Identification of Rotundone as a Potent Odor-Active Compound of Several Kinds of Fruits

Article abstract of DOI:10.1021/acs.jafc.7b00929

An investigation of the aromas of grapefruit, orange, apple, and mango revealed the presence of an odor-active compound that gave off a strong woody odor when assessed by gas chromatography-olfactometry. We isolated the compound from a high-boiling fraction of an orange essential oil, and subsequent nuclear magnetic resonance analyses of the isolated compound identified it as rotundone. Mass spectra and retention indices obtained from aroma concentrates of grapefruit, apple, and mango were identical to those of rotundone, which was therefore determined to be the common woody compound in these fruits. Sensory analyses were performed to assess the effects of rotundone on model beverages of the various fruits. It was revealed that rotundone added at even subthreshold levels to model beverages did not confer directly the woody odor, but had significant effects on the overall flavors of the beverages, helping them to better approximate the natural flavors of the fruits.

Full text of DOI:10.1021/acs.jafc.7b00929

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Article
Identification of rotundone as a potent odor-
active compound of several kinds of fruits
Akira Nakanishi, Yusuke Fukushima, Norio Miyazawa, Keisuke
Yoshikawa, Tomoko Maeda, and Yoshiko Kurobayashi
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 18 May 2017
Downloaded from http://pubs.acs.org on May 18, 2017
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Journal of Agricultural and Food Chemistry
Identification of Rotundone as a Potent OdorꢀActive
Compound of Several Kinds of Fruits
Akira Nakanishi,* Yusuke Fukushima, Norio Miyazawa, Keisuke Yoshikawa, Tomoko Maeda, and
Yoshiko Kurobayashi
R&D Center, T. Hasegawa Co., Ltd., 29ꢀ7, Kariyado, Nakaharaꢀku, Kawasakiꢀshi, 211ꢀ0022, Japan
AUTHOR ADDRESS: akira_nakanishi@tꢀhasegawa.co.jp
CORRESPONDING AUTHOR FOOTNOTE.
TEL: +81ꢀ44ꢀ411ꢀ0298; FAX: +81ꢀ44ꢀ434ꢀ5257; Eꢀmail: akira_nakanishi@tꢀhasegawa.co.jp
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ABSTRACT. An investigation of the aromas of grapefruit, orange, apple, and mango revealed the
presence of an odorꢀactive compound that gave off a strong woody odor when assessed by gas
chromatography–olfactometry. We isolated the compound from a highꢀboiling fraction of an orange
essential oil, and subsequent nuclear magnetic resonance analyses of the isolated compound identified it
as rotundone. Mass spectra and retention indices obtained from aroma concentrates of grapefruit, apple,
and mango were identical to those of rotundone, which was therefore determined to be the common
woody compound in these fruits. Sensory analyses were performed to assess the effects of rotundone on
model beverages of the various fruits. It was revealed that rotundone added at even subꢀthreshold levels
to model beverages didn’t confer directly the woody odor, but had significant effects on the overall
flavors of the beverages, helping them to better approximate the natural flavors of the fruits.
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KEYWORDS: rotundone; odorꢀactive compound; woody odor; fruits; grapefruit; orange; apple; mango;
isolation
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INTRODUCTION
Owing to the development of analytical technology, most of the key odorants, which exist even in
trace amounts in foods, have been identified. Recently, our group reported that (6Z,8E)ꢀundecaꢀ6,8,10ꢀ
trienꢀ3ꢀone, (4Z,7Z)ꢀtridecaꢀ4,7ꢀdienal, and cisꢀ3ꢀmethylꢀ4ꢀdecanolide were identified as novel odorꢀ
active compounds in yuzu (Citrus junos), dried bonito, and wasabi (Wasabia japonica) respectively and
these odorants had a great influence on the aroma profiles of these foods.1–3 Since these compounds
were present in only trace amounts in the volatiles of each food, they were difficult to elucidate the
structure, however, by application of repeated fractionations and/or multiꢀdimensional gas
chromatography–mass spectrometry (MDꢀGCꢀMS), clear mass spectra were obtained to propose the
structures, and the identification was successfully achieved by syntheses of the putative compounds.
