Journal of Oleo Science Copyright ©2015 by Japan Oil Chemists’ Society doi : 10.5650/jos.ess14225 J. Oleo Sci. 64, (5) 577-584 (2015)
Characteristic Odor Components of Essential Oils from Eurya japonica Ryota Motooka1, Atsushi Usami1, Hiroshi Nakahashi1, Satoshi Koutari1, Satoshi Nakaya1, Ryoyu Shimizu1, Kaoru Tsuji2, Shinsuke Marumoto3 and Mitsuo Miyazawa1＊ 1
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University (Kindai University) (3-4-1, Kowakae, Higashiosaka-shi, Osaka 577-8502, JAPAN) 2 Center for Ecological Research, Kyoto University (Hirano 2, Otsu-shi, 520-2113, JAPAN) 3 Joint Research Center, Kinki University (Kindai University) (3-4-1, Kowakae, Higashiosaka-shi, Osaka 577-8502, JAPAN)
Abstract: The chemical compositions of essential oils from the flower and aerial parts (i.e., leaf and branch) of Eurya japonica were determined and quantified using gas chromatography-mass spectrometry (GC-MS). A total of 87 and 50 compounds were detected in the oils from the flower and aerial parts, respectively. The main compounds of the flower oil were linalool (14.0%), (9Z)-tricosene (12.0%), and nonanal (7.4%). In the oil from the aerial parts, linalool (37.7%), a-terpineol (13.5%), and geraniol (9.6%) were detected. In the oils from the flower and aerial parts, 13 and 8 aroma-active compounds were identified by GC-olfactometry (GC-O) analysis, respectively. The key aroma-active compounds of the flower oil were heptanal [fatty, green, flavor dilution (FD) = 128, odor activity value (OAV) = 346], nonanal (sweet, citrus, FD = 128, OAV = 491), and eugenol (sweet, spicy, FD = 64, OAV = 62): in the oil from the aerial parts, the key aroma-active compounds were linalool (sweet, citrus, FD = 64, OAV = 95), (E)-b-damascenone (sweet, FD = 256, OAV = 4000), and (E)-b-ionone (floral, violet, FD = 128, OAV = 120). This study revealed that nonanal and eugenol impart the sweet, citrus, and spicy odor of the flower oil, while (E)-b-damascenone and (E)-b-ionone contribute the floral and sweet odor of the oil from the aerial parts. Key words: Eurya japonica, essential oil, GC-MS, GC-O, AEDA, OAV 1 INTRODUCTION Eurya japonica（Hisakaki in Japanese）is an evergreen broad-leaf understory tree from the Pentaphylaceae family; it is commonly known as Uyanggal laba in the Manipuri language. This plant generally grows in India, Southeast Asia, China, Korea, and Japan. This is an economically important species in Korea because the branches are widely used in floral tributes and wreaths1）. In China, the fruits and leaves of this plant are used as a traditional medicine called Lingmu, which is used for the treatment of rheumatoid arthritis, tympanites, and hemostasis of injuries2）. In eastern Japan, E. japonica has been used as a substitute for Cleyera japonica （Sakaki in Japanese）as an offering of the altar. In previous reports, lignans such as ovafolinin B-9’ -Oβ-D-glucopyranoside,（−）-2a-O-（β-D-glucopyranosyl）lyoniresinol,（＋）-3a-O-（β-D-glucopyranosyl）lyoniresinol, and aviculin, flavonoids such as epitaxifolin 3-O-β- D -
xylopyranoside, taxifolin 3-O-β-D-xylopyranoside,（2R,3R）（＋）-glucodistylin, and（ 2S,3S） （−） -glucodistylin, and phenyl glycoside 6’ -O-coumaroyl-1’ -O-［2-（3,4-dihydroxyphenyl）-ethyl］-β-D-glucopyranoside have been isolated from the stems and leaves of E. japonica. These compounds from extracts of this plant have been found to possess antioxidants1, 2） and antibacterial activities3）. In a previous study, the volatile fraction indicated that the essential oil from E. japonica obtained by steam distillation contained 3-hexenol and 2-hexenal4）. However, there have been very few detailed investigations of E. japonica. Thus, we collected the essential oils from the flower and aerial parts（i.e., leaf and branch）of E. japonica using hydrodistillation and investigated the volatile compounds that have aroma properties using gas chromatography mass spectrometry（ GC-MS）and GC-olfactometry（ GC-O）to gain further knowledge of the plant. For flavor analysis, GC-O is the most valuable technique
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Correspondence to: Mitsuo Miyazawa, Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University (Kindai University), 3-4-1, Kowakae, Higashiosaka-shi, Osaka 577-8502, JAPAN E-mail: [email protected] Accepted December 24, 2014 (received for review October 8, 2014)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
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R. Motooka, A. Usami and H. Nakahashi et al.
for identifying the key aroma-active compounds in plants. There are various methods for screening aroma-active compounds. One of the frequently used methods is aroma extract dilution analysis（AEDA）5）. The odor potency was estimated using the flavor dilution （FD） -factor6）. Moreover, the significant contribution of each odorant to the characteristic flavor is determined by the odor activity value （OAV）. The OAV is the ratio of the concentration to the odor threshold of the compound, and it is well accepted that compounds with high OAV values contribute more to （i） investithe aroma of food7）. The aim of this study was to gate the characteristic odor compounds of the essential oils obtained from E. japonica by a sensory evaluation and analysis of OAV and（ii） establish that the essential oils of E. japonica deserve further investigation in the food industry.
