Identification of Odor-Active Compounds Released from a Damaged

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Identification of Odor-Active Compounds Released from a Damaged Plant of the Asian Skunk Cabbage Symplocarpus renifolius Kensuke Sakamaki,*,† Suguru Oguri,‡ Yuko Katsumi,† Yasutaka Ohkubo,† Yoshiko Kurobayashi,† and Kikue Kubota§ †

Technical Research Institute R&D Center, T. Hasegawa Co., Ltd., 29-7, Kariyado, Nakahara-ku, Kawasaki-shi, 211-0022, Japan Department of Northern Biosphere Agriculture, Faculty of Bioindustry, and §Department of Food and Cosmetic Science, Graduate School of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri, Hokkaido 099-2493, Japan

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ABSTRACT: Symplocarpus renifolius (Asian skunk cabbage) is a perennial herb of the Araceae family. As its common name implies, this plant produces a strong garliclike irritant odor with a rotten note when the plant parts are crushed. To elucidate the odor characteristics, the volatile compounds released from crushed plant parts (rhizome, petioles, and leaf blades) of S. renifolius were identified by a dynamic headspace method. Fifteen sulfur compounds were identified as odoractive compounds by gas chromatography−mass spectrometry−olfactometry (GC−MS−O). The sulfur compounds may be responsible for the strong odor emitted by crushed skunk cabbage. Many of the compounds lack a carbon−carbon bond, and all of the carbon atoms are connected to sulfur. This is regarded as the characteristic structure of the sulfur compounds released from the damaged plant parts of the skunk cabbage. Nine of the sulfur compounds were detected in all three of the plant parts analyzed in this study: hydrogen sulfide, methanethiol, 1-hexanethiol, methyl dithioformate, 2,4dithiapentane, dimethyl trisulfide, methional, 2,3,5-trithiahexane, and tris(methylthio)methane. Methyl dithioformate and methylthiomethyl dithioformate were identified for the first time as natural products.

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preparation methods, namely, hydro distillation and solventassisted flavor evaporation.4 Their aroma extract dilution analysis (AEDA) identified a total of 15 compounds as important constituents [dimethyl disulfide, hex-2-enal, methylpyrazine, ethyl isovalerate, 2-butoxyethanol, ethyl pentanoate, 2-methoxythiazole, 2-pentylfuran, phenyl alcohol, phenylacetaldehyde, nonanal, maltol, p-vinylguaiacol, 5-methylthiazole, and (Z)-ligustilide].4 There have been no earlier descriptions, however, of a mephitic odor perceivable as a mixture of skunk, putrid meat, and garlic when the plant parts of the genus Symplocarpus spp. are injured. Between the two types of odors from the different plant organs of S. renifolius, our group analyzed floral volatiles from the intact inflorescences over three flowering phases in previous research.5 The inflorescences in the female phase had an earthy−rotten−minty odor, whereas those in the male phase changed to a rotten−oily odor. Forty compounds, 28 of them odor-active, were identified by GC−MS−O. Four of the compounds, namely, 3-methylbutyl 3-methylbutanoate, 1,8cineole, dimethyl disulfide, and sabinene, were commonly found in all phases, collectively accounting for 52−54% of the total volatiles.5 An odor emitted from the damaged part of skunk cabbages, remains to be investigated. Our group

he genus Symplocarpus (skunk cabbage) is a perennial rhizomatous herb of the Araceae family patchily distributed in eastern North America and eastern Asia. The spadix of the genus Symplocarpus has the rare ability to generate heat, a phenomenon known as thermogenesis.1−3 Symplocarpus renifolius (Asian skunk cabbage) is a Symplocarpus species found in damp woodlands throughout Japan. The inflorescences of the plants appear in fields with remnant snow in early spring before the leaves expand. The flowers of S. renifolius are protogynous, and the flowering process is divided into female, bisexual, and male sex phases.3 Heat generation from the spadix is most active during the female phase.3 The leaves rapidly expand after flowering, transforming into ovateshaped leaf blades of about 30 cm by mid-June. Two odors emitted from different plant organs of the Symplocarpus spp. have been described: a floral odor emitted from the inflorescence during flowering and an odor emitted from a damaged part of the plant. Uemura et al. have reported that the female or bisexual spadices smell faintly like carrion.3 Knutson, meanwhile, described the odor produced by the damaged Eastern skunk cabbage (S. foetidus) as a mixture of odors of skunk, putrid meat, and garlic.2 Only a few reports on the volatile compounds produced by a damaged plant of Symplocarpus spp. have been published.4 Miyazawa et al. studied the chemical composition of the essential oil from the aerial parts (leaves and stems) of S. foetidus by two sample © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 11, 2018

