Potent Odorants of Characteristic Floral/Sweet Odor in Chinese Chrysanthemum Flower Tea Infusion

Article abstract of DOI:10.1021/acs.jafc.7b04116

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.

Full text of DOI:10.1021/acs.jafc.7b04116

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Article
Potent Odorants of Characteristic Floral/Sweet Odor
of Chinese Chrysanthemum Flower Tea Infusion
Shu Kaneko, Jingxiu Chen, Jieming Wu, Yuto Suzuki, Lin Ma, and Kenji Kumazawa
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04116 • Publication Date (Web): 31 Oct 2017
Downloaded from http://pubs.acs.org on November 4, 2017
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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 15ꢀ7, 279ꢀ0032, Urayasu, Chiba, Japan
‡Ogawa Flavors & Fragrances (Shanghai) Co., Ltd., 85 Jiangtian Rd. (East),
Songjiang District, Shanghai 201613, China
* To whom correspondence should be addressed
PHONE
FAX
+81ꢀ47ꢀ305ꢀ1408
+81ꢀ47ꢀ305ꢀ1423
EꢀMAIL
kaneko.shu@ogawa.net
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ABSTRACT
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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 phenolꢀlike/spicy notes.
Among them, four aroma peaks having a floral/sweet were identified as verbenone,
ethyl 3ꢀphenylpropanoate, propyl 3ꢀphenylpropanoate, 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.
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Key Words: chrysanthemum flower tea; aroma extract dilution analysis; verbenone;
3ꢀphenylpropanoate; 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 2 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 (80ꢀ90 ˚ C, 5ꢀ8 hours), then packed after sterilization.
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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 1,8ꢀcineole, aromadendrene, βꢀselinene,
βꢀchamigrene, αꢀbergamotene, 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
chromatographyꢀolfactometry (GCꢀO) 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 GCꢀO 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,
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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
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Materials. Chrysanthemum flowers cropped in 2013 were purchased from
Datong Industry and Trade Co. Ltd. (Tongxiang, Zhejiang, China).
Chemicals. 3ꢀ(Methylthio)propanal, (E,Z)ꢀ2,6ꢀnonadienal, 3ꢀmethylbutanoic acid,
2ꢀmethylbutanoic acid, (ꢀ)ꢀverbenone, (E,E)ꢀ2,4ꢀdecadienal, 2ꢀmethoxyphenol, ethyl
3ꢀphenylpropanoate, 4ꢀnonanolide, 4ꢀmethylphenol (pꢀcresol), (E)ꢀisoeugenol,
5ꢀnonanolide, ethyl cinnamate, 4ꢀethylphenol, 5ꢀdecanolide, 2′ꢀaminoacetophenone,
4ꢀhydroxyꢀ3ꢀmethoxybenzaldehyde
(vanillin),
3ꢀphenylpropanoic
acid,
(1R)ꢀ(+)ꢀαꢀpinene, cobalt (III) acetylacetonate, and Nꢀhydroxyphthalimide were
purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
3ꢀHydroxyꢀ4,5ꢀdimethylꢀ2(5H)ꢀfuranone, phenylacetic acid, and cobalt (II) acetate
tetrahydrate were purchased from SigmaꢀAldrich Japan Co., Ltd. (Tokyo, Japan).
2ꢀAcetylꢀ1ꢀpyrroline, (Z)ꢀ1,5ꢀoctadienꢀ3ꢀone, transꢀ4,5ꢀepoxyꢀ(E)ꢀ2ꢀdecenal, and
transꢀ4,5ꢀepoxyꢀ(E,Z)ꢀ2,7ꢀdecadienal were synthesized according to the literature.8–11
(+)ꢀVerbenone and propyl 3ꢀphenylpropanoate were synthesized by the following
reactions.
(+)-Verbenone. (1R)ꢀ(+)ꢀαꢀPinene (2.00 g, 19.2 mmol) was dissolved in 5 mL of
acetonitrile in a 10ꢀmL glass test tube. Cobalt (II) acetate tetrahydrate (33.2 mg, 0.133
mmol), cobalt (III) acetylacetonate (68.5 mg, 0.192 mmol), and Nꢀhydroxyphthalimide
(217.7 mg, 1.33 mmol) were then added to the glass tube. The darkꢀgreen suspension
was then stirred at 40 °C for 6 hours 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 nꢀpentane (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 nꢀpentane slurry of 30 g of Wakosil Cꢀ300
(40−64 ꢁm, Wako Pure Chemical Industries, Ltd., Osaka, Japan). Separation was
performed with 200 mL of nꢀpentane, followed by the successive addition of 75 mL of
different ratios of nꢀpentane/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 nꢀpentane/diethyl ether=17/3 fraction was dried in
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vacuo. The isolated yield was 33%. H and 13C {1H} NMR data agreed with the data
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reported in the literature.12 MSꢀEI: m/z (%) 107 (100), 135 (72), 91 (68), 80 (46), 79
(45), 150 (43), 39 (40), 41 (30).
