Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. 2017, 65, 10058-10063
Potent Odorants of Characteristic Floral/Sweet Odor in Chinese Chrysanthemum Flower Tea Infusion Shu Kaneko,*,† Jingxiu Chen,‡ Jieming Wu,‡ Yuto Suzuki,‡ Lin Ma,‡ and Kenji Kumazawa† †
Ogawa & Co. Ltd., Chidori 15-7, 279-0032, Urayasu, Chiba, Japan Ogawa Flavors & Fragrances (Shanghai) Co., Ltd., 85 Jiangtian Road (East), Songjiang District, Shanghai 201613, China
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ABSTRACT: An investigation using the aroma extract dilution analysis (AEDA) technique applied to the aroma concentrates prepared from the tea infusions of two different types of Chinese chrysanthemum flowers (flower buds, blooming flowers) revealed that 29 aroma peaks were detected in the aroma concentrates, and 17 compounds were newly identified or tentatively identified in the chrysanthemum flower tea. AEDA also revealed that the aroma peaks having high flavor dilution factors mainly consisted of a floral/sweet note in addition to metallic and phenol-like/spicy notes. Among them, four aroma peaks having a floral/sweet were identified as verbenone, ethyl 3-phenylpropanoate, propyl 3-phenylpropanoate, and ethyl cinnamate, and a semiquantitative analysis revealed that the flower buds were rich in these compounds. Furthermore, a chiral analysis revealed that (−)-verbenone existed in both flowers at a 3 times higher concentration than (+)-verbenone. Additionally, because the detection threshold of (−)-verbenone was lower than that of the (+)-verbenone, it is concluded that the (−)-isomer was a main contributor of the aroma peak of verbenone in the chrysanthemum flower tea. KEYWORDS: chrysanthemum flower tea, aroma extract dilution analysis, verbenone, 3-phenylpropanoate, cinnamate
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INTRODUCTION The chrysanthemum flower is a kind of Chinese herb consumed as a tea infusion since ancient times. It is also used as a Chinese medicine having many medicinal benefits of reducing eye strain, an antipyretic effect, sedative effect, and reduction of blood pressure.1 There are several cultivars of chrysanthemum for consumption as a tea or medicine in China, and the major cultivar in the Chinese market is Chrysanthemum morifolium Ramat. cultivated in Tongxian city of the Zhejiang Province, central eastern China.2 There are two different commercial products of chrysanthemum flower teas produced in Tongxian. One is made from the flower buds called Taiju (TJ), and the other is made from the blooming flowers called Hangbaiju (HBJ). After being picked, the flowers are steamed for several minutes to deactivate the enzymes in the flower. The flowers are next spread on a wide tray and dried by heated air (80−90 °C, 5−8 h) and then packed after sterilization. Chrysanthemum tea generally has a unique floral/sweet aroma as well as a bitter and sweet aftertaste. Especially, TJ is a precious tea having a pleasant rich aroma. There have been many studies of the volatile components in the chrysanthemum flower. Ito et al. identified 1,8-cineole, aromadendrene, β-selinene, β-chamigrene, α-bergamotene, thymol, and eugenol as the major volatile components in a steam-distillate of C. morifolium in addition to chrysanthenone, which is a major typical volatile compound in the chrysanthemum flower.3 Furthermore, many sesqui/monoterpene hydrocarbons/alcohols/ketones and phenol compounds were identified as major volatile components in the steam distillates of C. morifolium.4,5 Zhou et al. reported that camphor was the major component in the head space volatiles of the C. morifolium flower by SPME analysis.6 Xiao et al. applied the gas chromatography−olfactometry (GC−O) © 2017 American Chemical Society
technique to several chrysanthemum essential oils, and revealed 38 aroma active compounds which were mainly hydrocarbons.7 Whereas there are many reports concerning the volatile components in the steam distillate, solvent extract, or headspace volatiles of the chrysanthemum flowers in addition to the GC−O study of commercially available essential oils, there are no investigations focusing on the potent odorants in a chrysanthemum flower tea infusion. The aim of this research is, therefore, to identify the potent odorants in the chrysanthemum flower tea infusion by aroma extract dilution analysis (AEDA), and to clarify the potent odorants exhibiting a typical floral/sweet odor in the tea. Furthermore, the stereoisomers of verbenone, which is one of the potent odorants having a floral/ sweet odor, were clarified by chiral analysis.