However, odorants having extremely low odor thresholds are still presented in the aromas of foods in a
concentration that is too low to detect mass spectra of the odorants, and can often play a critical role in
increasing the similarity of food aroma recombinants; the discovery of such highly potent odorants is
still a challenging field of research.4
All kinds of fruits (in this paper, “fruit” indicates “edible fruit”) have distinctive and pleasant aromas
that are complex mixtures of various characteristic odorants, and complete duplication of the aromas of
natural fruits is a very important task for the flavor industry. The aromas of many kinds of fruits have
been analyzed by gas chromatography–olfactometry (GCꢀO) based on the task in our laboratory, and,
through these GCꢀO analyses, it was found that one odorant was commonly detected only by GCꢀO.
Particularly in investigations of the aromas of grapefruit, orange, apple, and mango, this odorant was
very noticeable and gave off a strong woody odor even though its mass spectrum could not be obtained
at all in these fruits (data not shown). This odorant had a different type of woody odor from that of the
odorants previously known to have a woody odor, and the odor closely resembled the aroma of dried
wood chips when assessed by GCꢀO; however, the woody odor has never been sensed directly in fruit
aromas. From the retention indices (2300 in InertCap WAX and 1680 in InertCap 1MS) and the woody
odor quality of the odorant, this odorant may have been rotundone. Rotundone has been identified as an
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odorꢀactive component in patchouli oil,5 Shiraz wine (which is characterized by a pepper aroma),6
Shiraz grape (a special grape used as a raw material for Shiraz wine),6 spices (white and black peppers,
marjoram, oregano, etc.),6 oak aged spirits,7 frankincense oil,8 and cypriol oil.9 To the best of our
knowledge, rotundone has not been detected in fruits other than Shiraz grape. The odor qualities of
rotundone were certainly described as “woody”, but they were mainly characterized as "peppery/spicy"
in the above literatures. However, the unique "peppery/spicy" odors of rotundone were never perceived
from the sniffing port of our GCꢀO analyses of the aromas of the above fruits. From these facts, we
could not conclude to tentatively identify this woody compound as rotundone. Therefore, we were very
interested in the woody odor hidden in the aroma of these fruits, and in this study we aimed to elucidate
the structure of the “woody” unknown and reveal its presence in fruits by obtaining further analytical
data and to clarify its role as an odorꢀactive compound in fruit aromas by sensory analyses.
MATERIALS AND METHODS
Materials. The highꢀboiling fraction of orange essential oil was purchased from R. C. Treatt & Co.
Ltd. (Suffolk, UK). Grapefruit (marsh seedless) and apple (SunꢀFuji) were purchased in a local
supermarket in Kawasaki, Japan, and mango (Alphonso) puree was available commercially from O’will
Corporation (Tokyo, Japan).
Chemicals. The following chemicals were purchased commercially: (−)ꢀguaiol, cobalt(II) 2ꢀ
ethylhexanoate, cobalt(II) acetylacetonate, cobalt(II) naphthenate (SigmaꢀAldrich, St. Louis, USA),
toluene, pyridine, N,Nꢀdimethylꢀ4ꢀaminopyridine, 4ꢀmethylꢀ2ꢀpentanone (Junsei Chemical Co., Ltd.,
Tokyo, Japan), acetic anhydride (Daicel Corporation, Tokyo, Japan), silica gel 60N (spherical neutral,
particle size 63–210 ꢁm, Kanto Chemical Co., Inc., Tokyo, Japan), and Wakogel® Cꢀ100 (Wako Pure
Chemical Industries, Ltd., Osaka, Japan). Gaseous oxygen was generated from an oxygen concentrator
(LFYꢀIꢀ5F, Malus Corporation, Tokyo, Japan). All other reagents and solvents were of analytical grade.
The chemicals used in the sensory analyses were obtained from T. Hasegawa (Kawasaki, Japan).
