Characterization of epoxydecenal isomers as potent odorants in black tea (Dimbula) infusion

Article abstract of DOI:10.1021/jf0603127

In a black tea (Dimbula) infusion, the potent "sweet and/or juicy" odorants were identified as the cis-and trans-4,5-epoxy-(E)-2- decenals by comparison of their gas chromatography retention indices, mass spectra, and odor quality to those of the actual synthetic compounds. Of the two odorants, cis-4,5-epoxy-(E)-2-decenal has been identified for the first time in the black tea. On the basis of the aroma extract dilution analysis on the flavor distillate obtained using the solvent-assisted flavor evaporation technique from the black tea infusion, these isomers showed higher flavor dilution (FD) factors. The FD factors and concentrations of these odorants in the black tea infusion were observed to be much higher than those from Japanese green tea. In addition, the model studies showed that these odorants were generated from linoleic acid and its hydroperoxides by heating, but the generated amounts of these odorants from linoleic acid were much less than those of its hydroperoxides. It can be assumed from these results that the withering and fermentation, which are characteristic processes during the manufacturing of the black tea, which includes the enzymatic reaction such as lipoxygenase, is one of the most important factors for the formation of the epoxydecenal isomers.

Full text of DOI:10.1021/jf0603127

J. Agric. Food Chem. 2006, 54, 4795−4801 4795
Characterization of Epoxydecenal Isomers as Potent Odorants
in Black Tea (Dimbula) Infusion
KENJI KUMAZAWA,* YOSHIYUKI WADA, AND HIDEKI MASUDA
Ogawa & Company, Ltd., 15-7 Chidori Urayasushi, Chiba 279-0032, Japan
In a black tea (Dimbula) infusion, the potent “sweet and/or juicy” odorants were identified as the cis-
and trans-4,5-epoxy-(E)-2-decenals by comparison of their gas chromatography retention indices,
mass spectra, and odor quality to those of the actual synthetic compounds. Of the two odorants,
cis-4,5-epoxy-(E)-2-decenal has been identified for the first time in the black tea. On the basis of the
aroma extract dilution analysis on the flavor distillate obtained using the solvent-assisted flavor
evaporation technique from the black tea infusion, these isomers showed higher flavor dilution (FD)
factors. The FD factors and concentrations of these odorants in the black tea infusion were observed
to be much higher than those from Japanese green tea. In addition, the model studies showed that
these odorants were generated from linoleic acid and its hydroperoxides by heating, but the generated
amounts of these odorants from linoleic acid were much less than those of its hydroperoxides. It can
be assumed from these results that the withering and fermentation, which are characteristic processes
during the manufacturing of the black tea, which includes the enzymatic reaction such as lipoxygenase,
is one of the most important factors for the formation of the epoxydecenal isomers.
KEYWORDS: Dimbula; black tea; cis-4,5-epoxy-(E)-2-decenal; trans-4,5-epoxy-(E)-2-decenal; manufactur-
ing process; enzyme reaction
INTRODUCTION
because the previous flavor analysis of the Ceylon black tea
was not coupled with an aroma extract dilution analysis
(AEDA).
Black tea is one of the most widely consumed beverages in
the world. The high acceptability of black tea is due to many
factors, one of the most important being its flavor. Black tea is
produced in many places around the world, and many cultivars
are known. These cultivars have characteristic flavors, such as
the Darjeeling muscat flavor and Keemun smoky flavor, due
to various primary factors, for instance, the climate, soil,
manufacturing conditions, etc. On the other hand, the so-called
Ceylon black teas, which are produced in Sri Lanka, have a
typical floral and juicy-sweet note and have been preferred by
the Japanese for a long time.
The black tea flavor has already been the subject of much
research, and many of the volatile compounds have been
identified (1). A number of volatile components in the Ceylon
black tea flavor have also been identified by gas chromatography
(GC) and GC-mass spectrometry (MS) measurements (2-4).