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MATERIALS AND METHODS
Materials. Chrysanthemum flowers cropped in 2013 were purchased from Datong Industry and Trade Co. Ltd. (Tongxiang, Zhejiang, China). Chemicals. 3-(Methylthio)propanal, (E,Z)-2,6-nonadienal, 3methylbutanoic acid, 2-methylbutanoic acid, (−)-verbenone, (E,E)2,4-decadienal, 2-methoxyphenol, ethyl 3-phenylpropanoate, 4-nonanolide, 4-methylphenol (p-cresol), (E)-isoeugenol, 5-nonanolide, ethyl cinnamate, 4-ethylphenol, 5-decanolide, 2′-aminoacetophenone, 4-hydroxy-3-methoxybenzaldehyde (vanillin), 3-phenylpropanoic acid, (1R)-(+)-α-pinene, cobalt(III) acetylacetonate, and N-hydroxyphthalimide were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 3-Hydroxy-4,5-dimethyl-2(5H)-furanone, phenylacetic acid, and cobalt(II) acetate tetrahydrate were purchased from Sigma-Aldrich Japan Co., Ltd. (Tokyo, Japan). 2-Acetyl-1-pyrroline, Received: Revised: Accepted: Published: 10058
September 4, 2017 October 25, 2017 October 31, 2017 October 31, 2017 DOI: 10.1021/acs.jafc.7b04116 J. Agric. Food Chem. 2017, 65, 10058−10063
Article
Journal of Agricultural and Food Chemistry
anthemum Flower Tea. One hundred grams of the chrysanthemum flower was extracted with 5000 mL of boiled distilled water for 5 min. The tea brew was then rapidly cooled to room temperature, and the flower was removed by filtration. After adding 250 μL of 2.0 g/L of 2octanol in an ethanol solution as an internal standard material, the obtained pale yellow solution was passed through the glass column (100 mm × 10 mm i.d.) filled with 100 mL of SP 700 resin, followed by washing with distilled water (100 mL × 2) and eluting with 400 mL of dichloromethane. The dichloromethane fraction was dried with an excess amount of anhydrous sodium sulfate and then distilled by SAFE (40 °C, 210 °C by a ribbon heater. Moist air was pumped into the sniffing port at 100 mL/ min to quickly remove the odorant eluted from the TCD out of the sniffing port. The aroma concentrates underwent a GC−O analysis by three subjects. Determination of the odor qualities detected by sniffing was achieved by triplicate experiments for each subject. AEDA. The original aroma concentrate of the chrysanthemum flower tea was stepwise diluted with dichloromethane from 1:4 to 1:256, and aliquots (1 μL) of each fraction were analyzed by capillary GC on a DB-Wax column. AEDA was performed three times with respect to each sample by three subjects. The detection of each compound was defined as not less than two detections by all subjects, and the flavor dilution (FD) factor of each compound was determined as the maximum dilution degree of detection. GC−MS. An Agilent 7890A gas chromatograph coupled to an Agilent model 5975C inert XL series mass spectrometer was used. A fused silica column (60 m × 0.25 mm i.d. coated with a 0.25 μm film of DB-Wax, J&W Scientific; or 60 m × 0.25 mm i.d. coated with a 0.25 μm film of DB-1, J&W Scientific) was used for the analyses. Helium was used as the carrier gas at the flow rate of 1 mL/min, and the injector temperature was set to 250 °C. Aliquots (1 μL) of the sample were injected with the split ratio of 1:30 or splitless. The chromatography was performed from the oven temperature of 80 to 230 °C at the rate of 3 °C/min for the split injections, and from the oven temperature of 40 to 230 °C at the rate of 3 °C/min for the splitless injections. The mass spectrometer was used under the