Propyl 3-phenylpropanoate. 1ꢀPropanol (0.10 g, 1.66 mmol) and
3ꢀphenylpropanoic acid (0.25 g, 1.66 mmol) were dissolved in 1 mL of
dichloromethane in a 10ꢀmL beaker, and a drop of pꢀtoluenesulfonic acid and an
excess amount of anhydrous sodium sulfate were then added to the mixture. After
stirring for 1 minute, the mixture was filtered and the solvent of dichloromethane
replaced with nꢀpentane by evaporation (550 mmHg, 35˚C). the obtained nꢀpentane
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solution was separated by silica gel chromatography using a glass column (500 × 10
mm i.d.) filled with an nꢀpentane slurry of 5 g of Wakosil Cꢀ300. Separation was
performed with 50 mL of nꢀpentane, followed by the successive addition of 25 mL of
nꢀpentane/diethyl ether mixtures (19/1, 18/2, 10/10). The eluate of the
nꢀpentane/diethyl ether=19/1 fraction was dried in vacuo. The isolated yield was 95%.
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1H NMR (400MHz, 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, CDCl3) :
δ 10.1 (OCH2CH2CH3), 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)).
MSꢀEI: 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 ꢀL of 2.0 g/L of
2ꢀoctanol in an ethanol solution as an internal standard material, the obtained pale
yellow solution was passed through the glass column (100 mm x 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 x 4) and eluting with 20 mL of dichloromethane. The dichloromethane fraction was
dried with an excess amount of anhydrous sodium sulfate, then distilled by Solvent
Assisted Flavor Evaporation (SAFE) (40 °C, <1.0 x 10ꢀ4 Pa).14 The aroma concentrate
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was obtained by concentrating the dichloromethane layer by rotary evaporation,
followed by nitrogen steam evaporation to about 100 ꢀL. The respective aroma
concentrates were used for the GCꢀO and AEDA studies.
Identification and Semiquantitative analysis of the Key Aroma Compounds
in the Aroma Concentrates of Chrysanthemum Flower Tea. A hundred gram 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 ꢀL of 2.0 g/L of 2ꢀoctanol in an ethanol solution
as an internal standard material, the obtained pale yellow solution was passed
through the glass column (100 mm x 10 mm i.d.) filled with 100 mL of SP 700 resin,
followed by washing with distilled water (100 mL x 2) and eluting with 400 mL of
dichloromethane. The dichloromethane fraction was dried with an excess amount of
anhydrous sodium sulfate, then distilled by SAFE (40 °C, <1.0 x 10ꢀ4 Pa). The aroma
concentrate was obtained by concentrating the dichloromethane layer by rotary
evaporation, followed by nitrogen steam evaporation to about 100 ꢀL. After
replacement of the solvent from dichloromethane to nꢀpentane, 100 ꢀL of the aroma
concentrate was separated by silica gel chromatography using a glass column (200 x
10 mm i.d.) filled with a pentane slurry of 10 g of Wakogel Cꢀ300 (40ꢀ64mm, Wako
Pure Chemical Industries, Ltd., Osaka, Japan). Separation was performed with 50 mL
of nꢀpentane (Fraction I), followed by 50 mL of a nꢀpentane/diethyl ether mixture
(19:1) (Fraction II), 50 mL of a nꢀpentane/diethyl ether mixture (18:2) (Fraction III), 50
mL of a nꢀpentane/diethyl ether mixture (17:3) (Fraction IV) , 50 mL of a
nꢀpentane/diethyl ether mixture (16:4) (Fraction V) , and 50 mL of diethyl ether
(Fraction VI). The respective fractions were concentrated by nitrogen steam to about
50 ꢀL. Identification of each aroma compound was performed by comparing its Kovats
GC retention index (RI) and mass spectrum with the authentic compound by GCꢀMS
using the DBꢀWAX column, in addition to by comparing its RI and odor quality with the
authentic compound by GCꢀO analysis using the DBꢀWAX column.
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Semiquantitative data of the respective key aroma compounds were calculated
based on the internal standard material (2ꢀoctanol) using an estimated response
factor of 1 by GCꢀMS.