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Synthesis of Authentic Rotundone. Rotundone [(3S,5R,8S)ꢀ5ꢀisopropenylꢀ3,8ꢀdimethylꢀ3,4,5,6,7,8ꢀ
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hexahydroꢀ1(2H)ꢀazulenone] (1), was synthesized from (−)ꢀguaiol (2), using modified literature
procedures.6 (−)ꢀGuaiol was converted to rotundone by acetylation, allylic oxidation, and pyrolytic
elimination of acetic acid (Figure 1).
2ꢀ[(3S,5R,8S)ꢀ3,8ꢀDimethylꢀ1,2,3,4,5,6,7,8ꢀoctahydroazulenꢀ5ꢀyl]propanꢀ2ꢀyl acetate (3, guaiyl acetate)
A mixture of (−)ꢀguaiol (2, 445 mg, 2.00 mmol), acetic anhydride (225 mg, 2.20 mmol), and N,Nꢀ
dimethylꢀ4ꢀaminopyridine (12.2 mg, 0.100 mmol) was added dropwise to a mixture of pyridine (237 mg,
3.00 mmol) and toluene (5 mL) at 4 °C. After stirring at room temperature for 1.5 h, acetic anhydride
(5.17 g, 50.6 mmol), N,Nꢀdimethylꢀ4ꢀaminopyridine (598 mg, 4.90 mmol), and pyridine (3.08 g, 39.0
mmol) were added again and stirred at 50 °C for 6 h. The reaction mixture was poured into 2N HCl and
extracted with ethyl acetate. The organic layer was washed with saturated aq. NaHCO3 and brine, dried
over MgSO4, and concentrated in vacuo. The residue (731 mg) was purified by silica gel
chromatography (silica gel 60N: 10 g, hexane/ethyl acetate = 100/1, v/v) to give guaiyl acetate (3,
yellow oil, 453 mg, yield 86%).
2ꢀ[(3S,5R,8S)ꢀ3,8ꢀDimethylꢀ3,4,5,6,7,8ꢀhexahydroꢀ1(2H)ꢀazulenonꢀ5ꢀyl]propanꢀ2ꢀyl acetate (4)
Oxygen gas was bubbled into a mixture of guaiyl acetate (3, 353 mg, 1.33 mmol), cobalt(II) 2ꢀ
ethylhexanoate (22.9 mg), cobalt(II) acetylacetonate (20.0 mg), cobalt(II) naphthenate (48.5 mg), and 4ꢀ
methylꢀ2ꢀpentanone (10 mL) at 70 °C for 2.5 h. The reaction mixture was cooled to room temperature,
poured into saturated aq. Na2S2O3, and extracted with ethyl acetate. The organic layer was washed with
water and brine, dried over MgSO4, and concentrated in vacuo. The residue (571 mg) was purified by
silica gel chromatography (silica gel 60N: 20 g, hexane/ethyl acetate = 25/1, v/v) to give 2ꢀ[(3S,5R,8S)ꢀ
3,8ꢀdimethylꢀ3,4,5,6,7,8ꢀhexahydroꢀ1(2H)ꢀazulenonꢀ5ꢀyl]propanꢀ2ꢀyl acetate (4, yellow oil, 45.2 mg,
yield 12%).
(3S,5R,8S)ꢀ5ꢀIsopropenylꢀ3,8ꢀdimethylꢀ3,4,5,6,7,8ꢀhexahydroꢀ1(2H)ꢀazulenone (1, rotundone)
2ꢀ[(3S,5R,8S)ꢀ3,8ꢀDimethylꢀ3,4,5,6,7,8ꢀhexahydroꢀ1(2H)ꢀazulenonꢀ5ꢀyl]propanꢀ2ꢀyl acetate (4, 47.7
mg, 0.171 mmol) was agitated using a magnetic stirring bar of appropriately small dimension (2 mm dia.
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by 5 mm length) at 280–300 °C for 1 h. The reaction mixture was cooled to room temperature and
purified by silica gel chromatography (silica gel 60N: 1.0 g, hexane/ethyl acetate = 50/1, v/v) to give
rotundone (1, yellow oil, 15.0 mg, yield 40%).
