with 200 ml of distilled-deionized water, then properties (Owoyele et al., 2004). The Mexican sunflower may be cultured with stirring for 1 day at 26ºC in the dark (Fig. 2). The a useful plant for human life. Kato et al. (2005) has already culture medium was evaporated to dryness in vacuo at 38ºC. reported that sundiversifolide displays an anti-microbal activity The concentrates were then fractionated. and inhibitory activity related to auxin. They used the conidial germination test and cat’s-eyes seedling test for the bioassay in their study. The fungus assay is useful for studying allelopathy, because Neurospora crassa has been a well established fungus model in molecular studies with considerable information available in all fields of microbe biology (Galagan et al., 2003, Tomita-Yokotani et al., 2003, Parvez et al., 2004). The cat’s-eyes (Veronica persica Poiret) seedling test has been a standard procedure for the screening of bio-active substances, because it has a high sensitivity against those substances (Node et al., 2003). We have already reported a part of the results about that, furthermore the result was reconsidered for detail discussion (Kato et al., 2005). In this paper, we report the allelopathic function of the exudates from the Mexican sunflower achenes. Fig. 2 Method for collection of exudates from Mexican sunflower As a comparison to the other species plant, the L. paludosum and L. paludosum achenes.

78（34） Eco-Engineering The process of fractionation and analysis is shown in Fig. 3. temp. 300ºC; symmetry C18, f 2.1 × 150 mm, Waters, H2O- The concentrates were fractionated into the acetone-soluble and MeOH, 2:3 v/v, 0.2 ml min-1) which established the presence of -insoluble fractions. The acetone-soluble fraction was sundiversifolide based on the HPLC retention time and mass fractionated into ethylacetate-soluble and -insoluble fractions. spectral fragmentation. The ethylacetate-soluble fraction was purified using a silica gel 2.3 Bioassay of water exudates from achenes of Sep-pak cartridge (Waters) with an n-hexane-ethylacetate Mexican sunflower in conidial germination in cat’s- solvent system by increasing the ethylacetate concentration in a eyes seedlings series of 20% step (20 ml per step). The 40% ethylacetate Eight cat’s-eyes (Veronica persica Poiret) seeds were placed fraction was purified using a C18 Sep-pak cartridge (Waters) in a 27 mm Petri dish containing the test solution dissolved in with a water-methanol solvent system by increasing the water (400 µl ) and incubated in the dark at 25ºC for five days. methanol concentration in a series of 10% step (20 ml per step). After the incubation, their shoot and root lengths were The 10-60% methanol fraction was analyzed by HPLC (TSK gel measured.

ODS-80Ts f 4.6 × 250 mm, TOSOH, H2O-MeOH, 2:3, v/v, 0.8 2.4 Bioassay of water exudates from achenes of ml min-1, 280 nm detector), LC- ESI+ /MS (capillary, 3.3 kvolts; Mexican sunflower during conidial germination in cone voltage, 3.3 V; source temp. 120ºC, desolvation temp. Neurospora crassa

300ºC; symmetry C18, f 2.1 × 150 mm, Waters, H2O-MeOH, The conidia were inoculated on complete agar medium and 2:3 v/v, 0.2 ml min-1) and established the presence of incubated at 26ºC for 6 days in the dark, then exposed to sundiversifolide and other allelopathic substances by the HPLC fluorescent light (10 µmol m-2 s-1) for 1 day. The conidia of the retention time and mass spectral fragmentation. cultures were suspended in cold sterile water, and then filtered As authentic sundiversifolide in the water exudates of the through two layers of gauze to remove the mycelia fragment achenes, 700 ml of achenes of the Russia sunflower and 1000 contamination. The suspension was centrifuged at 1780 × g for ml of water were used. The subsequent method was modified as 10 min at 4ºC, and the pellet was then washed with sterile water. previously described (Ohno et al, 2001: Kato et al., 2005). The conidia were resuspended and then incubated in a 10 ml Sundiversifolide was analyzed by LC-ESI+/MS ( capillary, Erlenmeyer flask with 1 ml volume of Fries minimal medium 3.3 kV; cone voltage, 3.3 V; source temp. 120ºC, desolvation containing 1.5% sucrose with or without the test solutions (control) at an optimum concentration of 2 × 106 conidia ml-1 at 26ºC in the dark with 120 shakings per minute. The conidial germination was counted using by a counter 3 hours after the incubations. These conidial germination tests were carried out as previously described (Tomita et al. 1996; Tomita-Yokotani et al. 2003).