GC–O. An Agilent model 6850 series gas chromatograph equipped with a
thermal conductivity detector (TCD) was used for the GCꢀO analysis. A fused silica
column (30 m x 0.25 mm i.d. coated with a 0.25 ꢁm film of DBꢀWax, J&W Scientific; or
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30 m x 0.25 mm i.d. coated with a 0.25 ꢁm film of DBꢀ1, J&W Scientific) was used with
a 1.0 ꢁL splitless injection. Helium was used as the carrier gas at the flow rate of 1
mL/min. The injector temperature was set to 250 °C and the purge valve was opened
at 60 seconds. The chromatography was performed from the oven temperature of
40 °C to 210 °C at the rate of 5 °C/min. The detector temperature was set to 230 °C. A
glass sniffing port was connected to the outlet of the TCD and heated to >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 GCꢀO 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 ꢁL) of each
fraction were analyzed by capillary GC on a DBꢀWax 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.
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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 x
0.25 mm i.d. coated with a 0.25 ꢁm film of DBꢀWax, J&W Scientific; or 60 m x 0.25
mm i.d. coated with a 0.25 ꢁm film of DBꢀ1, 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 ꢁL) of the sample were injected with the
split ratio of 1:30 or splitless. The chromatography was performed from the oven
temperature of 80 °C to 230 °C at the rate of 3 °C/min for the split injections, and from
the oven temperature of 40 °C to 230 °C at the rate of 3 °C/min for the splitless
injections. The mass spectrometer was used under the following conditions: ionization
voltage, 70 eV (EI); ion source temperature, 150 °C.
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Nuclear Magnetic Resonance Spectroscopy (NMR). H, 13C, COSY, HMQC,
and HMBC spectroscopic experiments were done using a Bruker AVANCEꢀ400
spectrometer (Bruker Biospin, Yokohama, Japan). CDCl3 was used as the solvent.
Chiral Analysis of Verbenone in Chrysanthemum Flower Tea. An Agilent
7890A gas chromatograph coupled to an Agilent model 5975C inert XL series mass
spectrometer was used. A fused silica column (30 m x 0.25 mm i.d. coated with a 0.25
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ꢁm film of InertCapTM CHIRAMIX column, GL Sciences, Inc., Tokyo, Japan) 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 200 °C. Aliquots (1 ꢁL) of the sample were injected
with the split ratio of 1:30. The chromatography was performed from the oven
temperature of 40 °C to 200 °C at the rate of 1 °C/min. The mass spectrometer was
used under the following conditions: ionization voltage, 70 eV (EI); ion source
temperature, 150 °C. The (+) and (ꢀ)ꢀverbenones were identified by comparing their
retention times (91.99 min and 92.75 min, respectively) and mass spectra with the
authentic compounds. The enantiomeric ratio of verbenone was determined by
extracting an ion chromatogram of m/z=107 from the total ion chromatogram.
Determination of Recognition Odor Threshold Concentration of Verbenone.
The recognition thresholds of (+)ꢀverbenone and (ꢀ)ꢀverbenone were determined
using the GCꢀO apparatus according to the literature.15
Sensory Evaluation of Chrysanthemum Flower Tea. Seven panelists were
recruited from Ogawa Flavors & Fragrances (Shanghai) Co., Ltd., and trained for 6
months with inꢀhouse programs of recognition, description, and discrimination tests.
Descriptors of the sensory evaluation were given out by the assessors consolidated
from the collected discussion among the seven assessors for both tea samples. The
respective chrysanthemum flower tea samples (20 mL) were poured into a plastic cup
at 25 °C for the sensory assessors, who were asked to score the strengths of the
floral/sweet, hayꢀlike, herb cooling, and fresh aroma notes on a scale from 1 (weak) to
5 (strong). A twoꢀway layout analysis of variance without replication was applied to the
sensory evaluation results performed by a fiveꢀpoint scoring method.
RESULTS AND DISCUSSION
Aroma Profile of the Two Different Types of Chrysanthemum Flower Teas.
First, to determine the flavor profiles of the TJ and HBJ infusions, a sensory
evaluation was conducted by the trained assessors. The TJ infusion had strong
floral/sweet and fresh aroma notes as well as moderate herb cooling and hayꢀlike
aroma notes, while the HBJ infusion had slightly weaker aroma characters of
floral/sweet, fresh, herb cooling, and nearly the same hayꢀlike aroma note compared
to the TJ infusion (Fig. 1). TJ is generally known as the chrysanthemum tea having a
rich aroma, and it seems to be caused by the rich fresh and floral/sweet aroma notes
from the sensory evaluation in this study.
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Identification of the Potent Odorants in the Two Different Types of
Chrysanthemum Flower Teas. The aroma concentrates of the chrysanthemum
flower teas had the same aroma characteristics as the sensory evaluation when it was
dipped in a filter paper and sniffed.