2 EXPERIMENTAL PROCEDURES 2.1 Plant material Fresh flower and aerial parts（i.e., leaf and branch）of E. japonica were harvested in Kyoto prefecture in April 2011. Identification of the plant was performed at a biotechnology laboratory at Kinki University （Kindai University） . A voucher specimen was deposited at the biotechnology laboratory of Kinki University（ Kindai University）in Osaka, Japan. 2.2 Extraction of essential oils Samples of fresh flower and aerial parts（i.e., leaf and branch） （100 g each） of E. japonica were subjected to hydrodistillation for 3 h using a Likens-Nickerson-type apparatus with diethyl ether. The obtained oils were dried over anhydrous sodium sulfate and stored at 4℃ in a refrigerator prior to analysis. （GC-MS） 2.3 Gas chromatography mass spectrometry GC-MS was performed using an Agilent 6890N gas chromatography-5973 MSD mass spectrometer. The samples were analyzed using a fused-silica capillary column, i.e., HP-5MS（5％ phenyl 95％ polydimethylsiloxane, 30 m× 0.25 mm i.d., film thickness＝0.25 μm） and DB-WAX （polyethylene glycol, 15 m×0.25 mm i.d., film thickness＝0.25 μm）. The oven temperature was programmed to increase from 40 to 260℃ at a rate of 4℃/min and then held at 260℃ for 5 min. The flow rate of the carrier gas（He）was 1.5 mL/min. On the DB-WAX column, the oven temperature was programmed to increase from 40 to 240℃ at a rate of 4℃/min and then held at 240℃ for 5 min. The injector and transfer line were held at 230℃, and the ionization energy was 70 eV. The mass range was 39-450 amu. Each sample （1 μL） was injected at a split ratio of 1:40.
2.4 Sniffing test using GC-olfactometry （GC-O） A trained panel of sensory evaluation specialists measured the odor intensities of the main aromatic constituents of E. japonica. Fourteen panelists, aged 21 to 26 years ［11 males and 3 females, members of Kinki University （Kindai University）, Japan］, participated in this study. Sensory-analysis sessions were performed only after suitable training（ ＞30 h）. The sniffing test by GC-O was carried out using an Agilent Technologies-6890N gas chromatograph equipped with an Agilent 5973 MSD mass spectrometer and sniffing port ODP 2（Olfactory Detector Port 2, Gerstel）. The GC instrument was equipped with an HP-5MS column（30 m×0.25 mm i.d., film thickness＝0.25 μm）. The sample was injected into the GC in splitless mode. The GC effluent from the capillary column was split in at a 1:1 （v/v）ratio between the MS and sniffing port. The oven conditions, injector and transfer line temperatures, carrier gas, flow rate, and ionization mode were the same as those described above for GC-MS. 2.5 Aroma extract dilution analysis （AEDA） The results of AEDA were expressed as FD factors, which are the ratios of the concentration of the odorant in the initial essential oil to its concentration in the most diluted volatile oil in which the odor can be still detected by GC-O. The highest sample concentration（10 mg/mL） was assigned an FD factor of 1. The essential oil was stepwise diluted with diethyl ether（1:1, v/v）, and aliquots of the dilutions（ 1 mL）were evaluated. The process was ceased when no aromas were detected by the evaluators. 2.6 Determination of odor activity value （OAV） OAVs were determined by dividing the concentration of a component with its odor threshold. The odor threshold data was obtained from reported literature data. 2.7 Identification and quantification of compounds The compounds were identified by comparing their retention indices（RIs）and mass spectra with those from previous studies 8−10） digital libraries（ Mass Finder4 and NIST02）, and Aroma Office version 3.0（Nishikawa-Keisoku Co. Ltd.） , which includes 72,120 RI entries for aroma compounds and literature sources. The RIs were calculated using a homologous series of n-alkanes C 5-C 29 on two columns with different polarities. The relative amounts of the individual compounds were calculated based on the GC peak areas（ FID response）, without using correction factors. Quantitative analyses of the aroma-active compounds （FD≥1）of the oils were performed using calibration curves for hexanol（3） , heptanal（5） , α-pinene（6） , myrcene （23）,（E） （11）, octanal（14）,（Z）-linalool oxide（furanoid） -linalool oxide （furanoid） （25） , nonanal（29） , lilac aldehyde （35） , α-terpineol（45）, geraniol（59） , vitispirane（63）, p-vinylguaiacol（68）, eugenol（70）,（E）-β-damascenone（73）,
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and（E）-β-ionone（86）within the concentration range of 0.5-1000 μg/mL. Because of the lack of a proper standard, vitispirane（63）was quantified using the calibration curve for（E）-β-ionone（86）. The weight percent of each compound was calculated using the FID response factor.