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DOI: 10.1021/acs.jnatprod.8b00563 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

detected organoleptically from the petiole and rhizome after the same treatment. This garliclike odor was most intense immediately after these parts were cut and gradually weakened over time. To elucidate the odor characteristics, the volatile compounds released from the damaged plant parts (leaf blade, petiole, and rhizome) of S. renifolius were isolated by a dynamic headspace technique using Tenax TA as adsorbent and then analyzed by thermal-desorption gas chromatography−mass spectrometry−olfactometry (TD-GC−MS−O). In total, 33 odor-active areas were detected from the three plant parts examined (Table 1). A comparison of the retention indices (RIs), odor qualities, and mass spectra of the odorants with the respective data on the authentic standards helped us to identify 16 compounds 1−6, 8b, 9, 11, 13, 17, 20, 23, 24, 32, and 33. Another six compounds 7, 10, 14, 15, 19, and 31 were identified by matching the RIs and odor qualities with the authentic standards, though the mass spectrometric confirmations for these compounds failed. Compound 25 had the same mass spectrum (Figure 2) as that described in an aroma analysis of cooked petai beans.6 The authors tentatively identified the compound as 1,3,5-trithiahexane by the molecular formulas given for the molecular ion and the fragments obtained from accurate masses determined by the TOF instrument. On the basis of this, compound 25 was also tentatively identified as 1,3,5-trithiahexane in the present study. The nine compounds 12, 16, 18, 21, 22, 26, and 28−30 remained unidentified, because low peak intensity precluded obtaining unequivocal mass spectra. In the GC−MS−O analysis of the petiole and rhizome, odorants 8a and 8b were coeluted in the area where the sulfurous, garliclike odor was detected. While the sulfurous, garliclike odor was observed in the same area in the analysis of the leaf blade, 8a was detected but not 8b. This implicated 8a as a possible source of the sulfurous, garliclike odor. Compound 27 also possessed a sulfurous, garliclike odor. Identification of Odorant 8a. The mass spectrum of odorant 8a (Figure 3) suggested an Mr of 92 and an isotopologue molecular ion at m/z 94. The isotopologue ion m/z 94 had an intensity of 9.6% relative to m/z 92, which suggested the presence of two sulfur atoms in the molecule. The molecular formula, therefore, was estimated to be C2H4S2. The spectrum differed from the spectra of both ethanedithioic acid and 1,3-dithiethane. Fragments at m/z 77 and 45 corresponded to the loss of [CH3]• and [CH3S]•, respectively, thus suggesting that 8a is methyl dithioformate. In the analyses of floral scent volatiles from the intact inflorescences of S. renifolius, odorant 8a was detected as the odor-active component in GC−MS−O analyses and tentatively identified as methyl dithioformate.5 These findings in S. renifolius are the first to suggest the presence of methyl dithioformate as a natural product. The compound, however, was synthesized as an intermediate in organometallics.7−9 According to the literature, methyl dithioformate is too unstable to isolate. Attempts to synthesize simple alkyldithioformates resulted in the isolation of trimers with the trithiane structure.10,11 This outcome suggests that the major difficulty in isolating these dithioformates stems from their tendency to readily trimerize or polymerize because of their high reactivity. However, the difficulty is not unsurmountable as isolation has been achieved. Jagirdar et al. isolated free methyl dithioformate generated from a ruthenium complex by distillation and characterized the compound by 1H, 13C NMR, IR, and UV spectroscopy.8 However, the mass spectrum of methyl dithioformate was not

detected a strong garliclike odor with a rotten note in recent organoleptic observations of crushed S. renifolius. Compounds with garliclike odors have not been previously reported in Symplocarpus spp. The aim in the present study was to clarify the garliclike odor with the rotten smell released from crushed Asian skunk cabbage. The volatile compounds released from the damaged plant organs (rhizome, petiole, and leaf blade) of S. renifolius were analyzed by the headspace technique and then identified as odor-active compounds by GC−MS−O.