To identify the potent odorants in the two different types of chrysanthemum
flower tea infusions, the AEDA was applied to the aroma concentrates of the
respective teas. Twentyꢀfive aroma peaks were detected from the TJ aroma
concentrate and 24 aroma peaks were detected from the HBJ aroma concentrate,
among which 20 peaks were commonly detected from both aroma concentrates. It
suggested that most of aroma active compounds would be common in both of the
chrysanthemum flower teas. Summarizing the two AEDA experiments, a total of 29
aroma peaks was detected from at least either aroma concentrate, and among which
26 compounds were identified or tentatively identified in this study (Table 1). Only 9
compounds had already been identified, and the rest of the 17 compounds were
identified or tentatively identified in the chrysanthemum flower tea for the first time.
Among the detected peaks, verbenone (6), having a sweet/floral/camphorꢀlike
odor, ethyl cinnamate (17), having a sweet/honeyꢀlike odor, and an unknown
floral/woody aroma peak (23) had the highest FDꢀfactor of 64 in TJ, followed by the
detection of propyl 3ꢀphenylpropanoate (10), having a floral/sweet/honeyꢀlike odor,
transꢀ4,5ꢀepoxyꢀ(E)ꢀ2ꢀdecenal (11), having a metallic odor, and eugenol (18), having
a spicy/burnt odor as the next highest FDꢀfactor of 16 in TJ. These aroma active
compounds were also detected in the HBJ aroma concentrate with the same or one
slightly low FDꢀfactors. Based on the sensory analysis result, the aroma peaks can be
categorized as a floral/sweet note (verbenone (6), ethyl 3ꢀphenylpropanoate (9),
propyl 3ꢀphenylpropanoate (10), ethyl cinnamate (17)) in addition to a metallic note
((Z)ꢀ1,5ꢀoctadienꢀ3ꢀone
(2),
transꢀ4,5ꢀepoxyꢀ(E)ꢀ2ꢀdecenal
(11),
transꢀ4,5ꢀepoxyꢀ(E,Z)ꢀ2,7ꢀdecadienal (13)) and phenolꢀlike/spicy notes (pꢀcresol (14),
eugenol (18), 4ꢀethylphenol (19)) in both flowers.
Verbenone (6) is possibly formed by autoꢀoxidation or by plant or microbial
metabolites from αꢀpinene.16ꢀ18 A previous study reported that verbenone (6) was
detected in the fully opening flower.19 It indicated that verbenone (6) is generated in
the living flower as a plant metabolite. On the other hand, considering the production
process of the chrysanthemum flower as well as the model experiment of aerobic
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oxidation of αꢀpinene,16 a heavy heat history during airꢀdrying (80ꢀ90 ˚C, 5ꢀ8 hours)
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also causes generation of verbenone (6) from αꢀpinene in the flower. Ethyl cinnamate
(17), propyl 3ꢀphenylpropanoate (10), and ethyl 3ꢀphenylpropanoate (9) are thought to
be generated from the corresponding alcohol and carboxylic acid formed from
phenylalanine.20 Except for these phenylalka(e)noic acid esters, the other esters,
such as ethyl phenylacetate and propyl phenylacetate, were also detected in the
aroma concentrate (data not shown), but only three compounds were identified as
aroma active compounds due to those quantities in addition to their low odor detection
thresholds. transꢀ4,5ꢀEpoxyꢀ(E)ꢀ2ꢀdecenal (11) is thought to be generated during the
hot airꢀdrying process because it is one of the lipid oxidation products.21 Eugenol (18)
is thought to be generated via the biosynthetic pathway from phenylalanine.22
Relative Quantitation of Characteristic Floral/Sweet Odorants in Two
Different Types of Chrysanthemum Teas. The AEDA results of the aroma
concentrates of the two different chrysanthemum flower teas were compared to clarify
the differences in their aroma characteristics. Among the potent odorants having
FDꢀfactors of ≥16, whereas transꢀ4,5ꢀepoxyꢀ(E)ꢀ2ꢀdecenal (11), eugenol (18), and an
unknown aroma peak (23) were detected with the same FDꢀfactor in both aroma
concentrates, the aroma peaks having a floral/sweet odor note identified as
verbenone (6), propyl 3ꢀphenylpropanoate (10), and ethyl cinnamate (17) were
comparatively high in the TJ aroma concentrate (Table 1).