3 RESULTS AND DISCUSSION 3.1 Constituents of the essential oils from E. japonica The essential oil from fresh flowers of E. japonica was colorless with a sweet, citrus, and spicy odor, while that from the aerial parts（ i.e., leaf and branch）was slightly yellow, with a floral and sweet odor; the yields of oils were 0.066 and 0.012 ％（w/w）, respectively. The TICs of the essential oils from the flower and aerial parts of E. japonica are shown in Fig. 1. The chemical compositions of the essential oils from the flower and aerial parts of E. japonica are summarized in Table 1. The results revealed that 87 and 50 compounds were obtained from the oils derived from the flower（93.6％）and aerial parts（95.4％）of E. japonica, respectively. The essential oil from the flower of E. japonica mainly comprised linalool（28: 14.0％）,（9Z）-tricosene（ 118: 12.0％）, nonanal（29: 7.4％）, hexadecanal（100: 5.8％）, and α-pinene（6: 4.5％）. Furanoid monoterpenes derived from linalool, such as lime oxide T （15） ,（Z） -linalool oxide
（furanoid） （ 23）,（E）-linalool oxide（furanoid） （ 25）, and lilac aldehyde（35）, were also found in this oil; these compounds appeared to contribute to the characteristic odor of the essential oil of the flower. The main compounds of the oil from the aerial parts of E. japonica were linalool （28; 37.7％）, α-terpineol （45; 13.5％）, geraniol（59; 9.6％）, vitispirane（ 63; 8.5％）, and phytol（110; 3.2％）.（3E） -2-Methyl-3-octen-5-yne（8）was the characteristic compound of the oil from the aerial parts of E. japonica; this compound was isolated from Lomatium dissectum in the Apiaceae family11）. Also, this oil contains four norisopren, vitispirane （63） ,（E） -β-damascenone oids （C13 compounds） （73）, megastigmatrienone（ 83）, and（ E）-β-ionone（ 86）. These compounds contribute to the characteristic odor of the oil. The abundance of monoterpenoids in the flower oil was about half that in the oil from the aerial parts. In contrast, the flower oil contained approximately seven times as much sesquiterpenids than the oil from the aerial parts. Linalool（28）, which is the main compound, was detected in the oil from the flower and aerial parts; the amount of linalool （28）in the flower oil was about 2.5 times that in the oil from the aerial parts. Although（9Z）-tricosene（118）, nonanal（29）, and hexadecanal（100）were present in the flower oil, they were not detected in the oil from the aerial parts. Conversely, vitispirane（63）was only detected in the oil from the aerial parts.
Fig. 1 TICs of essential oils from flower (A) and aerial parts (i.e., leaf and branch) (B) of E. japonica. 579
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Table 1 Chemical compositions of the essential oils from frower and aerial parts (i.e., leaf and branch) of E. japonica.
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Table 2 Aroma-active compounds in the essential oils from E. japonica.
3.2 GC-O, AEDA, and OAV The characteristic aroma-active compounds of the essential oils from E. japonica were identified by GC-O combined with AEDA. Thirteen and eight compounds were identified in the oils from the flower and aerial parts, re. The FD-factors were plotted by their spectively（Table 2） respective peaks on the gas chromatogram shown in Figs. 2 and 3. On the basis of the FD-factor, heptanal（5: FD＝ 128, fatty, green） and nonanal （29: FD＝128, sweet, citrus） were the most intense key aroma-active compounds and octanal（14: FD＝64, citrus）, eugenol（70, FD＝64, sweet, spicy） , and linalool（28: FD＝64, sweet, citrus）contributed to the flower odor. It was determined that heptanal imparts the fatty and green odor, while nonanal and octanal give the sweet and citrus odor. These compounds are found in many flavors including fruit and cooked food12, 13）. The most characteristic aroma-active compounds of the oil from the
aerial parts were（E） -β-damascenone（73: FD＝256, sweet） followed by（E）-β-ionone（86: FD＝128, floral, violet）and linalool（28: FD＝32, sweet, citrus）.（E）-β-Damascenone and（E）-β-ionone are degradation products of β-carotene and contribute significantly to the key aroma of the aerial parts. These sweet aroma compounds have been identified in a wide range of processed food, fruits and vegetables14）. （E）-β-Damascenone and linalool produce the sweet odor, whereas（E）-β-ionone produces the floral odor. These compounds were determined to be the characteristic aromaactive compounds of the aerial parts of E. japonica. The OAV method was used to determine the relative contribution of each of the compounds to the aroma of E. japonica. The OAVs were obtained by taking into account the concentration and odor threshold of each compound. In the flower odor extract, nonanal（29）has the highest OAV（491）, followed by heptanal（5: 346）, linalool（28, 581
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Fig. 2 Gas chromatogram and FD-factor (aromagram) of the essential oil from flower of E. japonica: 1, octane; 5, heptanal; 14, octanal; 28, linalool; 29, nonanal; 118, (9Z)-tricosene; 128, nonacosane.
