Characteristic Odor Components of Essential Oil from Scutellaria Laeteviolacea

Characteristic Odor Components of Essential Oil from Scutellaria laeteviolacea Mitsuo Miyazawa1＊ , Machi Nomura1, 2, Shinsuke Marumoto1 and Kiyoshige Mori2 1 ‌Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University 3-4-1 Kowakae, Higashiosakashi, Osaka 577-8502, Japan 2 Ohsugi Pharmaceutical Co., Ltd, 1-1-2 Abeno-ku Tennoji-cho minami, Osaka 545-0002, Japan

Abstract: The essential oils from aerial parts of Scutellaria laeteviolacea was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The characteristic odor components were also detected in the oil using gas chromatography-olfactometry (GC-O) analysis and aroma extraction dilution analysis (AEDA). As a result, 100 components (accounting for 99.11 %) of S. laeteviolacea, were identified. The major components of S. laeteviolacea oil were found to be 1-octen-3-ol (27.72 %), germacrene D (21.67 %),and β-caryophyllene (9.18 %). The GC-O and AEDA results showed that 1-octen-3-ol, germacrene D, germacrene B, and β-caryophyllene were the most characteristic odor components of the oil. These compounds are thought to contribute to the unique flavor of this plant.

Key words: ‌essential oil, Scutellaria laeteviolacea, 1-octen-3-ol, aroma extraction dilution analysis, relative flavor activity

1 INTRODUCTION particular, gas chromatography-olfactometry（GC-O）analy- Scutellaria laeteviolacea is a perennial plant belonging sis, including aroma extract dilution analysi（s AEDA）and to the family Lamiaceae. In recent years, this aromatic CharmAnalysis, has been used to identify the potent odor- plant of the aerial parts has been used as a herb in Japan. ants in herbs. In addition, the plant will be applied in fields such as food In the present study, quantitative determination of the products, nonessential luxuries and cosmetic（s as a skin- volatile components of S. laeteviolacea was achieved by whitening agent）1, 2）. The importance of aromatic plants is means of S. laeteviolacea two internal standards, and their considerable because of their potential commercial value in characteristic flavor components were examined by GC-O various fields such as spices, beverages, perfumery, cos- analysis. metics, pharmaceuticals, and aromatherapy1－6）. There have been a few studies on S. laeteviolacea. It has been reported that cyanidin glycoside is the major pigment from the petal parts2）. In addition, the sugar donor specificity of 2 EXPERIMENTAL flavonoid glycosyl transferase in Lamiales plants has been 2.1 Plant materials reported1）. S. laeteviolacea was harvested in Nara Prefecture, Flavor dilution（FD）factors expressed from GC-O ratings Japan, in June 2011. The plant was identified in the bio- provide information on characteristic flavor differences in technology laboratory at Kinki Universit（y Osaka, Japan）. foods. Moreover, the GC-O data correlate concentrations with FD factors equivalent to threshold values for the de- 2.2 Extraction of essential oils terminenations of relative flavor activity. However, the FD Fresh plant materia（l 120 g, whole aerial parts）from a factor does not always correlate with the characteristic sample was subjected to hydrodistillation for 2 h using Lik- flavor, because it sometimes depends on the content. The ens-Nickerson-type apparatus7, 8）. The obtained essential relative flavor activity, in which both FD factor and content oil was dried over anhydrous sodium sulfate, and diluted in are considered, may be a good index to the flavor charac- diethylether for used in the GC and GC-MS measurements. teristics. We have reported new information on the charac- The oil yield was as follows: S. laeteviolacea 26 mg terization of the essential oils from S. laeteviolacea. In （0.02％）.

＊Correspondence to: Mitsuo Miyazawa, Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University 3-4- 1 Kowakae, Higashiosakashi, Osaka 577-8502, Japan E-mail: [email protected] Accepted August 21, 2012 (recieved for review April 4, 2012) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs

51 M. Miyazawa, M. Nomura, S. Marumoto et al.

2.3 Gas chromatography（GC）and gas chromatography- comparing mass spectral data from Wiley, Mass Finder 2.1 mass spectrometry（GC-MS） Libraries, published data28）, the results of our previous GC and GC-MS were performed with an Agilent Technol- studies13, 29－40）, and Kovats retention indice（s RIs）with ogies 6890 chromatograph equipped with a flame ionization those of the standards or RIs reported in the literature. RI detecto（r FID）on a capillary column（HP-5（30 m×0.25 mm were calculated using a series of n-alkane（s C8-C27）on two i.d., film thickness 0.25 μm）and DB-WAX（15 m×0.25 mm columns of different polarities. i.d., film thickness 0.25 μm））. The oven temperature was The quantitative analysis was performed by means of the programmed to change from 40 to 260℃ at 4℃ /min and internal standard addition method（alkanes C12 and C19）. held at 260℃ for 5 min. The injector and detector tempera- The volatile oil was diluted 100 times using diethylether to ture were 270℃ and 280℃, respectively, with the actual achieve a volume of 1 mL volume, and 5 μL of a C12 and temperature in the MS source reaching approximately C19 mixture solution（1 mg/mL）was added to the diluted 230℃, and the ionization energy was 70 eV. The mass oil. The prepared samples were subjected to GC-MS and range was 39-450 amu. After 6 mg of oil was diluted with GC analyses. The quantitative analysis was performed on 500 μL of diethyl ether, 1 mL of the solution was injected, the basis of calibration curves fo（r E）-3-hexen-1-o（l 2）, and the split ratio was 1:10. 1-octen-3-o（l 7）, 3-octano（l 8）, 3-carene（18）, α-copaene The flow-rate of the carrier ga（s helium）was 1.8 mL/min. （32）, β-caryophyllene（41）, germacrene D（50）, ger- Peak areas were quantified using a computer integrator9－11）. macrene B（52）, δ-cadinene（55）, caroto（l 67）, τ-muurolol （80）, and α-cadino（l 82）within the concentration range 2.4 Gas chromatography-mass spectrometry/olfactom- 0.5-1000 μg/mL. The weight-percent of each compound etry（GC-MS/O） was calculated with the response factors to the FID. GC-MS/O was carried out with an Agilent Technologies 6890-Agilent Technologies 5973A-Olfactory Detection Port 2, using a capillary column（HP-5MS, 30 m×0.25 mm i.d., film thickness 0.25 μm）. The column temperature was pro- 3 RESULTS AND DISCUSSION grammed to change from 40 to 260℃ at a rate of 4℃/min 3.1 Volatile components of essential oil from S. laetevio- and held at 260℃ for 5 min. The injector and detector lacea temperatures were 270 and 280℃, respectively. The flow In the GC and GC-MS analyses of the essential oil from S. rate of the carrier ga（s helium）was 1.8 mL/min, with the laeteviolacea 100 compounds, representing 99.11％ of the actual temperature in the MS source reaching approxi- total oil, were characterized. Seventeen peaks were con- mately 230℃, and the ionization voltage was 70 eV. The firmed by sniffing with GC-O. The detected constituents of acquisition mass range was 39-450 amu. The Chemistation the essential oil from S. laeteviolacea are shown in Table 1, software acquired two channel signals simultaneously: one together with their peak percentage（w/w）on the basis of for MS, and the other from the olfactometer signal board. A the HP-5 column and classification according to their func- signal sniffer, the author, recorded the character manually. tional groups. The data are the mean values of triplicate results. The components are listed in order of their elution 2.5 Aroma Extract Dilution Analysis（AEDA） on the HP-5 column. The gas chromatogram and FD chro- The flavor dilution（FD）- factor of the odorants in the es- matogram of the essential oil from S. laeteviolacea are sential oil was determined by aroma extract dilution shown in Fig. 1. analysi（s AEDA）of the following dilution series12, 15－20）. The 1-Octen-3-o（l 27.72％ of total oil）was the most abundant highest dilution was defined as an FD-factor of 1（10 mg/ compound, followed by germacrene D（21.67％）, mL）. The oil was diluted stepwis（e 1:1, v/v）through the ad- β-caryophyllene（9.18％）, 3-carene（8.16％）, and ger- dition of diethyl ether. Aliquots were then analyzed by macrene B（7.11％）. Terpene hydrocarbons were predomi- GC-MS/O on the capillary column HP-5MS9, 11）. The highest nant in the essential oil from S. laeteviolacea. Among the dilution at which an individual component could be detect- oxygenated compounds, 33 alcohols were identified. Mono- ed was defined as the FD-factor for that odorant. On the terpene alcohols accounted for 0.33％, of which basis of the AEDA results, relative flavor activit（y RFA）was α-terpineo（l 0.12％）and geranio（l 0.10％）were the major calculated using the equation reported by Song et al.:12, 19－22）. components. Sesquiterpene alcohols accounted for 4.66％, of which α-cadino（l 1.82％）, caroto（l 1.21％）, and δ-cadinol RFA＝log FD facto（r 2n）/S0.5 （0.99％）were the major components. Khusimone（0.39％） where 2n is the FD factor and S is the weight percentage of was the principal ketone component of the S. laeteviola- the component23－27）. cea volatiles. Two ester（s 0.04％）and two acid（s 0.12％） were detected from this oil. 2.6 Identi cation and quanti cation of components The identities of the components were confirmed by