To precisely confirm these differences, a relative quantitation analysis was
conducted for the two aroma concentrates. Verbenone (6) was richest in both
concentrates containing 1400 ꢀg/kg in TJ and 900 ꢀg/kg in HBJ, and TJ contained
about a 1.6 times higher content than HBJ (Table 2). The concentrations of ethyl
3ꢀphenylpropanoate (9) and propyl 3ꢀphenylpropanoate (10) in TJ were also 1.9 and
2.5 times higher than those in HBJ. Furthermore, there was a very noticeable
difference in the concentration of ethyl cinnamate (17) which was 5.6 times higher in
TJ compared to HBJ.
Combining the relative quantitation result and comparative AEDAs, it was
concluded that verbenone (6), ethyl 3ꢀphenylpropanoate (9), propyl
3ꢀphenylpropanoate (10), and ethyl cinnamate (17) might more strongly contribute to
the TJ aroma. These results also well agreed with the stronger floral/sweet aroma
note of the TJ tea by the sensory evaluation.
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Chiral Analysis of Verbenone. Verbenone (6) is a bicyclic monoterpene
compound having two chiral centers in the molecule, and there are 2 enantiomeric
isomers of the (+)ꢀform ((1R,5R)ꢀ4,6,6ꢀtrimethylbicyclo[3.1.1]heptꢀ3ꢀenꢀ2ꢀone) and
(ꢀ)ꢀform ((1S,5S)ꢀ4,6,6ꢀtrimethylbicyclo[3.1.1]heptꢀ3ꢀenꢀ2ꢀone) (Fig. 2). Whereas
verbenone (6) has already been identified in many plants,23,24 its aroma profile was
only clarified for the (ꢀ)ꢀisomer. Furthermore, the odor detection thresholds of the
enantiomers have not yet been clarified.
To clarify the main contributor of verbenone (6) detected by AEDA, the
enantiomeric ratios in the respective aroma concentrates were calculated by a chiral
analysis using GCꢀMS. The analysis revealed that the concentration of the (ꢀ)ꢀisomer
was approximately 3 times higher than that of the (+)ꢀisomer in both aroma
concentrates (Table 3). Furthermore, the recognition thresholds of the respective
enantiomers were calculated by a GCꢀO technique referred to in the literature.15
Whereas (ꢀ)ꢀverbenone ((ꢀ)ꢀ6) with a lower recognition threshold of 6.8 ng/L having a
sweet, floral, and camphorꢀlike odor, (+)ꢀverbenone ((+)ꢀ6) of 24 ng/L had a sweet,
spicy, and floral odor. Because (ꢀ)ꢀverbenone ((ꢀ)ꢀ6) was rich in both flowers and its
detection threshold was relatively lower than that of the (+)ꢀisomer, the (ꢀ)ꢀisomer is a
main contributor of the aroma active components in the concentrates of the
chrysanthemum flower tea.
In conclusion, the Chinese chrysanthemum flower tea infusion mainly consisted
of three aroma attributes of a floral/sweet odor potentially contributed by
(ꢀ)ꢀverbenone, (hydroxy)cinnamate derivatives, a metallic odor by lipid degradation
products, and a spicy odor by phenols. Especially, based on a sensory analysis in
addition to an AEDA study and semiquantitative analysis, the floral/sweet odor
potentially contributed by (ꢀ)ꢀverbenone, propyl 3ꢀphenylpropanoate, and ethyl
cinnamate is one of the most dominant aroma attributes to characterize the unique
aroma of the chrysanthemum flower tea. These findings focusing on potent odorants
will give us more advanced knowledge to understand aroma characteristics of
chrysanthemum flower tea. Whereas many newly identified compounds were clarified
by the AEDA analyses in this study, some of the aroma peaks are still unknown. To
obtain further knowledge about the chrysanthemum flower aroma, it is still under
investigation.
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REFERENCES
(1) Zhu, F. R.; Yang, C. Y. Components and health benefit of Hangbaiju. In
Hangbaiju Jianshang; Zhu, F. R.; Yang, C. Y. Ed.; Zhejiang photography
publisher: Hangzhou, China, 2007; pp. 101ꢀ112.
335
336
337
(2) Zhu, F. R.; Yang, C. Y. Origin of chrysanthemum flower. In Hangbaiju Jianshang;
Zhu, F. R.; Yang, C. Y. Ed.; Zhejiang photography publisher: Hangzhou, China,
2007; pp. 1ꢀ15.
338
339
(3) Ito, T.; Tada, S.; Sato, S. Aroma constituents of edible chrysanthemum. J. Fac.
Agric., Iwate Univ. 1990, 35ꢀ42.
340
341
342
(4) Wang, Y.; Yang, X. W. GCꢀMS analysis of essential oil of the flower of the
Chrysanthemum morifolium by the different processing methods. China J.