Fig. 3 Gas chromatogram and FD-factor (aromagram) of the essential oil from aerial parts of E. japonica: 2, (3Z)hexenol; 28, linalool; 45, a-terpineol; 59, geraniol; 73, (E)-b-damascenone; 86, (E)-b-ionone; 116, unknown (M+ = 278). 582
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Fig. 4 Structures of main aroma-active compounds in flowers of E. japonica.
Fig. 5 Structures of main aroma-active compounds in aerial parts of E. japonica. 165）,（E）-linalool oxide（furanoid） （25: 64）, and eugenol （70: 62）. In particular, linalool and eugenol had high FDfactors; thus, these compounds were considered to be the main aroma-active compounds of the flower odor. In the odor extract of the aerial parts,（E）-β-damascenone（73） has high OAV（4000）, followed by（E）-β-ionone（86: 120）. （E） -β-Damascenone and （E） -β-ionone had high FD-factors; thus, these compounds were considered to be the main aroma-active compounds of the aerial parts odor. The odor 15） is high threshold of（E）-β-damascenone odor （0.004 ppb） in all wines; therefore, this compound is normally considered to be a positive contributor to wine aroma16）. This norisoprenoid is also related to tobacco notes17） and is the principal carotenoid-derived compound in French oak18）. The flower oil contained more monoterpenoids than the oil derived from the compared with aerial parts. Therefore, the flowers oil odor was mainly determined by monoterpenoids. On the other hand, the oil derived from the aerial than parts contained more norisoprenoids （C13 compounds） the flower oil. Therefore, the odor of the oil from the aerial parts was mainly determined by norisoprenoids（C13 compounds） . In conclusion, we investigated the characteristic odor compounds of the flower and aerial parts of E. japonica by a sensory evaluation and analysis of OAVs. On the basis of
AEDA, OAVs, and sensory evaluations, linalool （28）was determined to be the main aroma-active compound of the oils. In the flower oil, nonanal（29）and octanal（14）produced the fatty and green odor, heptanal（5）imparted the sweet and citrus odor, and eugenol （70） contributed to the sweet and spicy odor.（E） -β-Damascenone（73）and linalool （28）produced the sweet odor and（E）-β-ionone（86）contributed to the floral odor in the oil derived from the aerial parts. To the best our knowledge, this is the first study that elucidates the characteristic odor compounds that are responsible for the odor of E. japonica. We expect that these results will be used in future investigations of food and medicinal plants.
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Eurya japonica. J. Nat. Prod. 76, 580-587 （2013）. 3）Rosalind, T.; Dutta, B. K.; Paul, S. B. Phytochemical screening and antibacterial studies of the leaf extract of Eurya japonica Thunb. and Ficus auriculata Lour. J. Pure Appl. Microbio. 5, 909-916 （2011） . 4）Takei, S.; Sakato, Y.; Ono, M.; Kuroiwa, Y., Leaf alcohol. I. Distribution of leaf alcohol in various plants. Nippon Nogei Kagaku Kaishi 14, 709-716 （1934） . 5）Grosch, W. Evaluation of the key odorant of foods by dilution experiments, aroma models and omission. Chem. Senses. 26, 533-545 （2001） . 6）Buettner, A.; Schieberle, P. Characterization of the most odor-active volatiles in fresh, hand-squeezed juice of grapefruit（Citrus paradisi Macfayden）. J. Agric. Food Chem. 47, 5189-5193 （1999） . 7）Guth, H.; Grosch, W. Evaluation of important odorants in foods by dilution techniques. In Flavor Chemistry. Thirty Years of Progress, Teranishi R, Wich EL, Horn. Kluwer Academic/Plenum: New York, stein （eds） I 377-386 （1999） . 8）Adams, R. P. Identification of essential oil components by gas chromatography/mass spectrometry, 4th edn. Allured Books: Carol Stream, IL.（2007） . 9）Noudogbessi, J. P.; Yedomonhan, P.; Sohouhloue, D. C. K.; Chalchat, J. C.; Figueredo G. Chemical composition of essential oil Syzygium guineense（Willd.）DC. var. guineense （Myrtaceae） from Benin. Rec. Nat. Prod. 2, 33-38 （2008） . 10）Miyazawa, M.; Kurose, K.; Itoh, A.; Hiraoka, N. Comparison of the essential oils of Glehnia littoralis from northern and southern Japan. J. Agric. Food Chem. 49, 5433-5436 （2001） . 11）Bairamian, S.; Beauchamp, P. S.; Descarzo, J. T.; Dev, B. C.; Dev, V.; Frost, S. C.; Nguyen, C. V. California Lomatiums Part III. Composition of the hydrodistilled oils from two varieties of Lomatium dissectum. isolation of a new hydrocarbon. J. Essent. Oil Res. 16, 461-468 （2004） . 12）Jorge, A. P.; Judith, M. Contibution of volatile compounds to mango（Mangifera indica L.）aroma. Frav. Fragr. J. 21, 207-213 （2006） . 13）Yang, D. S.; Lee, K.-S.; Jeong, O.-Y.; Kim, K.-J.; Kays, S.J. Characterization of volatile aroma compounds in
