Floral scent emissions from Asarum yaeyamense and related species

Floral scent emissions from Asarum yaeyamense and related species

Biochemical Systematics and Ecology 38 (2010) 548–553 Contents lists available at ScienceDirect Biochemical Systematics and Ecology journal homepage...

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Biochemical Systematics and Ecology 38 (2010) 548–553

Contents lists available at ScienceDirect

Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco

Floral scent emissions from Asarum yaeyamense and related species Hiroshi Azuma a, *, Jun-ichi Nagasawa b, Hiroaki Setoguchi c a b c

Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan Division of Horticulture, Agriculture and Forestry Technology Department, Kyoto Prefecture Government, Kameoka 621-0806, Japan Department of Natural Environmental Sciences, Faculty of Integrated Human Studies, Kyoto University, Kyoto 606-8501, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 February 2010 Accepted 5 June 2010

The flowers of Asarum are usually regarded as scentless or sometimes to have a foul odor. Recently, we noticed that Asarum yaeyamense, endemic to Iriomote Island, Japan, has a floral fragrance with a distinct “fruity note.” To determine the chemical characteristics of this fragrance and whether “non-scented” Asarum species emit any volatiles, we collected floral scents of A. yaeyamense and related species (A. lutchuense, A. hypogynum, A. fudsinoi, A dissitum, A. tokarense, and A. senkakuinsulare) using headspace methods and analyzed these scents by gas chromatography–mass spectrometry (GC–MS). The results indicated that A. yaeyamense mainly emitted a-cedrene (tentatively identified), an unidentified sesquiterpene, methyl tiglate, and manoyl oxide (tentatively identified). Methyl tiglate may be a source of the “fruity note” in the A. yaeyamense fragrance. We also detected emissions of volatiles, mainly sesquiterpenes, from some “non-scented” Asarum species. This study constitutes a rare case of the detection of the emission of a diterpene (manoyl oxide) as a floral scent volatile. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Volatile Headspace Heterotropa Aristolochiaceae Methyl tiglate Manoyl oxide

1. Introduction The genus Asarum L. (Aristolochiaceae) comprises evergreen understory herbs found in East Asia, North America, and Europe, and approximately 70–80 species are known worldwide (Stevens, 2001 onwards; Huang, 2003; Sugawara, 2006; Mabberley, 2008). Plants bear one or a few greenish to dark-purple cup-shaped flowers that open on or near the ground under the plants’ own leaves, and the plants are often covered by fallen leaves. The peculiar flowering features of Asarum have attracted the interest of many naturalists and scientists (e.g., Peattie, 1940; Wildman, 1950; Lu, 1982; Tanaka and Yahara, 1987; Sugawara, 1988; Mesler and Lu, 1993). The center of Asarum diversity is located in East Asia (Huang, 2003; Sugawara, 2006). The genus is divided into several groups (sections), which have often been treated as separate genera (e.g., Asiasarum, Heterotropa, and Hexastylis). Fifty species are recognized in Japan based on floral morphology and most belong to section Heterotropa (46 species) and all are endemic (Sugawara, 2006). Little is known about the floral biology of the members of section Heterotropa except for Asarum tamaense, which is known to be visited by a fungus gnat and some other small animals for pollination (Sugawara, 1988). Asarum yaeyamense Hatus. of section Heterotropa is a perennial herb endemic to Iriomote Island, Ryukyu Islands, in southern Japan. We recently noticed that the flowers of A. yaeyamense grown in the laboratory emitted a pleasant odor. Flowers of Asarum species are generally considered to be scentless or to have a foul or musty odor (Peattie, 1940; Lu, 1982; Sugawara, 1988; Mesler and Lu, 1993). However, the floral scent we detected from A. yaeyamense had a distinctly sharp fruity note. To our knowledge, no studies have described obvious floral scent emission (good smells) in Asarum species. Indeed,

* Corresponding author. Tel./fax: þ81 (0) 75 753 4125. E-mail address: [email protected] (H. Azuma). 0305-1978/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2010.06.002