However, there are few reports concerning the potent odorants
of the black tea flavor (5-9). In these reports, many potent
odorants have been identified, and the flavor difference of each
cultivar is assumed to be caused by the difference in the amount
of the potent odorants (6, 8). However, there is no report about
the potent odorants of the Ceylon black tea, and the contributors
to the typical flavor of this black tea have not been identified,
The steps involved in the processing of black tea include
withering, leaf disruption (rolling and/or cutting), fermentation,
drying, and grading. For the characteristic process during the
black tea production, it utilizes an enzyme reaction. Namely,
black tea leaves were produced by drying after making use of
the sufficient enzyme action during the withering and fermenta-
tion process. On the other hand, the green tea leaves inactivated
the enzyme by steaming or parching during the first process,
and it hardly utilized the action of the enzyme. The black tea
flavor was quite different from that of the green tea regardless
of being made from the same kind of plant. For the flavor
formation of the black tea, in particular, it was pointed out that
the enzyme action during the manufacturing process is very
important for the intense monoterpene alcohols with glycosidase
(10, 11), aldehydes with polyphenol oxidase (12, 13), and C6
aldehydes with lipoxygenase and hydroperoxide lyase (14).
However, the influence of the enzyme action during the
manufacturing process on the formation of the potent odorants
is still mostly unresolved.
The objective of the present investigation was to elucidate
the potent odorants of Dimbula, which is one of the typical
Ceylon black teas by an AEDA. Furthermore, to clarify the
generation mechanism of the epoxydecenal isomers during the
manufacturing of the black tea leaves, a comparison of the black
* To whom correspondence should be addressed. Tel: +81 47 305 1408.
Fax: +81 47 305 1424. E-mail: kumazawa.kenji@ogawa.net.
10.1021/jf0603127 CCC: $33.50
© 2006 American Chemical Society
Published on Web 06/06/2006

4796 J. Agric. Food Chem., Vol. 54, No. 13, 2006
Kumazawa et al.
68 (100), 55 (15), 43 (12), 41 (20), 39 (18). 1H NMR (400 MHz,
and green teas and model experiments using the linoleic acid
and its hydroperoxides were performed.
CDCl3): δ (ppm) 0.92 (t, J ) 7.1 Hz, 3H, C-10), 1.32-1.36 (m, 4H,
C-8,9), 1.49 (m, 2H, C-7), 1.64-1.66 (m, 2H, C-6), 2.96 (td, J5-6
)
5.6 Hz, J5-4 ) 2.0 Hz, 1H, C-5), 3.33 (dd, J4-3 ) 7 Hz, J4-5 ) 2 Hz,
1H, C-4), 6.38 (dd, J2-1 ) 7.6 Hz, J2-3 ) 15.8 Hz, 1H, C-2), 6.56 (dd,
J3-4 ) 7 Hz, J3-2 ) 15.8 Hz, 1H, C-3), 9.56 (d, J1-2 ) 7.6 Hz, 1H,
C-1). cis-4,5-Epoxy-(E)-2-decenal: MS/EI m/z (%) 152 (3, [M+]), 139
MATERIALS AND METHODS
Materials. Tea Samples. The black tea products are as follows:
Dimbula (Sri Lanka), Uva (Sri Lanka), Nuwara eliya (Sri Lanka),
Darjeeling (India), Assam (India), Java (Indonesia), and Keemun
(China). These black tea products were produced in 2004, and they
were purchased from Mitsui Norin Co., Ltd. (Tokyo, Japan). The
Japanese green tea (Sen-cha) product was produced in Shizuoka
prefecture (Japan) in 2004. These black and green tea leaves were stored
at -80 °C until needed.
Chemicals. (E,E)-2,4-hexadienal, (E,E)-2,4-heptadienal, (E,E)-2,4-
octadienal, (E,E)-2,4-nonadienal, (E,E)-2,4-decadienal, linalool, n-
hexanoic acid, and vanillin were obtained from Tokyo Kasei Kogyo
(Tokyo, Japan), â-damascenone was obtained from Nihon Firmenich
(Tokyo, Japan), and linoleic acid, grycerol trioctanoate, and soy bean
lipoxygnase were obtained from Wako Pure Chemical Industries
(Osaka, Japan).
1
(6), 81 (27), 69 (11), 68 (100), 55 (16), 43 (12), 41 (17), 39 (22). H
NMR (400 MHz, CDCl3): δ (ppm) 0.90 (t, J ) 7.1 Hz, 3H, C-10),
1.23-1.45 (m, 4H, C-8,9), 1.45-1.65 (m, 4H, C-6,7), 3.28 (m, 1H,
C-5), 3.63 (ddd, J4-3 ) 6.5 Hz, J4-5 ) 4.5 Hz, J4-2 ) 0.9 Hz 1H,
C-4), 6.40 (ddd, J2-1 ) 7.8 Hz, J2-3 ) 15.8 Hz, J2-4 ) 0.9 Hz 1H,
C-2), 6.69 (dd, J3-4 ) 6.5 Hz, J3-2 ) 15.8 Hz, 1H, C-3), 9.60 (d,
J1-2 ) 7.8 Hz, 1H, C-1).