cooked black rice, J. Agric. Food Chem. 56, 235-240 （2008）. 14）Arctander, S. Perfume and flavor chemicals 1, Montclair, N. J.（1969）. 15）Guth, H. Identification of character impact odorants of different white wine varieties. J. Agric. Food Chem. 45, 3027-3032（1997）. 16）Escudero, A.; Farina, E.; Campo, L.; Cacho, J.; Ferreira, V. Analytical characterization of the aroma of five premium red wines. Insights into the role of odor families and the concept of fruitiness of wines. J. Agric. Food Chem. 55, 4501-4510（2007）. 17）Flanzy, C. Ecologia Fundamentos Cientificosy Tecnologicos. AMV-Mundi Prensa, Madrid, pp.137-168 （2003）. 18）Sefton, M. A.; Francis, I. L.; Williams, P. J. Volatile norisoprenoid compounds as constituents of oak wood used in wine and spirit maturation. J. Agric. Food Chem. 38, 2045-2204（1990）. 19）Miyazawa, M.; Kawauchi, Y.; Utsumi, Y.; Takahashi, T. Character impact odorants of wild edible plant-Cacalia hastate L. var. orientalis-used in japanese traditional food. J. Oleo Sci. 59, 527-533（2010）. 20）Dharmawan, J.; Kasapis, S.; Sriramura, P.; Lear, M. J.; Curran, P. Evaluation of aroma-active compounds in pontianak orange peel oil（Citrus nobilis Lour. var. microcarpa Hassk.） by gas chromatography-olfactometry, Aroma reconstitution, and omission test. J. Agric. Food Chem. 57, 239-244（2009）. 21）Boonbumrung, S.; Tamura, H.; Mookdasanit, J.; Nakamoto, H.; Ishihara, M.; Yoshizawa, T.; Varanyanond, W. Characteristic aroma components of the volatile oil of yellow keaw mango fruits determined by limited odor unit method. Food Sci. Technol. Res. 7, 200-206 （2001）. 22）Castro-Vazquez, L.; Diaz-Maroto, M. C.; Perez-Coello, M.S. Aroma composition and new chemical markers of Spanish citrus honeys. Food Chem. 103, 601-606 （2007）. 23）Miyazawa, M.; Hashidume, S.; Takahashi, T.; Kikuchi, T. Aroma evaluation of gamazumi（Viburnum dilatatum） by aroma extract dilution analysis and odour activity value. Phytochem. Anal. 23, 208-213（2012）.
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Characteristic Odor Components of Essential Oils from Eurya japonica Ryota Motooka1, Atsushi Usami1, Hiroshi Nakahashi1, Satoshi Koutari1, Satoshi Nakaya1, Ryoyu Shimizu1, Kaoru Tsuji2, Shinsuke Marumoto3 and Mitsuo Miyazawa1＊ 1
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University (Kindai University) (3-4-1, Kowakae, Higashiosaka-shi, Osaka 577-8502, JAPAN) 2 Center for Ecological Research, Kyoto University (Hirano 2, Otsu-shi, 520-2113, JAPAN) 3 Joint Research Center, Kinki University (Kindai University) (3-4-1, Kowakae, Higashiosaka-shi, Osaka 577-8502, JAPAN)
Abstract: The chemical compositions of essential oils from the flower and aerial parts (i.e., leaf and branch) of Eurya japonica were determined and quantified using gas chromatography-mass spectrometry (GC-MS). A total of 87 and 50 compounds were detected in the oils from the flower and aerial parts, respectively. The main compounds of the flower oil were linalool (14.0%), (9Z)-tricosene (12.0%), and nonanal (7.4%). In the oil from the aerial parts, linalool (37.7%), a-terpineol (13.5%), and geraniol (9.6%) were detected. In the oils from the flower and aerial parts, 13 and 8 aroma-active compounds were identified by GC-olfactometry (GC-O) analysis, respectively. The key aroma-active compounds of the flower oil were heptanal [fatty, green, flavor dilution (FD) = 128, odor activity value (OAV) = 346], nonanal (sweet, citrus, FD = 128, OAV = 491), and eugenol (sweet, spicy, FD = 64, OAV = 62): in the oil from the aerial parts, the key aroma-active compounds were linalool (sweet, citrus, FD = 64, OAV = 95), (E)-b-damascenone (sweet, FD = 256, OAV = 4000), and (E)-b-ionone (floral, violet, FD = 128, OAV = 120). This study revealed that nonanal and eugenol impart the sweet, citrus, and spicy odor of the flower oil, while (E)-b-damascenone and (E)-b-ionone contribute the floral and sweet odor of the oil from the aerial parts. Key words: Eurya japonica, essential oil, GC-MS, GC-O, AEDA, OAV 1 INTRODUCTION Eurya japonica（Hisakaki in Japanese）is an evergreen broad-leaf understory tree from the Pentaphylaceae family; it is commonly known as Uyanggal laba in the Manipuri language. This plant generally grows in India, Southeast Asia, China, Korea, and Japan. This is an economically important species in Korea because the branches are widely used in floral tributes and wreaths1）. In China, the fruits and leaves of this plant are used as a traditional medicine called Lingmu, which is used for the treatment of rheumatoid arthritis, tympanites, and hemostasis of injuries2）. In eastern Japan, E. japonica has been used as a substitute for Cleyera japonica （Sakaki in Japanese）as an offering of the altar. In previous reports, lignans such as ovafolinin B-9’ -Oβ-D-glucopyranoside,（−）-2a-O-（β-D-glucopyranosyl）lyoniresinol,（＋）-3a-O-（β-D-glucopyranosyl）lyoniresinol, and aviculin, flavonoids such as epitaxifolin 3-O-β- D -