3. Results and Discussion 3.1 Isolation and identification of sundiversifolide in exudates from achenes of Mexican sunflower The exudate from the achenes of the Mexican sunflower was purified for the isolation and identification of sundiversifolide. Figure 4 shows the MS chromatogram and MS spectral fragmentation of the extract. The retention time and the ms fragmentation [m/z 247 (M + Na)+] coincided with the authentic sundiversifolide isolated from the sunflower (Fig. 4 (A) and (B)). It has already confirmed the existence of sundiversifolide Fig. 3 Scheme for purification of sundiversifolide in the exudates in the exudates from achenes of Russia form of sunflower (Kato from the sunflower, Mexican sunflower and L. paludosum achenes. et al., 2006). A stereoisomer of sundiversifolide, i.e.,

Eco-Engineering （35）79 Fig. 4 LC-ESI+/MS chromatogram (A) and mass spectrogram (B) of Fig. 5 Effect of water exudates (A), water-soluble fraction (B) and the purified extracts from the exudates of Mexican sunflower ethylacetate-soluble fraction (C) from Mexican sunflower achenes. Upper chromatogram and mass spectrogram show achenese on the growth of cat’s-eyes seedlings. Bar sundiversiflode. indicates average (n=8) ±SE. diversifolide, is expected to be detected in the other fractions according to the methods of extraction for diversifolide (Kuo et al., 1999). This is evidence of a second species that produces sundiversifolide. On the other hand, sundiversifolide has not been found in the exudates of the achenes of the L. paludosum using this method. This suggests that the amount of sundiversifolide in the L. paludosum was lower than the detection limit, or this species does not produce sundiversifolide at all. Phylogenically the Mexican sunflower and sunflower are close to each other, compared to L. paludosum. 3.2 Effect of exudates from achenes of the Mexican sun- flower on the cat’s-eyes seedling test. The effect of the exudates from the achenes of the Mexican Fig. 6 Effect of water exudates (A), water-soluble fraction (B) and ethylacetate-soluble fraction (C) from Mexican sunflower sunflower on the growth of cat’s-eyes seedlings was examined. achenes on the germination of Neurospora crassa. growth of The ethylacetate-solube fraction inhibited the growth of the cat’s-eyes seedlings. Bar indicates average results of three experiments ±SE. cat's-eyes seedlings even at the lowest concentrations tested. (Fig. 5 (C)). germination in Neurospora crassa (P < 0.01). This result agrees 3.3 Effect of exudates from achenes of Mexican sun- with the case of the sunflower. Chemicals in this fraction can be flower on the conidial germination of the fungus, used as a germicide in crop production. The water-soluble Neurospora crassa fraction has a slight promotive activity of the conidial The effects of sundiversifolide on the conidial germination germination at concentrations of 10 to 30 ppm. This promotion have already been reported for the sunflower (Kato et al., 2005). might be related to inorganic compounds. In the sunflower, In this study, the effects of the exudates from the achenes of the several inorganic compounds were identified in the water Mexican sunflower on the conidial germination in Neurospora exudates from its achenes (Ohno et al., 2006). In this fraction, crassa were studied. The germination rate was analyzed three the plant growth promoting allelopathic substances, such as times in the test solution. As shown in Fig. 6, 30 to 300 ppm of lepidimoide, might contribute to the promotive activity the ethylacetate-soluble fraction of the exudates from the (Yamada et al., 1997). These fractions will be able to be used achenes of the Mexican sunflower inhibited the conidial fertilizers in eco-systems.