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RESULTS AND DISCUSSION Identification of Odor-Active Compounds. The inflorescence of S. renifolius (Asian skunk cabbage) appears from the bud in early spring before the leaves develop (Figure 1A).

Figure 1. A. Flowering plant of Symplocarpus renifolius: (a) inflorescence, (b) bud. B. Overview of the S. renifolius plant used for this study (leaves expanded, early May): (c) leaf blade, (d) petiole, (e) roots. C. Cross-section of rhizome: left, outside; right, inside. Scale bars represent 5 cm.

The leaves begin to grow from the end of the flowering season and expand to their full size in the summer. The S. renifolius plant is divided into four organs throughout this growth phase from early spring to summer: inflorescence, leaves, roots, and rhizome. (Figures 1B and 1C). The expanded leaf differentiates into a leaf blade and petiole (Figure 1B). In the organoleptic observations, it was found that each plant part of S. renifolius (bud, inflorescence, leaf blade, petiole, rhizome, and roots) emitted garliclike odors when crushed. The odors emitted from the crushed petiole and rhizome were the most pronounced. The leaf blade, petiole, and rhizome of S. renifolius were cut into small pieces to evaluate the qualities of the odors they released. A leafy green odor with a slightly garliclike note was detected organoleptically from the leaf blade of S. renifolius. A strong garliclike odor with a rotten note was immediately B



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Table 1. Odor-Active Compounds, Odor Qualities Detected by GC−MS−O, and Peak Areas (Given as Percentages of Total Peak Areas from the TIC on a TC-WAX Column) relative odor intensityd a

b

no.

odorant

odor quality

1 2 3 4 5 6 7 8a 8b 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

hydrogen sulfide methanethiol dimethyl sulfide 1-penten-3-one 1-hexanethiol (Z)-hex-3-enal hept-1-en-3-onef methyl dithioformatee methylthiomethanethiol 2,4-dithiapentane oct-1-en-3-onef dimethyl trisulfide unknown (Z)-hex-3-en-1-ol 2-isopropyl-3-methoxypyrazinef methionalf unknown (Z)-dec-4-enal unknown (2E,6Z)-nona-2,6-dienalf 2,3,5-trithiahexane unknown unknown tris(methylthio)methane 3-phenylpropanal 1,3,5-trithiahexaneg unknown methylthiomethyl dithioformatee unknown unknown unknown β-iononef 2,4,5,7-tetrathiaoctane 1,2,4,6-tetrathiepane

boiled-egg-like rotten rotten metallic sulfurous green metallic garlic, sulfurous garlic, sulfurous mushroomlike sulfurous catty green earthy, green potato-like haylike fatty, green citrus cucumber-like garlic, sulfurous galbanum honey garlic, sulfurous cinnamic garlic, sulfurous violet garlic, sulfurous harbal cinnamic galbanum violet garlic, rotten rotten vegetable

RI

c

peak area from TIC (%)

leaf blades

petiole

rhizome

leaf blades

petiole

rhizome

726 774 831 1047 1148 1160 1211 1273

1.0 2.5

1.5 3.0 0.5

1.5 3.0 1.0

0.7 18.18 0.09

0.72 17.40 0.02

2.0

0.5

trace 0.61 0.07 0.41 0.18 0.78

0.58

0.12

2.0 3.0

0.5 3.0

1.23h

17.08i

11.10i

1293 1311 1378 1380 1384 1427 1461 1517 1534 1545 1587 1654 1697 1755 1771 1784 1818 1840 1847 1866 1880 1885 1930 2278 2478