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The 1H and 13C NMR spectra and mass spectrum in the EI mode were identical to those reported.4 The
retention indices of rotundone in a polar column (InertCap WAX) and a nonpolar column (InertCap
1MS) by GCꢀMS were calculated as 2300 and 1680, respectively.
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Elucidation of the Woody Odor Compound. Depending on the sample amount, fractionation was
sequentially conducted on the highꢀboiling fraction of orange essential oil by vacuum distillation, silica
gel column chromatography, and preparative HPLC.
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Vacuum Distillation. The highꢀboiling fraction of orange essential oil (801.3 g) was distilled under
reduced pressure to obtain distillate fractions (Fr.iꢀ1: 136 °C / 1.0 kPa, 282.4 g; Fr.iꢀ2: 136 °C / 1.0 kPa,
96.8 g; Fr.iꢀ3: 136–157 °C / 0.9 kPa, 165.4 g; Fr.iꢀ4: 157–169 °C / 0.9 kPa, 85.6 g; Fr.iꢀ5: 169–172 °C /
0.9 kPa, 18.4 g) and residue (140.8 g). The presence of the target woody odor compound was
determined by GCꢀO analysis. Accordingly, fractions iꢀ3 through iꢀ5 as well as the residue were
combined and used for the next step.
Silica Gel Column Chromatography. The combined fractions and residue (350.0 g) obtained by
distillation was fractionated by silica gel chromatography (silica gel 60N: 1800 g, hexane/ethyl acetate =
3/1, v/v) into five fractions (Fr.iiꢀ1–5), each of which was evaporated and subjected to GCꢀO. A 112.8 g
sample of concentrated effluent was collected from Fr.iiꢀ3, which was confirmed to have the target
aroma by GCꢀO. This concentrated effluent (112.8 g) was further fractionated by silica gel
chromatography (silica gel 60N: 2000 g, hexane/ethyl acetate = 5/1, v/v) into seven fractions (Fr.iiiꢀ1–
7), each of which was evaporated and subjected to GCꢀO. A 68.0 g sample of concentrated effluent was
collected from Fr.iiiꢀ3–5, which was confirmed to have the target aroma. A 38.5 g sample of the
concentrated effluent was further fractionated by silica gel chromatography (silica gel 60N: 1750 g,
toluene/ethyl acetate = 100/1–75/1, v/v), and fractions with Rf values of 0.58 checked by thin layer
chromatography (toluene/ethyl acetate = 4/1, v/v) were collected. A total of 10.0 g concentrated sample
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was obtained from their fractions and confirmed to have the target aroma by GCꢀO. Even for these
fractionations by silica gel chromatography, a clear mass spectrum of the target woody aroma compound
was not obtained.
Preparative HPLC. Two successive HPLC fractionations were then performed on the concentrate of the
final fraction obtained by silica gel column chromatography. For each HPLC fractionation, fractions
featuring the characteristic woody odor of the target compound from paper smelling strips were
collected, and the HPLC fraction (1.3 mg) was obtained finally. GCꢀMS analysis of the HPLC fraction
indicated that the main component was the target compound (36.7%). HRMS (EI) calculated for
C15H22O 218.16707, found 218.16680. These analytical data suggest that the target compound is
rotundone, as they match those of authentic rotundone.
Identification of Rotundone in Grapefruit, Apple, and Mango.
Grapefruit. The flavedo layers (the outer colored layer of the pericarp of a citrus fruit) of 25 fresh white
grapefruits were carefully peeled off using a knife. The peel (1.20 kg) was then cut into pieces and
extracted with pentane (2.4 L) for 30 min while stirring vigorously. After separating the pentane solution
by decantation, the residue was extracted again with pentane (1.2 L). After removing the fibrous parts of
the peel by decantation, the two obtained pentane fractions were combined. The collected pentane
solution was dried over Na2SO4 and concentrated to ca. 200 mL by distilling off the solvent over a
Vigreux column at 43 °C. The concentrate was distilled using the solvent assisted flavor evaporation
(SAFE) method10 at 35 °C. The distillate was concentrated by distilling off the solvent over a Vigreux
column at 43 °C to yield 50.9 g of white grapefruit peel oil (the pentane concentration of the peel oil was
68.0%, as determined by GCꢀFID). The peel oil (12.0 g) was concentrated in vacuo, and the residue (4.4
g) was pipetted into the top of a glass column filled with 40 g of silica gel (Wakogel® Cꢀ100) in hexane.