80（36） Eco-Engineering Proceedings and selected papers of the fourth world congress on 4. Conclusion allelopathy 407-409. The identification of sundiversifolide in the exudates from the Kato, T., Tomita-Yokotani, K., Suzuki, T. and Hasegawa, K., 2006 : achenes of the Mexican sunflower and biological properties of New biological effects of sundiversifolide on microbial and plant the water exudates were discussed. Sundiversifolide was growth, and its relation to auxin. Weed Biol. Manage. (accepted) isolated from the exudates from the achenes of the Mexican Konarev., A. V., Anisimova, I. N., Gavrilova, V. A., Vachrusheva, sunflower plant as the second reported plant species. In this T.E., Konechnaya, G. Yu., Lewis, M. and Shewry, P. R., 2002 : study, we also determined that the exudate from the achenes of Serine proteinase inhibitors in the Compositae: distribution, the Mexican sunflower has allelopathic properties. polymorphism and proterities. Phytochemistry 59, 279-291. Allelochemicals, such as shown in this study, in the Mexican Khalid, S., T. Ahmad and R. A. Shad, 2002 : Use of allelopathy in sunflower and other near species of plants would be used for agriculture. Asian .l of P. Sci. 1 , 292-297 elemental recycling in a sustainable eco-system based on the Kuo, Y., and Lin, Y., 1999 : A new dinorxanthane and chromone from information obtained from this study. the root of Tithonia diversifolia. Chem. Pharm. Bull. 47, 428-429. Ohno, S., Tomita-Yokotani, K., Suzuki, T., Node, M., Kosemura, S., Acknowledgement Yamamura, S. and Hasegawa, K., 2001 : A new species-selective The authors are grateful to Dr. Suthep Tongma (Lampang allelopathic substance from germinating sunflower (Helianthus Agricultural Research and Training Center, Thailand) for annuus L.) seeds. Phytochemistry 56, 577-581. collection of the Mexican sunflower seeds. This study was Ohno, S., Tomita-Yokotani, K., Kato, T., Izawa, N. and Hasegawa, supported, in part, by “Ground-based Research Announcement K., 2006 : Elemental recycle in the sustainable eco-system using for Space Utilization” fund by the Japan Space Forum to allelochemicals. Eco-Engineering (In preparation) K.Tomita-Yokotani. Owoyele, V. B., Wuraola,C.O., Soladoye, A. O. and Olaleye, S. B., 2004 : Studies on the anti-inflammatory and analgesic properties of References Tithonia diversifolia leaf extract. J. Ethnopharmacol., 90, 317-321. Galagan, J. E., Calvo, S. E., Borkovich, K. A., Selker, E. U., Read, N. Node, M., Tomita-Yokotani, K., Suzuki, T., Kosemura, S., Hirata, H., D., Jaffe, D., FitzHugh, W., Ma, Li-Jun, Smirnov, S., Purcell, S., Hirata, K., Nawamaki, T., Yamamura, S. and Hasegawa, K., 2003 : Rehman, B., Elkins, T., Engels, R., Wang, S., Nielsen, C. B., Allelopathy of pinecone in Japanese red pine (Pirus densiflora Butler, J., Endrizzi, M., Qui, D., Ianakiev, P., Bell-Pedersen, D., Sieb. Et Zucc. Weed Biol. Manag. 3, 111-116. Nelson, M. A., Werner-Washburne, M., Selitrennikoff, C. P., Parvez, M. M., Tomita-Yokotani, K., Fujii, Y., Konishi, T. and Kinsey, J. A., Braun, E. L., Zelter, A., Schulte, U., Kothe, G. O., Iwashina, T. M., 2004 : Effects of quercetin and its derivatives on Jedd, G., Mewes, W., Staben, C., Marcotte, E., Greenberg, D., Roy, the growth of Arabidopsis thaliana and Neurospora crassa. A., Foley, K., Naylor, J., Stange-Thomann, N., Barrett, R., Gnerre, Biochem. Syst. Ecol. 32, 631-635. S., Kamal, M., Kamvysselis, M., Mauceli, E., Bielke, C., Rudd, S., Rice, E. L., 1984 : Allelopathy. Second edition. Academic Press, INC. Frishman, D., Krystofova, S., Rasmussen, C., Metzenberg, R. L., . Perkins, D. D., Kroken, S., Cogoni, C., Macino, G., Catcheside, D., Tomita, K., Tamura, Y., Takashiro, K., Nakamura, T. Z. and Li, W., Pratt, R. J., Osmani, S. A., DeSouza, C. P. C., Glass, L., Nakamura, T., 1996 : The effect of cytokinins on the conidial Orbach, M. J., Berglund, J. A., Voelker, R., Yarden, O., Plamann, germination and the hyphal elongation in Neurospora crassa. J. M., Seiler, S., Dunlap, J., Radford, A., Aramayo, R., Natvig, D. O., Jpn. Women’s Univ., Fac. Science 4, 49-55. Alex, L. A., Mannhaupt, G., Ebbole, D. J., Freitag, M., Paulsen, I., Tomita-Yokotani, K., Kato, T., Parvez, M. M., Mori, Y., Goto, N. and Sachs, M. S., Lander, E. S., Nusbaum, C. and Birren, B., 2003 : Hasegawa, K., 2003 : Approach of allelopathy study with The genome sequence of the filamentous fungus Neurospora Arabidopsis thaliana (L.) Heynh. and Neurospora crassa. Weed crassa. Nature 422, 859-868. Biol. Manag. 3, 93-97. Kato, T., Tomita-Yokotani, K., Kosemura, S. and Hasegawa, K., 2005 : Yamada, K., Kosemura, S, Yamamura, S. and Hasegawa, K., 1997 : Allelopathy of fruits in compositae – Sunflower (Helianthus annuus Exudation of an allelopathic substance lepidimoide from seeds L.) and Mexican sunflower (Tithonia diversifolia (Hemsl.) A. Gray). during germination. Plant Growth Regul. 22, 189-192.

Eco-Engineering （37）81