1.0 1.0 2.0 1.0 2.0 1.5 1.5 0.5 1.0

1.0

2.0 0.5 1.0

0.37

0.98

1.00

0.02

0.13

0.19

0.24

0.80

5.67

0.49

2.45

2.11

0.06 0.14

0.06 0.24 1.38

0.10 0.17 0.58

0.60

0.46

0.37 1.08 0.08

1.47 0.35

1.0 1.5 1.0 2.5

2.0 1.0 1.0 0.5 0.5

1.5 1.0 1.0

1.5 1.5

12.79 1.0 1.0 1.5 0.5 0.5 2.5 2.5 1.5 0.5 1.5 0.5 2.5 2.0 0.5 1.0 1.0 1.5 0.5

0.5 1.0 0.5 2.0 2.0 2.0 3.0 1.5 0.5 0.5 0.5 0.5 1.5 1.5 0.5 2.0 1.0 1.5

a

The odorant was identified by matching the mass spectrum, retention index, and odor quality with the authentic standards. bOdor quality perceived at the sniffing port. cRI = retention index on TC-WAX. dRelative odor intensity represents the average of the intensities (1: weak, 2: medium, 3: strong) perceived by two panelists. eThe compound was synthesized during this study. fNo equivocal mass spectrum could be obtained; identification was based on the resting criteria detailed in footnote a. gTentatively identified odorant. hPeak area of 8a from TIC (%). iPeak area of 8a+8b from TIC (%).

Figure 2. Mass spectrum (MS−EI) of odorant 25.

Figure 3. Mass spectrum (MS−EI) of odorant 8a.

included in their report. Therefore, to confirm the mass spectrum and RI of methyl dithioformate, it was analyzed by GC−MS following its synthesis as in Figure 4. First, the dithioformate anion was prepared via the reaction of carbon

disulfide with lithium triethylborohydride. Next, the dithioformate anion was reacted with methyl iodide to give methyl dithioformate. 1H NMR analysis of an aliquot confirmed the formation of methyl dithioformate. After the addition of nC



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A leafy green odor was detected organoleptically from the leaf blade but not from the rhizome or petiole in an evaluation of the odor qualities released from the three parts after wound treatment. The GC−MS−O analysis of the leaf blade detected a green odor at two areas and identified the odorants as (Z)hex-3-enal (6) and (Z)-hex-3-enol (13), respectively. Compounds 6 and 13 might account for the leafy green note in the odor emitted from the damaged leaf blade of S. renifolius, as both were not detected in the GC−MS−O analyses of the rhizome and petiole. Hatanaka proposed that these compounds were biosynthesized in green leaves through lipoxygenase-mediated lipid oxidation.13 A strong garliclike odor with a rotten note was detected organoleptically from the rhizome and the petiole, and a slightly garliclike odor was detected from the leaf blade. The GC−MS−O analyses detected 9, 15, and 14 sulfur compounds from the leaf blade, petiole, and rhizome, respectively. The 9 sulfur compounds detected from the crushed leaf blade were common components among the volatiles from the crushed petiole and rhizome. The GC−MS−O analysis of the leaf blade detected a garliclike and/or sulfurous odor at six areas and identified the odorants as 1-hexanethiol (5), methyl dithioformate (8a), 2,4dithiapentane (9), dimethyl trisulfide (11), 2,3,5-trithiahexane (20), and tris(methylthio)methane (23), respectively. The same compounds were also detected in the GC−MS−O analyses of the petiole and rhizome. In the area where the most intense sulfurous, garliclike odor was detected, only 8a was identified in the leaf blade, while both 8a and 8b were coeluted and identified in the petiole and rhizome. As mentioned above, 8a was identified for the first time as a natural product, and the GC−MS−O analysis indicated that 8a had a strong garliclike odor. Odorant 8b was identified as methylthiomethanethiol by matching the RI, odor qualities, and mass spectra with those of reference compounds. Werkhoff et al. described methylthiomethanethiol in a heated model system containing cysteine and glutamate.14 Frérot et al. identified the compound as a constituent of cooked petai beans.6 They confirmed the structure of methylthiomethanethiol by chemical synthesis and found the compound to be remarkably odoriferous, with an extremely strong alliaceous note. For these reasons, methylthiomethanethiol is regarded as another contributor to the sulfurous, garliclike odor in the area where the odor was detected most strongly. Compound 8b was previously identified in a bat-pollinated plant15 and Tulbaghia violacea Harv.16 The mass spectrum of 8b was described as an unknown component of Marasmius alliaceus.17 Compounds 9, 20, and 23 are known to possess garliclike odor in cheese.18,19 The same three compounds were also previously identified in white truffle (9,20,23),20 Tulbaghia violacea Harv. (9,20,23),16 cooked petai beans (9,20),6 Scorodocarpus borneensis Becc. (9,23),21 bat-pollinated plants (9,20),15 and Marasmius alliaceus (9,20,23).17 Compound 11 was also reported as an odorant in cheese possessing garliclike odor.18 Thus, compounds 9, 11, 20, and 23 might be responsible for the garliclike odor emitted from crushed S. renifolius. In the GC− MS−O of the petiole and rhizome, garliclike odor was detected at three more areas. Two of the odorants were definitively identified as methylthiomethyl dithioformate (27) and 2,4,5,7tetrathiaoctane (32), and the third was tentatively identified as 1,3,5-trithiahexane (25). All three odorants were assumed to additionally contribute to the garliclike odor emitted from the crushed petiole and rhizome.