52 J. Oleo Sci. 62, (1) 51-56 (2013) Characteristic Odor Components of Essential Oil from Scutellaria laeteviolacea

Table 1 Compositions of the essential oil from S. laeteviolacea.

RIb) RIb) a) Peak% Identification a) Peak% Identification No. Compounds c) No. Compounds c) HP-5 DB-WAX (w/w) method HP-5 DB-WAX (w/w) method 1 ethyl acetate 818 898 0.03 RI, MS 57 zonarene 1524 － 1.86 RI, MS 2 (E)-3-hexen-1-ol 857 － 0.45 RI, MS 58 (E)-γ-bisabolene 1531 － 0.06 RI, MS 3 (E)-2-hexen-1-ol 870 － 0.08 RI, MS 59 cadina-1,4-diene 1534 － 0.09 RI, MS 4 3-thujene 926 － trd) RI, MS 60 α-cadinene 1539 － 0.04 RI, MS 5 tricyclene 933 1007 0.02 RI, MS 61 selina-3,7(11)-diene 1544 － 0.17 RI, MS 6 sabinene 973 1134 0.10 RI, MS 62 viridiflorene 1551 － 0.37 RI, MS 7 1-octen-3-ol 987 1447 27.72 RI, MS 63 nerolidol 1559 － 0.01 RI, MS 8 3-octanol 1001 1566 1.93 RI, MS 64 α-elemol 1562 2089 0.07 RI, MS 9 α-terpinene 1018 － 0.06 RI, MS 65 zierone 1570 － 0.07 RI, MS 10 o-cymene 1026 － 0.02 RI, MS 66 epi-longipinanol 1574 － 0.01 RI, MS 11 limonene 1030 1211 0.06 RI, MS 67 carotol 1578 － 1.21 RI, MS 12 (E)-β-ocimene 1039 1245 0.03 RI, MS 68 daucene 1586 － 0.30 RI, MS 13 phenyl acetaldehyde 1046 1649 0.16 RI, MS 69 guaiol 1591 － 0.06 RI, MS 14 γ-terpinene 1060 1262 0.03 RI, MS 70 khusimone 1595 － 0.39 RI, MS 15 p-mentha-3,8-diene 1070 － 0.05 RI, MS 71 ledol 1600 － 0.02 RI, MS 16 acetophenone 1074 － 0.07 RI, MS 72 globulol 1605 2061 0.14 RI, MS 17 isoterpinolene 1089 － 0.02 RI, MS 73 cis-isolongifolanone 1611 － 0.04 RI, MS 18 3-carene 1104 1134 8.16 RI, MS 74 cubenol 1616 － 0.02 RI, MS 19 6-camphenol 1124 － 0.03 RI, MS 75 junenol 1621 － 0.05 RI, MS 20 terpinen-4-ol 1180 1616 0.07 RI, MS 76 trans-isolongifolanone 1625 － 0.03 RI, MS 21 menthol 1187 1626 trd) RI, MS 77 1-epi-cubenol 1630 － 0.10 RI, MS 22 α-terpineol 1193 1711 0.12 RI, MS 78 γ-eudesmol 1634 2185 0.04 RI, MS 23 pulegol 1212 － 0.02 RI, MS 79 δ-cadinol 1645 2211 0.99 RI, MS 24 pulegone 1223 － trd) RI, MS 80 τ-muurolol 1648 － 0.15 RI, MS 25 carvone 1232 － 0.02 RI, MS 81 α-acorenol 1652 － 0.55 RI, MS 26 geraniol 1257 1862 0.10 RI, MS 82 α-cadinol 1659 2219 1.82 RI, MS 27 bornyl acetate 1286 － 0.01 RI, MS 83 α-eudesmol 1674 2246 