xylopyranoside, taxifolin 3-O-β-D-xylopyranoside,（2R,3R）（＋）-glucodistylin, and（ 2S,3S） （−） -glucodistylin, and phenyl glycoside 6’ -O-coumaroyl-1’ -O-［2-（3,4-dihydroxyphenyl）-ethyl］-β-D-glucopyranoside have been isolated from the stems and leaves of E. japonica. These compounds from extracts of this plant have been found to possess antioxidants1, 2） and antibacterial activities3）. In a previous study, the volatile fraction indicated that the essential oil from E. japonica obtained by steam distillation contained 3-hexenol and 2-hexenal4）. However, there have been very few detailed investigations of E. japonica. Thus, we collected the essential oils from the flower and aerial parts（i.e., leaf and branch）of E. japonica using hydrodistillation and investigated the volatile compounds that have aroma properties using gas chromatography mass spectrometry（ GC-MS）and GC-olfactometry（ GC-O）to gain further knowledge of the plant. For flavor analysis, GC-O is the most valuable technique
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Correspondence to: Mitsuo Miyazawa, Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University (Kindai University), 3-4-1, Kowakae, Higashiosaka-shi, Osaka 577-8502, JAPAN E-mail: [email protected] Accepted December 24, 2014 (received for review October 8, 2014)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
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for identifying the key aroma-active compounds in plants. There are various methods for screening aroma-active compounds. One of the frequently used methods is aroma extract dilution analysis（AEDA）5）. The odor potency was estimated using the flavor dilution （FD） -factor6）. Moreover, the significant contribution of each odorant to the characteristic flavor is determined by the odor activity value （OAV）. The OAV is the ratio of the concentration to the odor threshold of the compound, and it is well accepted that compounds with high OAV values contribute more to （i） investithe aroma of food7）. The aim of this study was to gate the characteristic odor compounds of the essential oils obtained from E. japonica by a sensory evaluation and analysis of OAV and（ii） establish that the essential oils of E. japonica deserve further investigation in the food industry.
2 EXPERIMENTAL PROCEDURES 2.1 Plant material Fresh flower and aerial parts（i.e., leaf and branch）of E. japonica were harvested in Kyoto prefecture in April 2011. Identification of the plant was performed at a biotechnology laboratory at Kinki University （Kindai University） . A voucher specimen was deposited at the biotechnology laboratory of Kinki University（ Kindai University）in Osaka, Japan. 2.2 Extraction of essential oils Samples of fresh flower and aerial parts（i.e., leaf and branch） （100 g each） of E. japonica were subjected to hydrodistillation for 3 h using a Likens-Nickerson-type apparatus with diethyl ether. The obtained oils were dried over anhydrous sodium sulfate and stored at 4℃ in a refrigerator prior to analysis. （GC-MS） 2.3 Gas chromatography mass spectrometry GC-MS was performed using an Agilent 6890N gas chromatography-5973 MSD mass spectrometer. The samples were analyzed using a fused-silica capillary column, i.e., HP-5MS（5％ phenyl 95％ polydimethylsiloxane, 30 m× 0.25 mm i.d., film thickness＝0.25 μm） and DB-WAX （polyethylene glycol, 15 m×0.25 mm i.d., film thickness＝0.25 μm）. The oven temperature was programmed to increase from 40 to 260℃ at a rate of 4℃/min and then held at 260℃ for 5 min. The flow rate of the carrier gas（He）was 1.5 mL/min. On the DB-WAX column, the oven temperature was programmed to increase from 40 to 240℃ at a rate of 4℃/min and then held at 240℃ for 5 min. The injector and transfer line were held at 230℃, and the ionization energy was 70 eV. The mass range was 39-450 amu. Each sample （1 μL） was injected at a split ratio of 1:40.