Chinese Materia Medica 2006, 31, 456ꢀ459.
343
344
345
346
347
(5) Cao, G.; Cai, H.; Cong, X.; Liu, X.; Ma, X.; Lou, Y.; Qin, K.; Cai, B. Global
detection and analysis of volatile components from sunꢀfried and
sulfurꢀfumigated herbal medicine by comprehensive twoꢀdimensional gas
chromatography/timeꢀofꢀflight mass spectrometry. Analyst, 2012, 137,
3828ꢀ3835.
348
349
350
351
(6) Zhou, H. M.; Xie, P. S.; Wang, W. H.; Ma, J. Q.; Li, P. Analysis of volatile
components from the flowers of Chrysanthemum morifolium by GCꢀMS with
solidꢀphase microextraction. China J. Chinese Materia Medica 2005, 30,
986ꢀ989.
352
353
354
355
(7) Xiao, Z.; Fan, B.; Niu, Y.; Wu, M.; Liu, J.; Ma, S. Characterization of odorꢀactive
compounds
of
various
Chrysanthemum
essential
oils
by
gas
chromatographyꢀolfactometry, gas chromatographyꢀmass spectrometry and their
correlation with sensory attributes. J. Chromatogr. B 2016, 1009-1010, 152ꢀ162.
356
357
(8) Schieberle, P. Primary odorants in popcorn, J. Agric. Food Chem. 1991, 39,
1141ꢀ1144.
358
359
360
(9) Swoboda, P. A.; Peers, K. E. Metallic odour caused by vinyl ketones formed in
the oxidation of butterfat. The identification of octaꢀ1,cisꢀ5ꢀdienꢀ3ꢀone. J. Sci.
Food Agric. 1977, 28, 1019ꢀ1024.
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Page 13 of 21
Journal of Agricultural and Food Chemistry
13
361
(10) Kumazawa, K.; Wada, Y.; Masuda, H. Characterization of epoxydecenal isomers
as potent odorants in black tea (Dimbula) infusion. J. Agric. Food Chem. 2006,
54, 4795–4801.
362
363
364
365
366
(11) Miyazato, H.; Hashimoto, S.; Hayashi, S. Identification of the odorꢀactive
aldehyde transꢀ4,5ꢀepoxyꢀ(E,Z)ꢀ2,7ꢀdecadienal in yuzu (Citrus junos Sieb. Ex
Tanaka). Eur. Food Res. Technol. 2012, 235, 881ꢀ891.
367
368
(12) Lajunene, M. K.; Maunula, T.; Koskinene, A. M. P. Co(II) catalyzed oxidation of
alphaꢀpinene by molecular oxygen. Part 2. Tetradedron 2000, 56, 8167ꢀ8171.
369
370
371
(13) Kaneko, S.; Kumazawa, K.; Nishimura, O. Comparison of the key aroma
compounds in five different types of Japanese soy sauces by Aroma Extract
Dilution Analysis (AEDA). J. Agric. Food Chem. 2012, 60, 3831ꢀ3836.
372
373
374
(14) Engel, W.; Bahr, W.; Schieberle, P. Solvent assisted flavour evaporation, a new
and versatile technique for the careful and direct isolation of aroma compounds
from complex food matrices. Eur. Food Res. Technol. 1999, 209, 237–241.
375
376
377
(15) Ulrich, F.; Grosch, W. Identification of the most intense volatile flavour
compounds formed during autoxidation of linoleic acid. Z. Lebensm. Unters.
Forsch., 1987, 184, 277ꢀ282.
378
379
(16) Neuenschwander, U.; Guignard, F.; Hermans, I. Mechanism of the aerobic
oxidation of alphaꢀpinene. ChemSusChem. 2010, 3, 75ꢀ84.
380
381
382
(17) Hirata, T.; Ikeda, Y.; Izumi, S.; Shimoda, K.; Hamada, H.; Kawamura, T.
Introduction of oxygenated functional groups into 3ꢀcarene and 2ꢀpinene by
cultured cells. Phytochemistry 1994, 37, 401ꢀ403.
383
384
385
(18) Wright, S. J.; Caunt, P.; Carter, D.; Baker, P. B. Microbial oxidation of
alphaꢀpinene by Serratia marcescens. Appl. Microbiol. Biotechnol. 1986, 23,
224ꢀ227.
386
387
388
(19) Xu, J.; Li, Y. Y.; Zheng, C. S.; Wang, C.; Yoo, Y. K. Studies of aroma compounds
in chrysanthemum in different florescence and in florescence parts and aroma
releasing. Acta. Bot. Boreal. Occident. Sin. 2012, 32, 722ꢀ730.