0.10 RI, MS 28 guaiacol 1319 － 0.02 RI, MS 84 epi-α-bisabolol 1683 － 0.02 RI, MS 29 α-cubebene 1328 1546 0.20 RI, MS 85 khusinol 1689 － 0.04 RI, MS 30 α-longipinene 1339 － 2.53 RI, MS 86 mayurone 1694 － 0.04 RI, MS 31 cyclosativene 1350 1522 0.09 RI, MS 87 curcuphenol 1709 － 0.03 RI, MS 32 α-copaene 1368 1496 0.74 RI, MS 88 isolongifolol 1715 － 0.02 RI, MS 33 α-isocomene 1379 1550 1.38 RI, MS 89 mintsulfide 1740 － 0.09 RI, MS 34 β-bourbonene 1385 1528 0.52 RI, MS 90 (Z)-isovalencenol 1801 － 0.04 RI, MS 35 β-cubebene 1391 － 0.38 RI, MS 91 palmitic acid 1964 1971 0.10 RI, MS 36 cycloseychellene 1397 － 0.01 RI, MS 92 heneicosane 2100 － 1.70 RI, MS 37 α-gurjunene 1401 － 0.01 RI, MS 93 linoleic acid 2131 2136 0.02 RI, MS 38 longifolene 1408 1400 0.03 RI, MS 94 tricosane 2300 2300 0.41 RI, MS 39 β-funebrene 1411 － 0.26 RI, MS 95 tetracosane 2400 2400 0.08 RI, MS 40 α-cedrene 1417 － 0.01 RI, MS 96 pentacosane 2500 2500 0.34 RI, MS 41 β-caryophyllene 1423 1608 9.18 RI, MS 97 hexacosane 2600 2600 0.01 RI, MS 42 β-copaene 1432 － 0.72 RI, MS 98 heptacosane 2700 2700 0.12 RI, MS 43 α-bergamotene 1436 － 0.63 RI, MS 99 octacosane 2800 2800 0.01 RI, MS 44 aromadendrene 1441 1660 0.05 RI, MS 100 nonacosane 2900 2900 0.03 RI, MS 45 γ-muurolene 1446 1684 0.37 RI, MS hydrocarbon 46 humulene 1452 1680 0.03 RI, MS aliphatic 2.69 47 β-santalene 1456 － 1.01 RI, MS aromatic 59.30 48 cadina-1(6),4-diene 1465 － 0.15 RI, MS alcohol 49 trans-muurola-4(14),5-diene 1468 － 0.08 RI, MS aliphatic 30.19 50 germacrene D 1486 1722 21.67 RI, MS aromatic 5.86 51 γ-cadinene 1496 1758 0.12 RI, MS ketone 0.66 52 germacrene B 1500 1738 7.11 RI, MS aldehyde 0.16 53 β-bisabolene 1508 1507 0.18 RI, MS ester 0.04 54 cubebol 1512 － 0.01 RI, MS acid 0.12 55 δ-cadinene 1515 － 0.04 RI, MS sulfur compound 0.09 56 α-selinene 1517 － 0.39 RI, MS 99.11 total 99.11 a) Compounds are listed in order of their elution time from a HP-5 MS column. b) RI = retention indices determined on HP-5MS and DB-WAX columns,using the homologous series of n -alkanes (C8-C27). c) Identification methods: RI, identification based on retention index ; MS, identification based on mass spectra matching. d) tr = trace (< 0.01 %)