Figure 4. Synthesis of methyl dithioformate (8a).

hexane and the removal of inorganic salts, the remaining solution was analyzed by GC−MS. The RI and mass spectrum of the synthesized compound matched those of 8a, thus odorant 8a was identified as methyl dithioformate. The GC− MS−O analysis of the compound revealed a strong garliclike odor. The compound was 50% decomposed after 3 days of storage at room temperature, even when stored in dilute nhexane solution. This rapid decomposition demonstrated the instability of 8a. Identification of Odorant 27. The mass spectrum of compound 27 (Figure 5) suggested an Mr of 138 and an

Figure 5. Mass spectrum (MS−EI) of odorant 27.

isotopologue molecular ion at m/z 140. The isotopologue ion m/z 140 had an intensity of 14.5% relative to m/z 138, which suggested the presence of three sulfur atoms in the molecule. Therefore, the molecular formula was estimated to be C3H6S3. A fragment at m/z 61 corresponded to [CH3SCH2]+, and fragments at m/z 77 and 45 corresponded to losses of [CH3SCH2]• and [CH3SCH2S]•, respectively. Hence, compound 27 was suggested to be methylthiomethyl dithioformate. Methylthiomethyl dithioformate has been identified as a primary photoproduct of 254 nm irradiation of an MeCN solution of 1,3,5-trithiane.12 However, the mass spectrum of methylthiomethyl dithioformate was not reported. Like methyl dithioformate, it was expected that methylthiomethyl dithioformate would also be unstable and difficult to isolate. Therefore, in the present study, after duplicating the literature procedure, the reaction mixture was analyzed by GC−MS−O to confirm the mass spectrum, RI, and odor quality of methylthiomethyl dithioformate. The mass spectrum, RI, and odor quality of the compound detected in the reaction mixture matched those of 27. From these results, odorant 27 was identified as methylthiomethyl dithioformate. It is a new compound as a natural product, and this is the first report of its odor. Sensory Characteristics of the Odor-Active Compounds. The GC−MS−O analyses revealed the presence of 33 odor-active areas from three parts of S. renifolius of which 24 could be identified definitively and one could be identified tentatively. Compounds 2, 3, 9, 11, 14, 15, and 20 shared common components with the volatiles from the intact inflorescences of S. renifolius.5 The remaining 17 compounds have not been previously identified in S. renifolius. D