Isolation of the Volatiles from Tea Infusion. Each tea (black and
green) infusion (1 L) was passed through a column packed with 10 g
of Porapak Q (80/100 mesh, Waters). The adsorbed compounds were
then eluted with 100 mL of methylene chloride. The eluate was dried
over anhydrous sodium sulfate (approximately 10 g), and to remove
the nonvolatile material, the eluate was distilled under reduced pressure
(40 °C at 5 × 10-3 Pa) using the solvent-assisted flavor evaporation
(SAFE) method (16). The distillate was dried over anhydrous sodium
sulfate, and the solvent was then removed by rotary evaporation (35
°C at 550 mmHg) to about 5 mL. Further concentration was conducted
in a nitrogen stream to about 150 µL. For the quantitative analysis, an
internal standard solution (10 µL) prepared from methyl undecanoate
(5.06 mg) in methylene chloride (10 mL) was added to the eluate before
the SAFE treatment, and then, the acids in the tea distillate were
removed using a saturated solution of NaHCO3 (2 × 50 mL), because
the epoxydecenal isomer peaks were overlapped with the hexenoic acid
peaks on the gas chromatogram using the DB-Wax stationary phase
column. The combined organic phases were washed with a saturated
solution of NaCl (2 × 100 mL) and then dried over anhydrous sodium
sulfate. The resulting concentrate was used as the sample for the AEDA
and the GC-MS analysis.
Enrichment of Epoxydecenal Isomers for Identification. For the
identification experiments, the black tea volatiles were isolated from
the black tea infusion (Dimbula) by combining the adsorptive column
method and the SAFE technique as described above. These procedures
were repeated, and all of the volatile fractions were combined (total:
6 L of deionized water was added to 450 g of black tea leaves). The
concentrated volatile fraction was applied to a water-cooled glass
column (15 °C, 20 cm × 0.7 cm i.d.) filled with silica gel (wakogel
C-200; Wako Pure Chemical Industries) in isopentane. After the column
had been flushed with isopentane (100 mL) and isopentane/methylene
chloride (150 mL, 80/20, v/v), the epoxydecenal isomers were eluted
with methylene chloride (100 mL). The solution was concentrated to
about 100 µL as already described.
Preparation of Linoleic Acid Hydroperoxides. Linoleic acid
hydroperoxides were obtained from the oxidation of linoleic acid by
soybean lipoxygenase. The hydroperoxides were isolated by column
chromatography and used as soon as possible to avoid the model
reaction. Linoleic acid (1 g) was emulsified with 0.8 mL of 0.1% Tween
80 in 500 mL of 20 mmol sodium borate buffer (pH 8.5). After
saturation with oxygen for 10 min, the substrate was incubated at room
temperature with 20 mg of lipoxygenase dissolved in 1 mL of the same
buffer. After 1 h, the mixture was acidified with concentrated HCl to
pH 3 and extracted with 3 × 250 mL of methylene chloride. The
combined organic phases were washed with distilled water (2 × 250
mL) and then dried over anhydrous sodium sulfate. The oxidation
product of linoleic acid by soybean lipoxygenase was subjected to thin-
layer chromatography (TLC) using Silica Gel 60 F254 plates (Merck
Japan Ltd., Tokyo, Japan). After two developments with the solvent
system, hexane, diethyl ether, and formic acid (50 + 50 + 1), the
hydroperoxides were detected on the TLC plates by UV irradiation,
and the major product gave the Rf of 0.54. After the solvent was
removed by evaporation, the reaction mixture was dissolved in about
2 mL of hexane and then purified by column chromatography on silica
gel. The reaction mixture, dissolved in hexane, was placed on the top
of a column (2 cm i.d.) packed with a slurry of 50 g silica gel (Wakogel
C-200) in hexane. Elution was performed using the following sol-
Tea Infusion. Deionized hot water (3 L) at 85-90 (black tea) or
70-75 °C (green tea) was added to 150 g of tea, and the leaves were
removed using coarse filter paper after the mixture stood for 5 min.
The filtrate (3 L) was immediately cooled to about 20 °C in tap water.