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some other Asarum species (section Heterotropa) grown in the same laboratory had no scent emissions detectable by the human nose. Chemical analyses of the floral scent and foul odors emitted by Asarum species have never been conducted. In this study we sampled the floral scent using the headspace method and used gas chromatography–mass spectrometry (GC–MS) analysis to reveal the chemical characteristics of the “fruity note” of A. yaeyamense. We also collected and analyzed the floral headspace of six additional “non-scented” Asarum species regionally related to A. yaeyamense to determine whether “non-scented” Asarum species emit any scent volatiles. 2. Materials and methods 2.1. Plant materials Floral scent volatiles emitted from the following seven Asarum species were collected using headspace methods: A. yaeyamense, A. lutchuense T. Ito ex Koidz., A. hypogynum Hayata, A. fudsinoi T. Ito, A. dissitum F. Maek. ex Hatus., A. tokarense Hatus., and A. senkakuinsulare Hatus. All species belong in section Heterotropa and are endemic to the southern part of Japan (Ryukyu Islands) except for A. hypogynum, which is found in Taiwan. Among these species, we only smelled a “fruity note” from the flower of A. yaeyamense. Plants were transplanted from their original habitats and grown in the laboratory. Two individuals of A. yaeyamense and one individual of each of the other six species were used for headspace sampling. 2.2. Scent collection A single newly opened intact flower was gently enclosed within aluminum foil (ca. 15 cm  7 cm), and a glass cartridge (7 mm  5 cm) containing 35 mg of adsorbent (Tenax GR 60/80 mesh; GL Sciences, Tokyo, Japan) was inserted into the aluminum foil. One side of the cartridge was placed in front of the flower and the other side was connected to a flow tube. The air containing volatiles emitted from the flower was suctioned with a mini-pump (MP-S30; Sibata Scientific Technology, Tokyo, Japan) at a flow rate of 100 ml/min for 6 (daytime) or 8 (night) h. Sampling took place in the laboratory from 10:00 to 16:00 for all species and from 22:00 to 06:00 for A. yaeyamense and A. lutchuense. At the same time, controls (empty aluminum foil placed in the same pot) were collected for all samplings. 2.3. GC–MS analysis The volatiles trapped on the adsorbent were eluted with 300 ml diethyl ether, and 2 ml of eluent was used for GC–MS analysis. The analysis was performed using a GC-17A gas chromatograph coupled with a GC–MS-QP5000 ver. 3 mass detector (Shimadzu, Kyoto, Japan). A DB-1 fused-silica capillary column (30 m; inner diameter, 0.25 mm; film thickness, 0.25 mm) was used. The injection temperature was maintained at 40  C for the first 5 min, programmed to increase by 5  C/min to 250  C, and held at 250  C for 5 min. Helium was used as the carrier gas. After each analysis a 1-ml aliquot of nonyl acetate solution (0.5 mg/ml dichloromethane) was added to the same sample as an internal standard to calculate the amounts of the volatiles and then immediately analyzed under the same conditions. The volatile compounds were identified by comparing their GC retention times and MS spectra with those of the authentic compounds, or tentatively identified by MS spectra in the NIST 02 mass spectral library and retention indexes reported elsewhere (Adams, 2001; NIST Chemistry WebBook, http://webbook. nist.gov/chemistry/). 3. Results The chemical profiles of the volatiles emitted by the seven Asarum species are presented in Table 1. The compounds listed in Table 1 are classified according to chemical classes proposed by Knudsen et al. (2006). In total, 49 compounds were detected in the headspaces of the seven Asarum species. The number of compounds detected in each species varied from 0 (A. tokarense) to 36 (A. yaeyamense). A. yaeyamense emitted the highest amount of volatiles, 533.4 ng/h during the day, among the examined species, while the headspace of A. senkakuinsulare contained trace amounts of a few compounds. A. lutchuense emitted a relatively high amount of volatiles, i.e., 202.5 ng/h during the day (13 compounds). Although only three compounds were detected in the headspace, A. hypogynum emitted a moderate amount of volatiles (69.6 ng/h during the day). A. fudsinoi and A. dissitum emitted 30.5 and 6.1 ng/h of volatiles during the day (12 and 3 compounds), respectively. The dominant compound class was sesquiterpenes (i.e., a-cedrene, caryophyllene, thujopsene, (E)-nerolidol, and many unidentified sesquiterpenes) including both hydrocarbon and oxygenated types, which constituted a large part of the floral headspaces of the Asarum species (Table 1). In A. yaeyamense, sesquiterpenes accounted for up to 69.7% of the total amount (flower-1, daytime) and 86% (31/36 compounds) of the total number of compounds. The contribution of sesquiterpenes reached almost 100% in A. lutchuense and A. hypogynum. Caryophyllene, a sesquiterpene, was the only compound found in more than three Asarum species (five species); all other compounds were found in fewer than three species. Therefore, the floral headspace of each species showed a characteristic composition of volatiles; i.e., the major compounds characterizing floral headspace differed for each Asarum species. The headspace volatiles of A. yaeyamense mainly comprised sesquiterpene-12, a-cedrene (tentatively identified), methyl tiglate, and manoyl oxide (tentatively identified), which together accounted for 75% of the total volatiles (flower-1, daytime; Table 1