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chemical shifts with TMS as an internal standard. The chemical shifts (δ) are expressed in parts per million (ppm). Plant Material. To avoid damage to the leaves during sampling, a complete winter bud of Asian skunk cabbage (Symplocarpus renifolius) was carefully collected from a community forest park in Abashiri city, Hokkaido, Japan in mid-April 2016 with the permission of the Abashiri City Council. The collected bud was transplanted to a pot and grown in a greenhouse equipped with inner shade curtains for 3 weeks until the leaf blades fully expanded. Synthesis of Methyl Dithioformate 8a. Methyl dithioformate (8a) was synthesized according to the procedure described by Sanchez et al.7 with slight modifications. A solution of CS2 (1.81 mL, 30.0 mmol) in 10 mL of anhydrous THF was added dropwise to a 1 M THF solution of LiEt3BH (30.0 mL, 30.0 mmol) under a nitrogen atmosphere at −78 °C. After being stirred at the same temperature for 0.5 h, a solution of MeI (1.87 mL, 30.0 mmol) in 10 mL of anhydrous THF was added dropwise. The mixture was stirred for a further 0.5 h, warmed to 0 °C, and stirred again for 0.5 h. 1H NMR analysis of an aliquot confirmed the formation of methyl dithioformate 8a. Jagirdar et al. reported two singlets at δ 2.68 (MeS) and 11.33 [HC(S)],8 and the current data showed two singlets at δ 2.47 (MeS) and 11.29 [HC(S)]. The resulting solution was analyzed by GC−MS after adding n-hexane (30 mL) and removing inorganic salt. Methyl dithioformate (8a): 1H NMR (400 MHz, CDCl3) δ 2.47 (s, 3H); 11.29 (s, 1H); MS−EI (m/z: intensity in %) 94 (9), 92 (98), 77 (20), 64 (9), 47 (20), 45 (100). Dynamic Headspace Sampling. Dynamic headspace samplings were performed for three parts of S. renifolius, namely, the rhizome, petiole, and leaf blade. Each part (6.3 g) was cut into small pieces and promptly placed into a two-necked pear-shaped flask (100 mL). Gerstel TDS tubes packed with Tenax TA were used as the adsorbent. The flask was purged with nitrogen (200 mL/min) for 10 min at room temperature. The adsorbed volatiles were thermally desorbed and analyzed by GC−MS−O. Thermal-Desorption Gas Chromatography−Mass Spectrometry−Olfactometry (TD-GC−MS−O). The TD-GC−MS−O analyses were performed with a Gerstel TDS3 thermal-desorption system equipped with a Gerstel CIS4 programmed temperature vaporization (PTV) inlet and a Gerstel CTS2 cryo-trap system (Gerstel, Mulheim an der Ruhr, Germany) installed on an Agilent 7890A gas chromatograph with a 5975C MSD mass selective detector (Agilent Technologies, Palo Alto, CA, U.S.) and Gerstel ODP 3 olfactory detection port (Gerstel). Each sample was thermally desorbed through a TDS3 ramped up from 20 (held for 0.5 min) to 260 °C (held for 2 min) at a rate of 60 °C/min. The desorbed compounds were cryo-focused at −50 °C on a Tenax TA packed liner in the PTV. After desorption, the temperature at the PTV inlet was ramped up from −50 to 250 °C (held for 5 min) at a rate of 12 °C/sec. The injector was operated in the splitless mode. The CTS 2 was also cooled to −150 °C during the thermal desorption and injection steps. After injection for 1 min, the CTS 2 was ramped to 250 °C at a rate of 20 °C/sec and the analytes were introduced into the capillary column. Capillary GC analyses were performed in a TC-WAX capillary column (0.25 mm i.d. × 30 m, 0.5 μm film thickness; GL Sciences Co.) with a constant carrier helium gas flow of 1.8 mL/min. The oven was ramped up from 40 (5 min) to 230 °C at a rate of 4 °C/min. The effluent of the column at the end of the capillary was branched into two and routed by deactivated fused silica capillaries to the sniffing port and MSD, respectively. Mass spectra in the electron impact (EI) mode were recorded at 70 eV ionization energy. The linear retention indices (RIs) of the compounds were calculated from the retention times of n-alkane standards. The GC−O analyses were performed by two panelists, both of whom were extensively experienced in olfactometry and unimpaired by anosmias. The panelists assessed the odor intensities perceived at the sniffing port according to a scale: 1 for weak, 2 for medium, and 3 for strong. Table 1 presents the average intensities obtained from the assessments by the two panelists.