Chromatography was performed using hexane (900 mL) to give a nonpolar fraction, followed by ethyl
acetate (900 mL) to give a polar fraction. The polar fraction was concentrated in vacuo to yield 44.3 mg
of a polar part. Two successive HPLC fractionations were performed on the polar part. The HPLC
fraction (10.7 mg, diethyl ether solution) was subjected to GCꢀO to obtain the mass spectrum, retention
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indices on two columns (InertCap WAX and InertCap 1MS), and odor quality of rotundone. These
analytical data obtained with GCꢀO were matched with those of the authentic rotundone analyzed under
the same conditions.
Apple. Ten apples were cut into four pieces, the stems and cores were removed, and the remaining
material was grated. Diethyl ether (1.6 L) and NaCl (240 g) were added to the grated apples (2400 g),
and the mixture was shaken. After leaving overnight at 0 °C, the diethyl ether solution was separated by
decantation and concentrated to ca. 100 mL. The concentrate was distilled using the SAFE method at 60
°C. The distillate was concentrated in vacuo to obtain an apple aroma concentrate (25.5 mg). Two
successive HPLC fractionations were performed on the aroma concentrate. The HPLC fraction (7.8 mg,
diethyl ether solution) was subjected to MDꢀGCꢀMS/O to obtain the mass spectrum, retention indices on
two columns (InertCap WAX and InertCap 1MS), and odor quality of rotundone. These analytical data
obtained with GCꢀO were matched with those of the authentic rotundone analyzed under the same
conditions.
Mango. Sodium sulfate (1000 g) was added to mango puree (1000 g) at room temperature. After mixing
sufficiently, diethyl ether (1.5 L) was added and the mixture was stirred at 0 °C for 1 h. The mixture was
filtered and the diethyl ether solution was concentrated to ca. 200 mL by distilling off the solvent over a
Vigreux column at 43 °C. Half of the concentrate was distilled using the SAFE method at 60 °C. The
distillate was concentrated in vacuo to obtain a mango aroma concentrate (20.4 mg). Two successive
HPLC fractionations were performed on the aroma concentrate. The HPLC fraction (11.8 mg, diethyl
ether solution) was subjected to GCꢀO to obtain the mass spectrum, retention indices on two columns
(InertCap WAX and InertCap 1MS), and odor quality of rotundone. These analytical data obtained with
GCꢀO were matched with those of the authentic rotundone analyzed under the same conditions.
High Performance Liquid Chromatography (HPLC) Fractionation. Two instrument systems were
used in this study. Instrument system A comprised a Waters 600 controller, a Waters 600 pump, a
Waters 2707 autosampler, and a Waters 2487 dual λ absorbance detector (Waters Corporation, Milford,
USA). Instrument system B comprised a Shimadzu LCꢀ20AT, a Shimadzu SILꢀ20AC, a Shimadzu
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CTOꢀ20A, a Shimadzu SPDꢀM20A, and a Shimadzu CBMꢀ20A (Shimadzu Corporation, Kyoto, Japan).
In both elucidation of the woody odor compound and identification of rotundone mentioned below, two
successive HPLC fractionations were performed using these instrument systems.
(a) Elucidation of the woody odor compound
The first HPLC conditions were as follows: an Inertsil ODSꢀ3 column (5 ꢁm particle size, 250 mm × 4.6
mm i.d., GL Sciences Co.) was fitted in instrument system A. The injected sample was diluted to ca.