Species

550

Table 1 Floral scent volatiles detected in the headspaces of Asarum species by GC–MS analyses. yaeyamense

lutchuense

flower-1

hypogynum

fudsinoi

dissitum

flower-2

senkakuinsulare

tokarense

Daytime (10:00–16:00) or Night (22:00–06:00)

D

N

D

N

D

N

D

D

D

D

D

Sampling hours

6

8

6

8

6

8

6

6

6

6

6

533.4

461.9

225.9

172.0

202.5

48.7

69.6

30.5

6.1

0.0

0.0

94.1

49.3

tr

25.1

–e













Total ng/h

IDa

C5-Branched Chain Compounds Methyl tiglate SD

RTb

RIc

6.80

852

d

SD MS RI

18.16 21.09 31.11

1166 1261 1643

– – –

– – tr

– – 4.5

2.2 – 2.1

– – –

– – –

– – –

– – –

– 2.8 –

tr tr –

– – –

Monoterpenes Linalool Bornyl acetate

SD SD

15.57 21.27

1086 1267

– –

– –

– tr

– 1.9

– –

– –

– –

– –

– –

– –

– –

RI SD RI

22.73 23.19 23.32 24.23 24.32 24.39 24.46 24.59 24.74 24.89 25.06 25.18 25.32 25.6 25.86 25.95 26.00 26.08 26.18 26.29 26.53 26.54 26.74 27.11 27.94

1317 1334 1339 1371 1374 1377 1379 1384 1389 1394 1400 1404 1410 1421 1431 1435 1437 1440 1444 1448 1458 1458 1465 1479 1511

– – – – – 10.2 – – tr tr 27.2 101.2 8.1 36.9 138.7 6.9 1.9 27.3 – 5.4 tr tr 2.7 tr –

– – – – – 9.7 – – 1.7 1.3 22.6 88.6 7.2 30.8 113.6 5.8 1.7 23.5 tr 4.7 tr 1.6 1.9 1.8 –

– – – – – 2.6 – – – – 6.4 28.5 9.3 6.6 32.4 2.2 tr 6.2 tr 2.4 – tr 2.1 tr –

– – – – – 2.3 – – tr – 6.8 25.0 10.1 9.9 33.4 2.2 tr 6.7 tr tr – tr tr tr –

23.3 3.5 3.0 9.9 64.2 – 7.2 4.9 – – – – 62.9 – – – – – 15.0 – 6.3 – – – –

7.1 tr tr 4.5 21.8 – 1.8 tr – – – – 8.0 – – – – – 1.9 – 1.7 – – – –

– – – – – – – – – – – – 13.2 – – – – – – – – – – – 5.9

– – – – – – – – – 2.3 5.0 2.5 1.9 – tr – tr tr – tr – – – – –

– – – – – – – – – – – – tr – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – –

28.82 29.80 30.43 30.48 30.74

1548 1587 1613 1616 1627

– – tr 1.9 tr

– – tr 3.2 1.5

– – – tr tr

– – tr – –

– – – – –

– – – – –

50.5 – – – –

– 4.5 – – –

– – – – –

– – – – –

– – – – –

Sesquiterpenes (ST) Hydrocarbon-type ST-1 ST-2 ST-3 ST-4 ST-5f ST-6 ST-7 ST-8 ST-9 ST-10 ST-11 a-Cedrene Caryophyllene Thujopsene ST-12g ST-13 ST-14 ST-15 a-Humulene ST-16 ST-17 ST-18 ST-19 ST-20 ST-21 Oxygenated-type (E)-Nerolidol ST-22 ST-23 ST-24 ST-25

SD

SD

H. Azuma et al. / Biochemical Systematics and Ecology 38 (2010) 548–553

Benzenoids Methyl salicylate Isosafrole Apiol

ST-26 ST-27 ST-28 ST-29 ST-30

31.10 31.32 31.52 31.61 31.86

1643 1652 1661 1665 1675

2.7 – tr 2.3 –

3.1 tr tr 2.6 –

tr 3.6 tr tr 3.2

– tr – – tr

– – – – –

– – – – –

– – – – –

– – – – –

– – – – –

– – – – –

– – – – –

RI

38.77

1997

64.0

78.5

113.3

44.3

2.4

1.9











Irregular Terpenes Geranylacetone

RI

25.78

1429

tr

tr

tr

tr

tr





tr

3.3





26.44 29.97 30.24 31.24 31.39 36.83

1454 1594 1605 1649 1655 1901

1.9 tr – tr – –

2.4 1.3 tr 3.6 – –

tr tr 2.8 tr tr –

tr – tr – tr –

– – – – tr –

– – – – – –

– – – – – –

tr – – – – 14.3

– – – – – –

– – – – – –

– – – – – –

Unidentified Compounds unidentified-1 unidentified-2 unidentified-3 unidentified-4 unidenfitied-5 unidentified-6h a b c d e f g h