Hydrogen sulfide (1) was identified in an area with a detectable boiled-egg-like odor, and methanethiol (2) and dimethyl sulfide (3) were identified in areas with a detectable rotten odor. All three odorants were thought to contribute to the rotten odor. Figure 6 shows the structures of the 15 sulfur compounds identified either definitively or tentatively as odor-active

Figure 6. Sulfur compounds identified in skunk cabbage for the first time as odorants responsible for the garliclike, rotten odor. *Tentatively identified compounds.

compounds in this study. All of the sulfur compounds but 1hexanethiol (5) and methional (15) lacked a carbon−carbon bond, and all of the carbon atoms were bonded to sulfur atoms. This structure is thought to be characteristic of the sulfur compounds released from the damaged plant parts of Asian skunk cabbage S. renifolius. In conclusion, a headspace analysis of the volatile compounds released from the damaged plant parts of S. renifolius was used to elucidate the odor quality of the strong garliclike odor with the rotten note that characterizes the plant. The GC−O analyses detected 15 odor-active sulfur compounds that had not been identified in S. renifolius. Two of the compounds, methyl dithioformate (8a) and methylthiomethyl dithioformate (27), have been identified for the first time as natural products. The 15 compounds possessed sulfurous, garliclike, or rotten odor and thus were determined to be responsible for the odor released from the damaged plant parts of S. renifolius.

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EXPERIMENTAL SECTION

General Experimental Procedures. Chemicals and solvents used in the experiments were dimethyl sulfide, pent-1-en-3-one, 1hexanethiol, (Z)-hex-3-enal, 2,4-dithiapentane, oct-1-en-3-one, dimethyl trisulfide, (Z)-hex-3-enol, 2-isopropyl-3-methoxypyrazine, methional, (Z)-dec-4-enal, (2E,6Z)-nona-2,6-dienal, tris(methylthio)methane, 3-phenylpropionaldehyde, β-ionone, 1 M lithium triethylborohydride in THF (Sigma-Aldrich Japan, Tokyo, Japan), carbon disulfide, THF (Junsei Chemical Co., Ltd., Tokyo, Japan), and methyl iodide (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). The following compounds were synthesized by using reported methods: hydrogen sulfide and methanethiol,22 hept-1-en-3-one,23 methylthiomethanethiol,5 2,3,5-trithiahexane,21 methylthiomethyl dithioformate,12 2,4,5,7-tetrathiaoctane,21 and 1,2,4,6-tetrathiepane.24 Methyl dithioformate was synthesized as described below. 1H NMR experiments were performed on a JNM-ECX400 spectrometer (JEOL Ltd., Tokyo, Japan). Using CDCl3 as solvent, we measured E



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Gas Chromatography−Mass Spectrometry (GC−MS). The GC−MS analyses were performed with an Agilent 7890 gas chromatograph (GC) combined with an Agilent MSD5975 quadrupole mass spectrometer equipped with a TC-WAX capillary column (0.25 mm i.d. × 60 m, 0.25 μm film thickness; GL Sciences Co.). A 0.2 μL volume of each sample was injected in a split mode (30:1) at a constant temperature of 250 °C. The oven temperature was held at 40 °C for the initial 3 min and then ramped up to 250 °C at a rate of 3 °C/min, with a constant carrier helium gas flow of 1.8 mL/min. Mass spectra in the EI mode were recorded at 70 eV ionization energy. The linear RIs of the compounds were calculated from the retention times of the n-alkanes. Gas Chromatography−Mass Spectrometry−Olfactometry (GC−MS−O). The GC−MS−O analyses were performed with an Agilent 7890 GC combined with a 5975 mass selective detector and sniffing port. The effluent of the column at the end of the capillary was branched into two and routed by deactivated fused silica capillaries to the mass detector and sniffing port, respectively. The column type, sample volume, split ratio, injection temperature, oven temperature program, carrier gas, flow rate, and ionization mode all matched those set for the GC−MS analyses described above.

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(21) Kubota, K.; Ohhira, S.; Kobayashi, A. Biosci., Biotechnol., Biochem. 1994, 58, 644−646. (22) Li, J. X.; Schieberle, P.; Steinhaus, M. J. Agric. Food Chem. 2012, 60, 11253−11262. (23) Buettner, A.; Schieberle, P. J. Agric. Food Chem. 1999, 47, 5189−5193. (24) Morita, K.; Kobayashi, S. Chem. Pharm. Bull. 1967, 15, 988− 993.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +81-44-411̅ 0298. Fax: +81-44-434-5257. ORCID

Kensuke Sakamaki: 0000-0002-1629-9014 Notes

The authors declare no competing financial interest.

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