Syntheses of cis-4,5-Epoxy-(E)-2-decenal and trans-4,5-Epoxy-
(E)-2-decenal. The target compounds were synthesized from cis- or
trans-2-octenol as the starting materials by epoxidation with m-
chloroperbenzoic acid (m-CPBA), followed by oxidation with Dess-
Martin periodinane and the Wittig reaction (15). In the first step, cis-
or trans-2,3-epoxyoctanol was synthesized using m-CPBA. The m-
CPBA (purity > 65%, 31.9 g) was then dissolved in methylene chloride
(500 mL), and this solution was dropped into (Z)-2-octenol (purity >
95%, 0.1 mol, Eiweiss Chemical Corp., Shizuoka, Japan) or (E)-2-
octenol (purity > 95%, 0.1 mol, Sigma-Aldrich Japan, Tokyo, Japan)
solution, which was dissolved in methylene chloride (1000 mL) at 0-5
°C, and the mixture was stirred for 2 h at room temperature. When the
epoxidation stopped, saturated solutions of Na2S2O3 and NaHCO3 were
added and then extracted with Et2O (3 × 100 mL). The combined
organic phases were washed with a saturated solution of NaCl (2 ×
100 mL) and then dried over anhydrous sodium sulfate. After the solvent
was removed by evaporation, the compounds were purified by column
chromatography on silica gel, and then, 13.5 g of cis-2,3-epoxyoctanol
or 13.5 g of trans-2,3-epoxyoctanol, each with a purity of >97%, was
obtained. The obtained cis- or trans-2,3-epoxyoctanol was then oxidized
into the corresponding aldehyde, cis- or trans-2,3-epoxyoctanal, using
Dess-Martin periodinane. The cis- or trans-2,3-epoxyoctanol (2.9 g)
was dissolved in methylene chloride (300 mL), and then, a 15%-Dess-
Martin periodinane solution (dissolved in methylene chloride, 85 g)
was added, and the solution was stirred for 2 h at room temperature.
When the oxidation stopped, saturated solutions of Na2S2O3 and
NaHCO3 were added and then extracted with Et2O (3 × 100 mL). The
combined organic phases were washed with a saturated solution of NaCl
(2 × 100 mL) and then dried over anhydrous sodium sulfate. After the
solvent was removed by evaporation, the compounds were purified by
column chromatography on silica gel; then, 2.4 g of cis-2,3-epoxy-
octanal or 2.5 g of trans-2,3-epoxyoctanal, each with a purity of >97%,
was obtained. In the third step, the target compounds were obtained
by the Wittig reaction, in which the cis-2,3-epoxyoctanal (2.4 g) or
trans-2,3-epoxyoctanal (2.4 g) and formylmethylene triphenylphos-
phorane (5.2 g) were dissolved in toluene (110 mL), and the reaction
mixture was refluxed for 8 h. After it was cooled, the reaction mixture
was evaporated to remove the solvent, and the residue was extracted
with hexane. After the solvent was removed by evaporation, the
compounds were purified by column chromatography on silica gel; then,
1.7 g of cis-4,5-epoxy-(E)-2-decenal or 1.8 g of trans-4,5-epoxy-(E)-
2-decenal, each with a purity of > 97%, was obtained. These structures
were confirmed by mass spectrometry (electron impact [EI] mode) and
1H nuclear magnetic resonance. The 1H NMR characterization afforded
the following data: δ [multiplicity, coupling constant (in hertz), and
relevant H at carbon (numbering refers to Figure 3)]. trans-4,5-Epoxy-
(E)-2-decenal: MS/EI m/z (%) 152 (2, [M+]), 139 (4), 81 (27), 69 (11),

Characterization of Epoxydecenal Isomers in Black Tea
J. Agric. Food Chem., Vol. 54, No. 13, 2006 4797
Figure 1. Flavor dilution chromatograms of the black tea (Dimbula) infusions (a, linalool; b, (E,E)-2,4-decadienal; c,
and e, vanillin).
â-damascenone; d, hexanoic acid;
Table 1. Selected Ion and Calibration Factors for Mass
Chromatography (SIM)
vents: hexane (100 mL), hexane/diethyl ether (8/2, 100 mL), hexane/
diethyl ether (7/3, 100 mL), hexane/diethyl ether (6/4, 100 mL), and
hexane/diethyl ether (1/1, 100 mL). The separated fractions (30 mL
each) were monitored for hydroperoxides by UV absorption on the
TLC plates, and the major product was collected. Immediately after
the solvent was removed by rotary evaporation, 380 mg of the linoleic
acid hydroperoxides was dissolved in 10 mL of methylene chloride.