389
390
391
(20) Koukol, J.; Conn, E. E. The metabolism of aromatic compounds in higher plants.
IV. Purification and properties of the phenylalanine deaminase of Hordeum
vulgare. J. Biol. Chem. 1961, 236, 2692ꢀ2698.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 21
14
392
393
394
(21) Buettner, A.; Schieberle, P. Aroma properties of a homologous series of
2,3ꢀepoxyalkenals and transꢀ4,5ꢀepoxyalkꢀ2ꢀenals. J. Agric. Food Chem. 2001,
49, 3881ꢀ3884.
395
396
397
(22) Manitto, P.; Gramatica, P.; Monti, D. Biosynthesis of phenylpropanoid
compounds. Part II. Incorporation of specifically labelled cinnamic acids into
eugenol. J. Chem. Soc., Perkin Trans. 1 1975, 1548ꢀ1551.
398
399
400
(23) Reglero, G.; Herraiz, M.; Sanz, J. Capillary gas chromatographic determination
of volatiles in solid matrices by direct introduction using a programmableꢀ
temperature vaporizer. J. Chromatogr. 1989, 483, 43ꢀ50.
401
402
(24) Papageorgiou, V. P.; Mellidis, A. S.; Argyriadou, N. The chemical composition of
the essential oil of mastic gum. J. Ess. Oil Res. 1991, 3, 107ꢀ110.
403
404
405
406
(25) Han, T.; Mao, J.; Ji, Z. W.; Huang, G. D. Analysis of volatile components in
Chrysanthemum morifolium flowers by comprehensive twoꢀdimensional gas
chromatography/timeꢀofꢀflight mass spectrometry. Shipin Kexue, 2013, 34,
159ꢀ164.
407
408
409
410
(26) Tamura, H.; Sakakibara, H.; Yanai, T.; Takagi, Y.; Kitahara, T.; Mori, K.
Synthesis of both the enantiomers of transꢀchrysanthenol and transꢀ
chrysanthenyl acetate as the characteristic major constituents of kougiku. Biosci.
Biotech. Biochem. 1995, 59, 1985ꢀ1986.
411
412
413
414
(27) Guo, Q. S.; Wang, Y. J.; Yang, X. W.; Xu, W. B. Characterization of chemical
components of essential oil from cupitulum of Chrysanthemum morifolium
cultivated in Tongxiang city. China J. Chinese Materia Medica 2008, 33,
624ꢀ627.
415
416
417
(28) Xie, X.; Liu, M.; Zhou, Q.; Wen, C.; Liu, Y. An analytical study on composition of
volatile oils in flower of Hebei Xiangju. Shizhen Guoyi Guoyao 2008, 19,
3052ꢀ3054.
418
419
420
421
(29) Xie, Y.; Yu, H.; Wang, J. F.; Zhang, Z. L.; Chen, S. Y.; Xiao, X.; Li, X. L.
Simultaneous determination of nine phenolic acids in Dendranthema morifolium
(Ramat) Tzvel. cv. Chuju samples by high performance liquid chromatography.
China J. Anal. Chem. 2013, 41, 383ꢀ388.
422
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FIGURE LEGEND
Figure 1.
Figure 2.
Aroma profile of two different types of Chinese chrysanthemum
flower teas.
Chemical structure of (+)ꢀ and (ꢀ)ꢀverbenone.
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Table 1. Key Aroma Compounds in Two Different Kinds of Chinese Chrysanthemum Flower
Teas
RIa
FD factorc
No.