(Z)-1,5-octadien-3-one, trans-4,5-epoxy-(E)-2-decenal, and trans-4,5epoxy-(E,Z)-2,7-decadienal were synthesized according to the literature.8−11 (+)-Verbenone and propyl 3-phenylpropanoate were synthesized by the following reactions. (+)-Verbenone. (1R)-(+)-α-Pinene (2.00 g, 19.2 mmol) was dissolved in 5 mL of acetonitrile in a 10 mL glass test tube. Cobalt(II) acetate tetrahydrate (33.2 mg, 0.133 mmol), cobalt(III) acetylacetonate (68.5 mg, 0.192 mmol), and N-hydroxyphthalimide (217.7 mg, 1.33 mmol) were then added to the glass tube. The dark-green suspension was then stirred at 40 °C for 6 h with air bubbling (12 mL/ min) using a glass filter dipped in a reaction solution. After 1 mL of distilled water was added to the reaction mixture, it was extracted with n-pentane (20 mL × 5) and dried with an excess amount of anhydrous sodium sulfate. After concentration to approximately 10 mL by rotary evaporation, the extract was separated by silica gel chromatography using a glass column (500 × 20 mm i.d.) filled with an n-pentane slurry of 30 g of Wakosil C-300 (40−64 μm, Wako Pure Chemical Industries, Ltd., Osaka, Japan). Separation was performed with 200 mL of n-pentane, followed by the successive addition of 75 mL of different ratios of n-pentane/diethyl ether mixtures (19/1, 18/2, 17/3, 16/4, 15/5, 14/6, 13/7, 12/8, 10/10). The eluate of the n-pentane/diethyl ether = 17/3 fraction was dried in vacuo. The isolated yield was 33%. 1 H and 13C {1H} NMR data agreed with the data reported in the literature.12 MS-EI: m/z (%) 107 (100), 135 (72), 91 (68), 80 (46), 79 (45), 150 (43), 39 (40), 41 (30). Propyl 3-Phenylpropanoate. 1-Propanol (0.10 g, 1.66 mmol) and 3-phenylpropanoic acid (0.25 g, 1.66 mmol) were dissolved in 1 mL of dichloromethane in a 10 mL beaker, and a drop of p-toluenesulfonic acid and an excess amount of anhydrous sodium sulfate were then added to the mixture. After stirring for 1 min, the mixture was filtered and the solvent of dichloromethane replaced with n-pentane by evaporation (550 mmHg, 35 °C). The obtained n-pentane solution was separated by silica gel chromatography using a glass column (500 × 10 mm i.d.) filled with an n-pentane slurry of 5 g of Wakosil C-300. Separation was performed with 50 mL of n-pentane, followed by the successive addition of 25 mL of n-pentane/diethyl ether mixtures (19/ 1, 18/2, 10/10). The eluate of the n-pentane/diethyl ether = 19/1 fraction was dried in vacuo. The isolated yield was 95%. 1H NMR (400 MHz, CDCl3): δ 0.90 (dt, 3, J = 7.4 Hz, J = 0.9 Hz, OCH2CH2CH3), 1.58 (m, 2, OCH2CH2CH3), 2.66 (t, 2, J = 7.8 Hz, PhCH2CH2), 2.95 (t, 2, J = 7.8 Hz, PhCH2CH2), 3.60 (dt, 2, J = 6.7 Hz, J = 0.9 Hz, OCH2CH2CH3), 7.20 (m, 3, Ph(o, p)), 7.28 (m, 2, Ph(m)). 13C{1H} NMR (100 MHz, CDCl 3 ): δ 10.1 (OCH 2 CH 2 CH 3 ), 25.3 (OCH2CH2CH3), 30.7 (PhCH2CH2), 35.7 (PhCH2CH2), 64.7 (OCH2CH2CH3), 126.4 (Ph(p)), 128.6 (Ph(o)), 128.6 (Ph(m)), 140.4 (Ph(gem)), 178.2 (PhCH2CH2C(O)). MS-EI: m/z (%) 104 (100), 91 (67), 105 (45), 107 (41), 192 (39), 133 (24), 77 (16), 79 (13), 103 (12), 78 (12), 150 (10). Preparation of the Aroma Concentrates of Chrysanthemum Flower Tea for GC−O and AEDA Analyses. The aroma concentrates of the chrysanthemum flower tea were obtained by the concentrating method described in the literature with a slight modification.13 Two grams of the chrysanthemum flower was extracted with 100 mL of boiled distilled water for 5 min. The tea brew was then rapidly cooled to room temperature, and the flower was removed by filtration. After adding 5 μL of 2.0 g/L of 2-octanol in an ethanol solution as an internal standard material, the obtained pale yellow solution was passed through the glass column (100 mm × 10 mm i.d.) filled with 5 mL of SP 700 resin (Mitsubishi Chemical Corp., Tokyo, Japan), which was conditioned with distilled water before use, followed by washing with distilled water (5 mL × 4) and eluting with 20 mL of dichloromethane. The dichloromethane fraction was dried with an excess amount of anhydrous sodium sulfate and then distilled by solvent assisted flavor evaporation (SAFE) (40 °C,