Model Experiment for Epoxydecenal Isomer Formation from
Linolenic Acid. The linoleic acid hydroperoxides (31.25 µmol) or
linoleic acid (31.25 µmol) was dissolved in 5 g of glycerol trioctanoate
containing FeSO4‚2H2O (2 mg) and then heated for 60 min at 150 °C
using a GC oven in a closed vessel. After the mixture was cooled,
methylene chloride (50 mL), containing methyl undecanoate (101 µg)
as the internal standard, was added, and the flavor compounds and
internal standard were isolated by the SAFE treatment. The epoxy-
decenal isomers were then quantified by GC-MS.
Gas Chromatography-Olfactometry (GC-O). A Hewlett-Packard
(HP) 5890 series gas chromatograph equipped with a thermal conduc-
tivity detector (TCD) and fused silica column (30 m × 0.25 mm i.d.,
coated with a 0.25 µm film of DB-Wax; J & W Scientific) was used
in the splitless injection mode (splitless time, 1 min). The column
temperature was programmed from 40 to 210 °C at the rate of 5 °C/
min for all runs. The injector and detector temperatures were 250 and
230 °C, respectively. Helium was used as the carrier gas at the flow
rate of 1 mL/min. A glass sniffing port was connected to the outlet of
the TCD and heated by a ribbon heater with moist air being pumped
into the sniffing port at about 100 mL/min to quickly remove the odorant
from the sniffing port that had been eluted from the TCD.
compound
selected ion (m/z)
calibration factor
(E,E)-2,4-hexadienal
(E,E)-2,4-heptadienal
(E,E)-2,4-octadienal
(E,E)-2,4-nonadienal
(E,E)-2,4-decadienal
cis-4,5-epoxy-(E)-2-decenal
trans-4,5-epoxy-(E)-2-decenal
methyl undecanoatea
96
110
124
138
152
68
0.22
0.28
0.34
0.45
0.58
0.10
0.08
1
68
200
a Internal standard.
GC-MS. An Agilent 6890 N gas chromatograph coupled to an
Agilent 5973 N series mass spectrometer was used. The column was
a 60 m × 0.25 mm i.d. DB-Wax fused silica capillary type (J & W
Scientific) with a film thickness of 0.25 µm. The column temperature
was programmed from 80 to 210 °C or from 40 to 210 °C at the rate
of 3 °C/min. The injector temperature was 250 °C, and the flow rate
of the helium carrier gas was 1 mL/min. An injection volume of 1 or
0.2 µL was applied using the split (the split ratio was 1:30) or splitless
technique. The mass spectrometer was used with an ionization voltage
of 70 eV (EI) and an ion source temperature of 150 °C. The quantities
of the components in each volatile fraction of the tea infusions were
determined from the extracted ion peak areas obtained by mass
chromatography. The GC-MS was operated in the selected ion mode
(SIM), and the extracted ions were monitored in the ranges listed in
Table 1. The calibration factors were determined in a mixture of equal
amounts by weight of an odorant and internal standard compound and
were calculated as the ratio of the extracted ion peak area of the internal
standard to the extracted ion peak area of the odorant. These extracted
ion peak areas were the mean values of the triplicate results. The
calibration factors were used to calculate the amount of each odorant
on the basis of the internal standard.
AEDA. The original odor concentrate of the tea infusion was
stepwise diluted with methylene chloride to 4n (n ) 3-8), and aliquots
(1 µL) of each fraction were analyzed by capillary GC using a DB-
Wax column. The odorants were then detected by GC eluate sniffing
(GC-O). The flavor dilution (FD) factors of the odorants were
determined by AEDA (17). Before the FD factor measurement, two
panelists repeatedly checked the retention time and the odor quality of
the odorants using each diluted sample (1:64), and then, the FD factor
of the odorants was determined by being detected at the dilution step
by both panelists.
Identification of the Components. Each component was identified
by comparing its Kovats GC retention index (RI), mass spectrum, and
odor quality with those of an authentic compound.

4798 J. Agric. Food Chem., Vol. 54, No. 13, 2006
Kumazawa et al.
Figure 2. Mass chromatogram of the volatile concentrate of a black tea (Dimbula) infusion showing the extracted ion, which is the typical fragment ion
of the homologous series of 4,5-epoxy-2-alkenals [peak 1, cis-4,5-epoxy-(E)-2-decenal; peak 2, trans-4,5-epoxy-(E)-2-decenal].