Identified based on mass spectrum and GC-retention time of authentic compound (SD) or by comparison with mass spectra in the NIST library (MS) and retention indexes (RI). Retention time. Retention index relative to n-alkanes (DB-1 column). Trace amount. Not detected. m/z (relative intensity): 41(60), 55(27), 69(21), 79(24), 91(34), 105(62), 119(92), 133(36), 161(21), 175(100), 189(22), 204(30). m/z (relative intensity): 41(58), 55(39), 67(27), 69(27), 77(26), 79(49), 81(70), 91(37), 93(100), 94(59), 95(70), 96(90), 107(23), 108(83), 109(27), 111(32), 119(10), 133(7), 161(4), 175(5), 189(8), 204(3). m/z (relative intensity): 41(75), 55(48), 67(29), 79(40), 81(100), 91(48), 93(37), 95(33), 105(89), 107(45), 119(62), 121(38), 133(25), 161(80), 187(10), 202(10), 229(20), 272(21).

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Diterpene Manoyl oxide

551

552

H. Azuma et al. / Biochemical Systematics and Ecology 38 (2010) 548–553

Fig. 1. Chromatograms of the headspace volatiles of Asarum yaeyamense and the control, and mass spectra indicating the presence of methyl tiglate and manoyl oxide. Peak number 1: ST-6, 2: ST-11, 3: a-cedrene, 4: caryophyllene, 5: thujopsene, 6: ST-12, 7: ST-13, 8: ST-14, 9: ST-15, 10: ST-16, 11: unidentified-1 (see Table 1).

and Fig. 1), although a second sample contained emission of a trace amount of methyl tiglate (flower-2, daytime; Table 1). In A. lutchuense, three sesquiterpenes, sesquiterpene-5, caryophyllene, and sesquiterpene-1, were the main volatiles (74% in daytime, 76% at night; Table 1), and (E)-nerolidol was the main compound in A. hypogynum (73%). Identification of manoyl oxide was tentatively conducted using the mass spectrum and retention index. Manoyl oxide and 13-epi-manoyl oxide showed the same mass spectrum, but the published retention index of manoyl oxide (1998) (Adams, 2001; 2017 for 13-epi-manoyl oxide) more closely corresponded to the calculated value (1997) of the compound detected here. 4. Discussion Among the Asarum species studied, we only smelled a “floral scent” from A. yaeyamense. However, contrary to our expectations, GC–MS analyses of the headspace volatiles emitted by flowers of the seven study species revealed emissions of various combinations of floral volatiles from not only A. yaeyamense but other Asarum species as well, especially A. lutchuense and A. hypogynum (Table 1). A chemical class of sesquiterpenes was commonly found among the headspace volatiles of some Asarum species including A. yaeyamense, but the other species did not have scent profiles detectable by the human nose, suggesting that sesquiterpenes may not cause the specific “fruity note” characterizing the scent profile of A. yaeyamense. However, most of the sesquiterpenes found in A. yaeyamense were specific (unique); therefore, we cannot eliminate the possibility that these sesquiterpenes were the source of the scent profile. Aside from the sesquiterpenes, GC–MS analyses indicated that methyl tiglate caused the characteristic scent profile of A. yaeyamense because the compound was unique to this species and present at relatively high amounts in one sample (flower1; Table 1 and Fig. 1), although another sample emitted low level of this compound (flower-2; Table 1). To the human nose, methyl tiglate has a distinctly fruity fragrance and therefore seems to have contributed to the floral scent of A. yaeyamense. Methyl tiglate, a scent compound classified as a “C5-branched chain compound” (Knudsen et al., 2006), has been reported as a main or minor floral scent volatile in several well-known scented plants, e.g., Nymphaeaceae (Kite et al., 1991), Orchidaceae (Kaiser, 1993), Magnoliaceae (Azuma et al., 1997), and Rubiaceae (Kaiser, 2004), indicating that A. yaeyamense is a “scented” plant species. Notably, some “non-scented” Asarum species were found to emit distinct combinations of volatile compounds (Table 1). Because flowers of Asarum species (especially section Heterotropa) are inconspicuous (greenish or dark-purple in color and located at ground level) and often covered by fallen leaves, and the pollination biology of most species is still uncertain, emission of a floral scent having a specific blend of volatiles may play an important role in attracting pollinators and may have resulted in the regional diversification observed in Asarum section Heterotropa. However, we used potted plants in this study; therefore, we need to confirm the floral scent emissions from individuals in the wild and to study the floral biology involved with the floral scent of Asarum species. Another important finding of this study was that two species (A. yaeyamense and A. lutchuense) emitted a class of diterpenes, manoyl oxide (tentatively identified). Diterpenes are rarely detected in floral scents and manoyl oxide has never been