53 J. Oleo Sci. 62, (1) 51-56 (2013) M. Miyazawa, M. Nomura, S. Marumoto et al.

Fig. 1 Gas chromatogram and aromagram (FD factor) of essential oil from S. laeteviolacea. 2, (E)-3-hexen-l-ol; 7, l- octen-3-ol; 18, 3-carene; 32, α-copaene; 41, β-caryophyllene; 50, germacrene B; 80, τ-muurolol.

3.2 Aroma Extract Dilution Analysis（AEDA）. an estimation of the relative flavor activity10, 26, 27）, these AEDA is a human bioassay the for determination of the compounds are regarded as important contributors to the odor activity of each compound in a mixture by sniffing the flavor of S. laeteviolacea. These results suggest that minor GC effluent through a series of dilutions. Each volatile components often contribute significantly to the character- component is separated by GC, and the odors are deter- istic flavor. mined at the sniffing port of the GC-O apparatus. Odor de- Sniff testing is used not only for AEDA but also for ex- scriptions of the compounds detected with GC-O are given pressing the aroma character of each component. In in Table 2. As shown in this table, the FD factor range of general, the organoleptic response to a compound depends each peak was between 1 and 7. on its concentration. The FD factor or relative flavor activi- 1-Octen-3-ol, β-caryophyllene, germacrene D, and ger- ty has proven to be useful criteria for the reconstruction of macrene B showed the highest FD factor of 7, and 3-carene the original aroma from the odor-active compounds detect- and α-copaene showed an FD factor of 6. The odor-active ed by AEDA. The concept of relative flavor activity used in volatiles in the S. laeteviolacea volatiles are given in Table this study was defined as a new odor unit calculated 2. Very small peaks such as those of β-caryophyllene, ger- through the use of FD factors instead of odor threshold macrene D, and germacrene B on the gas chromatogram values. However, the FD factor and relative flavor activity were detected as predominant peak（s FD factor of 7）on the often have no relation to the aroma character of a com- FD chromatogram in Fig. 1. pound. In other words, even if the FD factor of one com- The high FD factors of these compounds may result from pound is not comparatively high, it often contributes signif- the high concentrations of the compounds in the essential icantly to the original odor. Thus, the sniff test of the oil from S. laeteviolacea. original essential oil by on-line GC is an effective means of The GC-O technique of AEDA is based on the determina- determining the character impact odorants of an aroma. As tion of odor threshold values of the volatile components shown in Table 2, 1-octen-3-o（l peak 7）, α-copaene（peak eluted from the GC column. Higher FD factors are often 29）, β-caryophyllene（peak 41）, germacrene D（peak 50）, related to the top note of the aroma. However, the FD and germacrene B（peak 52）were estimated as having an S. factor also depends on the concentration. Therefore, the laeteviolacea-like odor in the sniff test. relative flavor activity was also determined as a more real- In this study the character impact odorants of S. laetevi- istic expression because the FD factor does not always co- olacea were screened first by FD factor then, the relative incide with the characteristic odor-active compounds. flavor activity was not used for the determination of the 1-Octen-3-ol is the most predominant componen（t 59.8 mg/ characteristic flavor components but for consideration of kg of fresh weight）, and its FD factor is as high as 7. the relative contributions to the flavor activity. Thus, the However, 1-octen-3-ol showed a low relative flavor activity sniff test of essential oil from S. laeteviolacea was adopted of 0.4, which means that it is of little importance in the es- t h r o u g h t h e u s e o f o n - l i n e G C . 1 - O c t e n - 3 - o l , sential oil from S. laeteviolacea. α-Copaene and τ-muurolol β-caryophyllene, germacrene D, and germacrene B, which showed high relative flavor activitie（s ≥ 1）. On the basis of were evaluated as S. laeteviolacea-like odors by the sniff

54 J. Oleo Sci. 62, (1) 51-56 (2013) Characteristic Odor Components of Essential Oil from Scutellaria laeteviolacea

Table 2 Odor-active volatiles in essential oil from S. laete- ni, M.; Iuchi-Okada, A.; Ishiguro, M.; Kiso, Y.; Nakaya- violacea as detected by GC-O. ma, T.; Ono, E. Local differentiation of sugar donor