Figure 3. Mass spectra of cis-4,5-epoxy-(E)-2-decenal (1) and trans-4,5-epoxy-(E)-2-decenal (2), which were obtained from the enriched fraction.
1
Proton Magnetic Resonance Spectrometry (1H NMR). The H
trans-4,5-Epoxy-(E)-2-decenal (2) has already been found to
NMR spectra were recorded in CDCl3 solution using a Bruker
AVANCE 400 spectrometer operating at 400 MHz and using tetra-
methylsilane as the internal standard.
be a potent odorant of many foodstuffs (18-20), and peak 2
can be presumed to correspond to 2 by comparison of the
retention index in the literature (21). The mass spectrum of 2
gave typical fragment ions derived from its structure. On the
other hand, peak 1 also gave similar fragment ions as did peak
RESULTS AND DISCUSSION
2. Therefore, peak 1 is thought to be an isomer of 2 and is
assumed to be cis-4,5-epoxy-(E)-2-decenal (1).
Potent Odorants in Ceylon Black Tea Flavor. The flavor
extract of the Ceylon black tea infusion was prepared by
To confirm the structures of 1 and 2, both epoxy isomers
were synthesized, and identification of the odorants in the black
tea was confirmed by comparison of their retention indices. The
geometric structure of the epoxy and double bond part of the
combining the adsorptive column method and the SAFE
technique. The flavor extract well-reproduced the characteristic
odor of the Ceylon black tea infusions. The AEDA applied to
the volatile fraction, which had been prepared from freshly
filtered Ceylon black tea infusion, revealed 61 odor active peaks
with FD factors between 43 and 47 (Figure 1). Among the
perceived odorants, seven peaks whose higher FD factors (g46)
have been proved to be the most important components of the
Ceylon black tea flavor, and these were identified by comparing
their Kovats indices, mass spectra, and odor quality with those
of the authentic compounds, as detailed in Figure 1. Five
compounds were identified from seven peaks by GC-MS [a,
linalool (floral); b, (E,E)-2,4-decadienal (fatty); c, â-dama-
scenone (sweet); d, hexanoic acid (acidity); and e, vanillin
(vanilla-like)], and these odorants have been shown to be some
of the most important compounds for the other kinds of black
tea cultivars (5-9). In Figure 2, the mass chromatogram of
the volatile concentrate of a Ceylon black tea infusion shows
the extracted ion with m/z 68. Peaks 1 (RI DBWax ) 1984)
and 2 (RI DBWax ) 2001) with the characteristic juicy and
sweet notes were identified as 4,5-epoxy-2-decenals by their
mass spectra obtained from the enriched fraction (Figure 3).
1
synthesized 1 and 2 was verified using the H NMR coupling
constant, and the structure of these isomers was characterized
as cis-epoxy, trans-ene (1) and trans-epoxy, trans-ene (2). The
retention indices of each of the 4,5-epoxy-(E)-2-decenals were
calculated by GC and compared to the peaks of the black tea.
As a result, the synthesized 1 and 2 closely agreed with peaks
1 and 2 in the volatile fraction of the black tea infusion. Of the
two odorants, 1 is reported here for the first time as a component
of the black tea flavor. Odorant 1 was first identified in fresh
field tomatoes (22, 23); however, this epoxydecenal has not yet
been identified in other kinds of aromas arising from natural
sources. Each odorant (1, 2) has a common juicy, sweet, and
metallic note. However, in addition to the common note, 1 has
a fatty note, and 2 has a fresh citrus-like note; thus, the odor of
these isomers slightly differed. Figure 4 shows the amounts of
the epoxydecenal isomers in the different black tea cultivars.
These data indicated that the contents of 1 and 2 differ in every
cultivar, and the amount of these odorants in Dimbula was more

Characterization of Epoxydecenal Isomers in Black Tea
J. Agric. Food Chem., Vol. 54, No. 13, 2006 4799
Table 2. Amounts of the 2,4-Alkadienals in Black Tea (Dimbula)
Infusion
RIa
compound
amount (µg/L)
1406
1414
1471
1506
1569
1601
1668
1712
1770
1820
2,4-hexadienal (isomer)
(E,E)-2,4-hexadienal
2,4-heptadienal (isomer)
(E,E)-2,4-heptadienal
2,4-octadienal (isomer)
(E,E)-2,4-octadienal
2,4-nonadienal (isomer)
(E,E)-2,4-nonadienal
(E,E)-2,4-decadienal (isomer)
(E,E)-2,4-decadienal
1.36b
7.72
27.23b
52.38
traceb
0.99
0.12b
0.87
0.9b
1.76
a Retention index in the DB-Wax column observed for GC-MS. b These values
were estimated by the calibration factor of corresponding 2,4-alkadienals.
octadecenoic acid decomposes by R-cleavage (24). Therefore,
in route A, if the other 2,4-alkadienals are included, the
corresponding 4,5-epoxy-2-alkenals may also be formed, whereas
in route B, 4,5-epoxy-2-decenal could mainly be expected.