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reported as a floral scent volatile in angiosperms (Knudsen et al., 2006), although this compound has often been detected in leaf and wood essential oils (e.g., Adams, 1998; Rezzi et al., 2001; Demetzos et al., 2002). Because we did not detect it in any of the control samples (Fig. 1), we consider it to have been emitted from the flower as a scent volatile. Acknowledgments We thank Ms. Keiko Yasuda for providing samples of Asarum used in this study. References Adams, R.P., 1998. The leaf essential oils and chemotaxonomy of Juniperus sect. Juniperus. Biochem. Syst. Ecol. 26, 637–645. Adams, R.P., 2001. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry. Allured Publishing Corporation, Carol Stream, IL. Azuma, H., Toyota, M., Asakawa, Y., Yamaoka, R., García-Franco, J.G., Dieringer, G., Thien, L.B., Kawano, S., 1997. Chemical divergence in floral scents of Magnolia and allied genera (Magnoliaceae). Pl. Species Biol. 12, 69–83. Demetzos, C., Angelopoulou, D., Perdetzoglou, D., 2002. A comparative study of the essential oils of Cistus salviifolius in several populations of Crete (Greece). Biochem. Syst. Ecol. 30, 651–665. Huang, S., 2003. Aristolochiaceae. In: Wu, Z.Y., Raven, P.H., Hond, D.Y. (Eds.), Flora of China, vol. 5. Science Press, Beijing, and Missouri Botanical Garden Press, St. Louis, pp. 246–269. Kaiser, R., 1993. The Scents of Orchids: Olfactory and Chemical Investigations. Elsevier, Amsterdam. Kaiser, R., 2004. Vanishing flora - lost chemistry: the scents of endangered plants around the world. Chem. Biodivers 1, 13–27. Kite, G., Reynolds, T., Prance, G.T., 1991. Potential pollinator-attracting chemicals from Victoria (Nymphaeaceae). Biochem. Syst. Ecol. 19, 535–539. Knudsen, J.T., Eriksson, R., Gershenzon, J., Ståhl, B., 2006. Diversity and distribution of floral scent. Bot. Rev. 72, 1–120. Lu, K.L., 1982. Pollination biology of Asarum caudatum (Aristolochiaceae) in Northern California. Syst. Bot. 7, 150–157. Mabberley, D.J., 2008. The Plant-Book, third ed. Cambridge University Press, Cambridge. Mesler, M.R., Lu, K.L., 1993. Pollination biology of Asarum hartwegii (Aristolochiaceae): an evaluation of Vogel’s mushroom-fly hypothesis. Madroño 40, 117– 125. Peattie, D.C., 1940. How is Asarum pollinated? Castanea 5, 24–29. Rezzi, S., Bighelli, A., Mouillot, D., Casanova, J., 2001. Composition and chemical variability of the needle essential oil of Pinus nigra subsp. laricio from corsica. Flavour Fragr. J. 16, 379–383. Stevens, P.F., 2001. Angiosperm Phylogeny Website. Version 9. onwards. http://www.mobot.org/MOBOT/research/APweb/ June 2008. Sugawara, T., 1988. Floral biology of Heterotropa tamaensis (Aristolochiaceae) in Japan. Pl. Species Biol. 3, 7–12. Sugawara, T., 2006. Asarum. In: Iwatsuki, K., Boufford, D.E., Ohba, H. (Eds.), Flora of Japan IIa. Kodansha, Tokyo, pp. 368–386. Tanaka, H., Yahara, T., 1987. Self-pollination of Asarum caulescens Maxim. (Aristolochiaceae) in Japan. Pl. Species Biol. 2, 133–136. Wildman, H.E., 1950. Pollination of Asarum canadense L. Science 111, 551.