odor qualities
compoundb
fraction
Reff
DBꢀW
DBꢀ1
TJd HBJe
AX
1
2
1337 892 popcornꢀlike
1369 957 metallic
2ꢀacetylꢀ1ꢀpyrrolineg
(Z)ꢀ1,5ꢀoctadienꢀ3ꢀoneg
methionalg
(E,Z)ꢀ2,6ꢀnonadienalg
3ꢀmethylbutanoic acid
2ꢀmethylbutanoic acid
III
II, III
IV, V
III
1
4
ndf
1
3
1451 864 cooked potatoꢀlike
1585 1121 cucumberꢀlike
4
ndf
1
4
1
25
25
25
26
27
5a
5b
6
1663
sour, powdery
V
4
4
1727
1809
sweet, floral, camphorꢀlike verbenone
IV, V
III
64
1
16
1
7
fatty
(E,E)ꢀ2,4ꢀdecadienal
2ꢀmethoxyphenolg
8
1856 1071 phenolic, burnt
1882 floral, sweet, honeyꢀlike
floral, sweet, honeyꢀlike
11 2003 1342 metallic
12 2031 fatty
IV
II
1
1
9
ethyl 3ꢀphenylpropanoate
propyl 3ꢀphenylpropanoate
transꢀ4,5ꢀepoxyꢀ(E)ꢀ2ꢀdecenalg III, IV
4
4
10 1971
II
16
16
1
4
16
1
4ꢀnonanolide
IV
transꢀ4,5ꢀepoxyꢀ(E,Z)ꢀ2,7ꢀdeca
13 2059 1336 metallic
III, IV
4
ndf
dienalg
14 2077
animalꢀlike
pꢀcresol
IV
IV
III
4
4
1
1
25
3
15 2085 1349 fatty, milky
5ꢀnonanolideg
ndf
ndf
16 2120
floral, herbal, sweet
unknown
sweet, honeyꢀlike,
cinnamonꢀlike
17 2131
ethyl cinnamate
II
64
16
18 2161
19 2175
spicy, burnt
eugenol
III, IV
IV
16
4
16
4
animalꢀlike, burnt
4ꢀethylphenol
3ꢀhydroxyꢀ4,5ꢀdimethylꢀ2(5H)ꢀf
20 2191 1084 seasoningꢀlike
21 2205 fatty, milky
22 2218 1274 grapeꢀlike
23 2278 woody, floral
24 2341 1426 spicy, floral
V
1
4
uranoneg
5ꢀdecanolide
2'ꢀaminoacetophenoneg
III, IV
IV
IV
IV
IV
V
1
1
1
1
unknown
64
ndf
1
64
1
ndf
(E)ꢀisoeugenolg
unknown
25 2453
26 2549
27 2556
28 2581
floral
honeyꢀlike
vanillaꢀlike
floral, woody
phenylacetic acid
vanillin
1
4
28
29
III
1
1
unknown
3ꢀphenylpropanonic acidg
V
1
ndf
ndf
29 2611 1324 floral
V
1
aRI, retention index on the DBꢀWAX column. bThe compound was identified by comparing its RI and mass
spectrum with the authentic compound by GCꢀMS in addition to comparison of its RI and odor quality with the
c
d
e
f
authentic compound by GCꢀO. FD factor, flavor dilution factor. TJ, Taiju. HBJ, Hangbaiju. Previous reports
identified as volatile components in chrysanthemum flower. gThe compound was tentatively identified by GCꢀO
analysis using DBꢀWAX and DBꢀ1 columns by comparison to the authentic compound, but no unequivocal mass
spectrum was available by GCꢀMS. fnd, not detected.
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Table 2. Relative Quantitation of Verbenone, Ethyl 3-Phenylpropanoate,
Propyl 3-Phenylpropanoate, and Ethyl Cinnamate in the Chrysanthemum
Flowers
concentration in flowerab (ꢀg/kg)
No.
compound
verbenone
ratio
TJc
1400 ±200
4.9 ±0.4
19 ±3
HBJd
6
9
900 ±60
2.6 ±0.2
7.7 ±1.2
4.5 ±0.4
1.6
1.9
2.5
5.6
ethyl 3ꢀphenylpropanoate
propyl 3ꢀphenylpropanoate
ethyl cinnamate
10
17
25 ±1
aThe relative concentration of each compound was calculated based on the ratio of the
peak area of the respective compounds and that of 5.0 mg/kg of 2ꢀoctanol using an
b
estimated response factor of 1. Data are the mean value of triplicate measurements.
cTJ, Taiju. dHBJ, Hangbaiju.
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Table 3. Aroma descriptions, Recognition Threshold, and Enantiomeric Ratio of (+)- and
(-)-Verbenone in the Chrysanthemum Flower
enantiomeric ratiob, %
recognition
No.
compound
aroma description
threshold in
TJc
HBJd
aira, ng/L
(+)ꢀ6 (+)ꢀverbenone
(ꢀ)ꢀ6 (ꢀ)ꢀverbenone
sweet, spicy, floral, woody
sweet, floral, camphorꢀlike
24
28
24
6.8
72
76
aRecognition threshold in air was determined using the GCꢀO instrument in accordance with the methodology previously
b
reported by Ulrich et.al. (ref. 15). Enantiomeric ratio was determined by extracting an ion chromatogram of m/z=107 from a
total ion chromatogram using the InertCap® CHIRAMIX® column. cTJ, Taiju. dHBJ, Hangbaiju.
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Figure 1.
floral/sweet
Hangbaiju (HBJ)
Taiju (TJ)
5
4
3
2
1
fresh
hay-like
herb cooling
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Figure 2.
O
O
(+)ꢀverbenone
(+)ꢀ6
(−)ꢀverbenone
(ꢀ)ꢀ6
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