The homologous series of 2,4-alkadienals and 4,5-epoxy-2-
alkenals in the volatile fraction of the black tea infusion was
then screened by GC-MS, and the formation pathway of the
epoxydecenal isomers was assumed from both results. In the
black tea infusion, the variety of the detected 2,4-alkadienals
ranged from six to 10 carbons (Table 2). Among these
compounds, the 2,4-heptadienals had the highest content, and
the contents decreased in the order of the 2,4-hexadienals, then
the 2,4-decadienals. However, a considerable amount of the 2,4-
octadienals and 2,4-nonadienals were found in the black tea
infusion. On the basis of the model experiments, all of the 2,4-
alkadienals from six to 10 carbons reacted with the hydroper-
oxides, which were prepared from linoleic acid with soybean
lipoxygenase and produced all of the corresponding 4,5-epoxy-
2-alkenals (data not shown). In other words, if the 4,5-epoxy-
(E)-2-decenal isomers are formed from the 2,4-decadienals as
intermediates, it is expected that the homologous series of the
4,5-epoxy-2-alkenals are detected in the black tea. In Figure
2, the chromatogram recorded with m/z 68, which is the typical
fragment ion of the homologous series of 4,5-epoxy-2-alkenals,
is shown. As a result, only the 4,5-epoxy-2-heptenals and 4,5-
epoxy-(E)-2-decenals were detected in the black tea, while the
other 4,5-epoxy-2-alkenals could not be found. These findings
Figure 4. Amounts of 4,5-epoxy-(E)-2-decenal isomers in the different
black tea cultivars.
than in the other kinds of black teas. Therefore, the epoxydecenal
isomers (1, 2) seem to be responsible for the characteristic flavor
of the Ceylon black tea, especially the juicy and sweet note.
Formation Mechanism of Epoxydecenal Isomers in the
Black Tea. trans-4,5-Epoxy-(E)-2-decenal (2) has been pro-
posed to originate from linoleic acid by oxidation (24, 25). It
has been reported that the tea leaves contain from 3 to 5% fatty
acids, and linoleic acid is one of the principal fatty acids (26).
Therefore, 2 in black tea would be expected to be generated
from the linoleic acid in the tea leaves. On the other hand, the
formation mechanism of cis-4,5-epoxy-(E)-2-decenal (1) has not
yet been clarified. However, it seems likely that the origin and
formation mechanism of 1 in the black tea are similar to that of
2, because, for several black tea cultivars, the contents of 1 have
a close relation to that of 2 (Figure 4). The formation pathway
of 2 formed from linoleic acid by oxidation has already been
reported, and two routes (A and B) have been proposed (Figure
5). Namely, route A is the pathway in which 2,4-decadienal as
the key intermediate reacts with peroxide (25), and route B is
the pathway in which the 12,13-epoxy-9-hydroperoxy-10-
Figure 5. Proposed pathway for the formation of trans-4,5-epoxy-(E)-2-decenal from linoleic acid [according to Gardner and Selke (24) and Gassenmeier
and Schieberle (25)].

4800 J. Agric. Food Chem., Vol. 54, No. 13, 2006
Kumazawa et al.
Table 4. Comparison of Generated Amount of 4,5-Epoxy-(E)-2-decenal
Isomers in Linoleic Acid and Its Hydroperoxides
amount (µg)
nonheated
linoleic
heated
linoleic
acid
odorant
acid
LHa
LHa
cis-4,5-epoxy-(E)-2-decanal
trans-4,5-epoxy-(E)-2-decaal
<0.01
<0.01
<0.01
<0.01
0.13
0.43
2.56
6.53
a Linoleic acid hydroperoxides.
model reactions that simulated the enzyme reaction during the
withering and fermentation process of the black tea production
were carried out using linoleic acid and its hydroperoxides
obtained by the soybean lipoxygenase treatment. As a result, it
was shown that 1 and 2 were generated from linoleic acid and
its hydroperoxides by heating, and it was observed that the
generated amounts of these odorants from the linoleic acid
hydroperoxides were much greater than that of linoleic acid
(Table 4). This finding explained the different amounts of 1
and 2 in the black and green teas and strongly suggested that
the enzyme reaction plays an important role in forming 1 and
2 in the black tea.