2.4 Sniffing test using GC-olfactometry （GC-O） A trained panel of sensory evaluation specialists measured the odor intensities of the main aromatic constituents of E. japonica. Fourteen panelists, aged 21 to 26 years ［11 males and 3 females, members of Kinki University （Kindai University）, Japan］, participated in this study. Sensory-analysis sessions were performed only after suitable training（ ＞30 h）. The sniffing test by GC-O was carried out using an Agilent Technologies-6890N gas chromatograph equipped with an Agilent 5973 MSD mass spectrometer and sniffing port ODP 2（Olfactory Detector Port 2, Gerstel）. The GC instrument was equipped with an HP-5MS column（30 m×0.25 mm i.d., film thickness＝0.25 μm）. The sample was injected into the GC in splitless mode. The GC effluent from the capillary column was split in at a 1:1 （v/v）ratio between the MS and sniffing port. The oven conditions, injector and transfer line temperatures, carrier gas, flow rate, and ionization mode were the same as those described above for GC-MS. 2.5 Aroma extract dilution analysis （AEDA） The results of AEDA were expressed as FD factors, which are the ratios of the concentration of the odorant in the initial essential oil to its concentration in the most diluted volatile oil in which the odor can be still detected by GC-O. The highest sample concentration（10 mg/mL） was assigned an FD factor of 1. The essential oil was stepwise diluted with diethyl ether（1:1, v/v）, and aliquots of the dilutions（ 1 mL）were evaluated. The process was ceased when no aromas were detected by the evaluators. 2.6 Determination of odor activity value （OAV） OAVs were determined by dividing the concentration of a component with its odor threshold. The odor threshold data was obtained from reported literature data. 2.7 Identification and quantification of compounds The compounds were identified by comparing their retention indices（RIs）and mass spectra with those from previous studies 8−10） digital libraries（ Mass Finder4 and NIST02）, and Aroma Office version 3.0（Nishikawa-Keisoku Co. Ltd.） , which includes 72,120 RI entries for aroma compounds and literature sources. The RIs were calculated using a homologous series of n-alkanes C 5-C 29 on two columns with different polarities. The relative amounts of the individual compounds were calculated based on the GC peak areas（ FID response）, without using correction factors. Quantitative analyses of the aroma-active compounds （FD≥1）of the oils were performed using calibration curves for hexanol（3） , heptanal（5） , α-pinene（6） , myrcene （23）,（E） （11）, octanal（14）,（Z）-linalool oxide（furanoid） -linalool oxide （furanoid） （25） , nonanal（29） , lilac aldehyde （35） , α-terpineol（45）, geraniol（59） , vitispirane（63）, p-vinylguaiacol（68）, eugenol（70）,（E）-β-damascenone（73）,
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and（E）-β-ionone（86）within the concentration range of 0.5-1000 μg/mL. Because of the lack of a proper standard, vitispirane（63）was quantified using the calibration curve for（E）-β-ionone（86）. The weight percent of each compound was calculated using the FID response factor.
3 RESULTS AND DISCUSSION 3.1 Constituents of the essential oils from E. japonica The essential oil from fresh flowers of E. japonica was colorless with a sweet, citrus, and spicy odor, while that from the aerial parts（ i.e., leaf and branch）was slightly yellow, with a floral and sweet odor; the yields of oils were 0.066 and 0.012 ％（w/w）, respectively. The TICs of the essential oils from the flower and aerial parts of E. japonica are shown in Fig. 1. The chemical compositions of the essential oils from the flower and aerial parts of E. japonica are summarized in Table 1. The results revealed that 87 and 50 compounds were obtained from the oils derived from the flower（93.6％）and aerial parts（95.4％）of E. japonica, respectively. The essential oil from the flower of E. japonica mainly comprised linalool（28: 14.0％）,（9Z）-tricosene（ 118: 12.0％）, nonanal（29: 7.4％）, hexadecanal（100: 5.8％）, and α-pinene（6: 4.5％）. Furanoid monoterpenes derived from linalool, such as lime oxide T （15） ,（Z） -linalool oxide
（furanoid） （ 23）,（E）-linalool oxide（furanoid） （ 25）, and lilac aldehyde（35）, were also found in this oil; these compounds appeared to contribute to the characteristic odor of the essential oil of the flower. The main compounds of the oil from the aerial parts of E. japonica were linalool （28; 37.7％）, α-terpineol （45; 13.5％）, geraniol（59; 9.6％）, vitispirane（ 63; 8.5％）, and phytol（110; 3.2％）.（3E） -2-Methyl-3-octen-5-yne（8）was the characteristic compound of the oil from the aerial parts of E. japonica; this compound was isolated from Lomatium dissectum in the Apiaceae family11）. Also, this oil contains four norisopren, vitispirane （63） ,（E） -β-damascenone oids （C13 compounds） （73）, megastigmatrienone（ 83）, and（ E）-β-ionone（ 86）. These compounds contribute to the characteristic odor of the oil. The abundance of monoterpenoids in the flower oil was about half that in the oil from the aerial parts. In contrast, the flower oil contained approximately seven times as much sesquiterpenids than the oil from the aerial parts. Linalool（28）, which is the main compound, was detected in the oil from the flower and aerial parts; the amount of linalool （28）in the flower oil was about 2.5 times that in the oil from the aerial parts. Although（9Z）-tricosene（118）, nonanal（29）, and hexadecanal（100）were present in the flower oil, they were not detected in the oil from the aerial parts. Conversely, vitispirane（63）was only detected in the oil from the aerial parts.
