Journal of Oleo Science Copyright ©2013 by Japan Oil Chemists’ Society J. Oleo Sci. 62, (1) 51-56 (2013) 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）.