Figure 6. Hypothetical generation mechanism of cis- and trans-epoxides.
Table 3. Comparison of 4,5-Epoxy-(E)-2-decenal Isomers in Black Tea
(Dimbula) and Japanese Green Tea (Sen-cha)
FD factor (4n)
black tea green tea
(Dimbula) (Sen-cha) (Dimbula) (Sen-cha)
amount (
µ
g/L)
black tea
green tea
odorant
cis-4,5-epoxy-(E)-2-decanal
trans-4,5-epoxy-(E)-2-decaal
6
7
1
2
0.28
0.60
<0.01
0.02
On the basis of these results, it is suggested that 1 and 2
were generated from linoleic acid contained in the tea leaf during
the manufacturing process of the black tea, and it can be
assumed that the formation mechanism is as follows. First, the
withering and fermentation, which is a characteristic process
of the black tea production, converts linoleic acid to its
hydroperoxide in which the oxygen adds to the 13 carbon at
the S- configuration by the lipoxygenase activity, since it is well-
known that the tea lipoxygenase has a high regio- and
stereospecificity to form the 13(S)-hydroperoxide of linoleic acid
(29, 30). In addition, by heating during the drying process of
the black tea production, the 13(S)-hydroperoxide of linoleic
acid produced 1 and 2 via the cis- and trans-epoxyallylic
radicals. Therefore, further investigation of the stereochemistry
of 1 and 2 in the black tea should serve to clarify the formation
mechanism of these odorants. Thus, the formation of 1 and 2
has a close relation to the manufacturing process; especially,
the enzyme reaction in the black tea seems to be one of the
most important factors for the formation of these isomers.
suggested that the formation of the 4,5-epoxy-(E)-2-decenals
cannot be explained by route A via 2,4-decadienal as the
intermediate, and it presumed that both isomers in the black
tea were indeed formed from linoleic acid in the tea leaves via
the 12,13-epoxy-9-hydroperoxy-10-octadecenoic acid.
In addition, the difference in the content of both isomers could
be recognized, and the content of 1 was less than that of 2.
This chemical behavior regarding the difference in the contents
1 and 2 can be assumed to affect the equilibration of the
alkoxydiene radicals, which are formed from the 13-hydroper-
oxide of linoleic acid as the intermediate. Namely, the alkoxy-
diene radicals, which are capable of forming the extended and
hindered conformers of the carbon 12,13 bond (Figure 6), and
the extended conformer are more dominant than the hindered
conformer. Therefore, it seems that the content of the cis-
epoxyallylic radical was less than that of the trans-epoxyallylic
radical formed from these alkoxydiene radicals. The influence
of the equilibrium of the alkoxydiene radical on the formation
of the epoxide has already been proposed on the basis of the
oxidation mechanism of the lipid (27, 28). Therefore, 1 and 2
in the black tea were expected to be generated from the cis- or
trans-epoxyallylic radicals via the 12,13-epoxy-9-hydroperoxy-
10-octadecenoic acid, which oxidized carbon 9 of epoxyallylic
radicals with O2.
The enzyme reactions during the withering and fermentation
process were characteristic processes of the black tea production.
On the other hand, the green tea leaves inactivated the enzyme
by steaming or parching, and it hardly utilizes the action of the
enzyme. For the flavor formation of the black tea, in particular,
it was pointed out that the enzyme action during the manufac-
turing process is very important (10-14). To clarify the
relationship of the manufacturing method of the black tea and
the formation of the 4,5-epoxy-(E)-2-decenal isomers, a com-
parison of the contents and FD factors of these isomers in the
black and green teas was done. It appeared that the FD factor
and amount of these odorants in the black tea were much greater
in comparison with that of the green tea (Table 3). Therefore,
1 and 2 seem to be characteristic compounds for the black tea
flavor, and it can be presumed that the black tea production
process was very important for the formation of 1 and 2. The
ACKNOWLEDGMENT
We are grateful to S. Fujikawa for the synthesis of the authentic
compounds.
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Received for review February 2, 2006. Revised manuscript received
April 27, 2006. Accepted April 29, 2006.
JF0603127