d) specificity of flavonoid glycosyltransferase in Lamia- Peak a) b) Odor FD-factor e) Compounds μg/kg c) n RFA no. description (2 ) les., The Plant Cell. 21, 1556-1572（2009）. f) 2） Yoshitama, K.; Ishii, K,; Yasuda, H. A chromatographic 2 (E)-3-hexen-1-ol 971 moss 1 0.4 survey of anthocyanins in the flora of Japan I. J. Fac- 7 1-octen-3-ol 59820 earthy 7 0.4 ulty of Science, Shinshu University. 15, 19-26 8 3-octanol 4165 earthy 4 0.9 （1980）. 18 3-carene 17609 citrus 6 0.6 3） Lei, W.; Zhang, J.; Oiao, A. Bioinformatic data mining 29 a-copaene 1597 woody 6 2.1 on UDP-glucose: flavonoid 7-O-glucosyltransferase 41 b-caryophyllene 19810 spicy 7 0.7 （UBGAT）genes and their encoding proteins in two 50 germacrene D 46764 spicy, woody 7 0.5 plants of genus Scutellaria. Afr. J. Biotechnol. 10 52 germacrene B 15343 spicy, woody 7 0.8 （21）, 4339-4346（2011）. 4 Fukuhara, K.; Fujimori, T.; Shigematsu, H.; Ohnishi, A. 55 d-cadinol 86 herb 3 0.9 ） Essential oil of Scutellaria baicalensis G. Agric. 67 carotol 2611 mild 3 0.9 Biol. Chem. 51（5）, 1449-1451（1987）. 80 t-muurolol 2136 spicy 3 2.3 5） Rosselli, S.; Bruno, M.; Simmonds, M. S. J.; Senatore, F.; 82 a-cadinol 3927 woody 3 0.7 Rigano, D.; Formisano, C. Volatile constituents of Scu- a) Compounds are listed in order of their elution time from a HP-5MS tellaria rubicunda Hornem subsp. linnaeana（Caru- column. b) el Rech. Lamiaceae endemic in Sicily. Biochem. Sys- μg/kg given for 1 kg plant materials. These values w ere computed from the ）（） volatile oil yield and GC peak area. tem. Ecol. 35, 797-800（2007）. c) Odor description at the GC-sniffing port. 6） Yua, J.; Leib, J.; Yua, H.; Caib, H.; Zoua, G. Chemical d) FD-factor : flavor dilution factor using AEDA method (FD-factor1-5mg/ composition and antimicrobial activity of the essential ml). e) RFA; relative flavor activity = log FD factor (2n)/S0.5, w here S is the w oil of Scutellaria barbata. Phytochem. 65, 881-884 eight percentage. （2004）. f) The sample concentration (12 mg/ml) w as assigned an FD factor of 1. 7） Bailly, S.; Jerkovis, V.; Marchand, B. J.; Collin, S.; Aro- ma extraction aroma analysis of Sauternes wines. Key test, had the highest FD factor of 7. α-Copaene, having an role of polyfunctional thiols. J. Agric. Food. Chem. FD factor of 6 and a relative flavor activity of 2.1, was also 54, 7227-7234（2006）. regarded as an odor-active compound of the essential oil 8） Grosch, W.; Evaluation of the key odorant of foods by from S. laeteviolacea. From the results of careful sniff dilution experiments, aroma models and omission., testing, it was found that germacrene D had the odor most Chem. Senses. 26, 33-545（2001）. similar to that of S. laeteviolacea, although its relative 9） Miyazawa, M.; Utsumi, Y.; and Kawata, J. Aroma-active flavor activity was low（0.5）. From these experiments, it is compounds of Elatostema laetevirens and Elat- concluded that comprehensive evaluation of the flavor ostema umbellatum var. majus. J. Oleo. Sci. 58（4）, should be accomplished by simultaneous chemical and 163-169（2009）. sensory analyses. 10） Miyazawa, M.; Marumoto, S.; Kobayashi, T.; Yoshida, S.; The results reported here suggest that 1-octen-3-ol, Utsumi, U. Determination of characteristic compo- α-copaene, β-caryophyllene, germacrene D, germacrene B nents in essential oils from Wisteria branchybotrys can be regarded as the character-impact odorants of the using gas chromatography olfactmetry incremental di- essential oil from S. laeteviolacea, and that germacrene D lution technique. Rec. Nat. Prod. 5（3）, 221-227 has the most odor-active character of the S. laeteviolacea （2011）. aroma. 11） Miyazawa, M.; Takahashi, T.; Mizui, K.; Volatile compounds with characteristic odour in moso-bamboo stem（s Phyllostachys pubescens Mazel ex Houz. De ehaie）. Phytochem. Anal. 21, 489-495（2010）. ACKNOWLEDGEMENTS 12） Tu, N. T. M.; Onishi, Y.; Son, U. S.; Ogawa, E.; Ukeda, H.; This work was supported by Grant-in-Aid from the Japan Sawamura, M. Characteristic odour components of Society for the Promotion of Science（No.24658055）. Citrus inflata Hort. ex Tanak（a Mochiyu）cold-pressed peel oil. Flav. Fragr. J. 18, 454-459（2003）. 13） Miyazawa, M.; Utsunomiya, H.; Kawata, J.; Chanoki, W.; Shirakawa, N. Components of essential oil from woods REFERENCE of Prunus mume Sieb. et Zucc. J. Oleo. Sci., 54, 609- 1） Noguchi, A.; Horikawa, M.; Fukui, Y.; Fukuchi-Mizuta- 612（2005）.

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