Fig. 1 TICs of essential oils from flower (A) and aerial parts (i.e., leaf and branch) (B) of E. japonica. 579
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Table 1 Chemical compositions of the essential oils from frower and aerial parts (i.e., leaf and branch) of E. japonica.
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Table 2 Aroma-active compounds in the essential oils from E. japonica.
3.2 GC-O, AEDA, and OAV The characteristic aroma-active compounds of the essential oils from E. japonica were identified by GC-O combined with AEDA. Thirteen and eight compounds were identified in the oils from the flower and aerial parts, re. The FD-factors were plotted by their spectively（Table 2） respective peaks on the gas chromatogram shown in Figs. 2 and 3. On the basis of the FD-factor, heptanal（5: FD＝ 128, fatty, green） and nonanal （29: FD＝128, sweet, citrus） were the most intense key aroma-active compounds and octanal（14: FD＝64, citrus）, eugenol（70, FD＝64, sweet, spicy） , and linalool（28: FD＝64, sweet, citrus）contributed to the flower odor. It was determined that heptanal imparts the fatty and green odor, while nonanal and octanal give the sweet and citrus odor. These compounds are found in many flavors including fruit and cooked food12, 13）. The most characteristic aroma-active compounds of the oil from the
aerial parts were（E） -β-damascenone（73: FD＝256, sweet） followed by（E）-β-ionone（86: FD＝128, floral, violet）and linalool（28: FD＝32, sweet, citrus）.（E）-β-Damascenone and（E）-β-ionone are degradation products of β-carotene and contribute significantly to the key aroma of the aerial parts. These sweet aroma compounds have been identified in a wide range of processed food, fruits and vegetables14）. （E）-β-Damascenone and linalool produce the sweet odor, whereas（E）-β-ionone produces the floral odor. These compounds were determined to be the characteristic aromaactive compounds of the aerial parts of E. japonica. The OAV method was used to determine the relative contribution of each of the compounds to the aroma of E. japonica. The OAVs were obtained by taking into account the concentration and odor threshold of each compound. In the flower odor extract, nonanal（29）has the highest OAV（491）, followed by heptanal（5: 346）, linalool（28, 581
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Fig. 2 Gas chromatogram and FD-factor (aromagram) of the essential oil from flower of E. japonica: 1, octane; 5, heptanal; 14, octanal; 28, linalool; 29, nonanal; 118, (9Z)-tricosene; 128, nonacosane.
Fig. 3 Gas chromatogram and FD-factor (aromagram) of the essential oil from aerial parts of E. japonica: 2, (3Z)hexenol; 28, linalool; 45, a-terpineol; 59, geraniol; 73, (E)-b-damascenone; 86, (E)-b-ionone; 116, unknown (M+ = 278). 582
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Fig. 4 Structures of main aroma-active compounds in flowers of E. japonica.
Fig. 5 Structures of main aroma-active compounds in aerial parts of E. japonica. 165）,（E）-linalool oxide（furanoid） （25: 64）, and eugenol （70: 62）. In particular, linalool and eugenol had high FDfactors; thus, these compounds were considered to be the main aroma-active compounds of the flower odor. In the odor extract of the aerial parts,（E）-β-damascenone（73） has high OAV（4000）, followed by（E）-β-ionone（86: 120）. （E） -β-Damascenone and （E） -β-ionone had high FD-factors; thus, these compounds were considered to be the main aroma-active compounds of the aerial parts odor. The odor 15） is high threshold of（E）-β-damascenone odor （0.004 ppb） in all wines; therefore, this compound is normally considered to be a positive contributor to wine aroma16）. This norisoprenoid is also related to tobacco notes17） and is the principal carotenoid-derived compound in French oak18）. The flower oil contained more monoterpenoids than the oil derived from the compared with aerial parts. Therefore, the flowers oil odor was mainly determined by monoterpenoids. On the other hand, the oil derived from the aerial than parts contained more norisoprenoids （C13 compounds） the flower oil. Therefore, the odor of the oil from the aerial parts was mainly determined by norisoprenoids（C13 compounds） . In conclusion, we investigated the characteristic odor compounds of the flower and aerial parts of E. japonica by a sensory evaluation and analysis of OAVs. On the basis of
AEDA, OAVs, and sensory evaluations, linalool （28）was determined to be the main aroma-active compound of the oils. In the flower oil, nonanal（29）and octanal（14）produced the fatty and green odor, heptanal（5）imparted the sweet and citrus odor, and eugenol （70） contributed to the sweet and spicy odor.（E） -β-Damascenone（73）and linalool （28）produced the sweet odor and（E）-β-ionone（86）contributed to the floral odor in the oil derived from the aerial parts. To the best our knowledge, this is the first study that elucidates the characteristic odor compounds that are responsible for the odor of E. japonica. We expect that these results will be used in future investigations of food and medicinal plants.
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