20% with methanol. The gradient elution was set as follows: 0–10 min, methanol/water = 70/30 →
80/20; 10–15 min, methanol/water = 90/10. The solvent flow rate was 2.0 mL/min, and the column
temperature was 45 °C. At retention times from 9.3 to 10.2 min, the eluent was collected, and then
methanol was removed from it by evaporation. NaCl (15% w/w) was added to the residue before
extraction with diethyl ether, and the diethyl ether layer was then dried over MgSO4 and concentrated in
vacuo. A second HPLC fractionation was performed on the concentrate. The second HPLC conditions
were as follows: an Inertsil 80A column (5 ꢁm particle size, 250 mm × 4.6 mm i.d., GL Sciences Co.)
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and a Sunrise C28 column (5 ꢁm particle size, 250 mm × 4.6 mm i.d., ChromaNik Technologies Inc.,
Osaka, Japan) were fitted in series in instrument system B. A ca. 10% acetonitrile solution of the above
concentrate was injected. The isocratic elution was set as follows: 0–20 min, acetonitrile/water = 80/20.
The solvent flow rate was 1.0 mL/min, and the column temperature was 40 °C. At retention times from
13.0 to 13.6 min, the eluent was collected and then concentrated using the same procedure as described
above.
(b) Identification of rotundone in grapefruit, mango, and apple
The first HPLC conditions were as follows: an Inertsil ODSꢀ3 column (5 ꢁm particle size, 250 mm × 4.6
mm i.d., GL Sciences Co.) was fitted in instrument system B. A ca. 20% methanol solution of sample
was injected. The gradient elution was set as follows: 0–10 min, methanol/water = 70/30 → 80/20; 10–
15 min, methanol/water = 90/10. The solvent flow rate was 2.0 mL/min, and the column temperature
was 45 °C. At retention times from 9.3 to 10.2 min, the eluent was collected and then concentrated using
the same procedure as described above. A second HPLC fractionation was performed on the concentrate.
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The second HPLC conditions were as follows: a Sunrise C28 column (5 ꢁm particle size, 250 mm × 4.6
mm i.d., ChromaNik Technologies Inc.) was fitted in series in instrument system B. A ca. 10%
acetonitrile solution of the above concentrate was injected. The isocratic elution was set as follows: 0–
20 min, acetonitrile/water = 80/20. The solvent flow rate was 1.0 mL/min, and the column temperature
was 40 °C. At retention times from 6.2 to 7.2 min, the eluent was collected and then concentrated using
the same procedure as described above.
Gas Chromatography–Mass Spectrometry (GC-MS). GCꢀMS analyses were performed using an
Agilent 7890 gas chromatograph combined with an Agilent MSD5975 quadrupole mass spectrometer
(Agilent Technologies, Santa Clara, USA) and a flame ionization detector (FID) equipped with an
InertCap WAX capillary column (0.25 mm i.d. × 60 m, 0.25 ꢁm film thickness, GL Sciences Co., Tokyo,
Japan). The effluent of the column at the end of the capillary was divided into two branches and routed
by deactivated fused silica capillaries to the mass spectrometer and the FID. The injection port was held
at 250 °C. The split ratio was 50:1, and 1 ꢁL of sample was injected. The oven temperature was held at
40 °C for the first 3 min and then increased to 230 °C at a rate of 3 °C/min, with a constant helium
carrier gas flow of 1.0 mL/min. Mass spectra in the electron impact (EI) mode were recorded at 70 eV
ionization energy. The linear retention indices of the compounds were calculated from the retention
times of nꢀalkanes. The purities of the synthesized compounds were calculated by integration of the
chromatogram obtained using the FID.
Gas Chromatography–Olfactometry (GC-O). GCꢀO analyses were performed using an Agilent
7890 GC combined with an Agilent MSD5975 quadrupole mass spectrometer and a Gerstel ODP3
sniffing port (Gerstel GmbH & Co. KG, Mülheim an der Ruhr, Germany) equipped with an InertCap
WAX capillary column (0.25 mm i.d. × 60 m, 0.25 ꢁm film thickness, GL Sciences Co.). The effluent of
the column at the end of the capillary was divided into two branches and routed by deactivated fused
silica capillaries to the mass spectrometer and the sniffing port. The sample volume, split ratio, injection
temperature, oven temperature program, carrier gas, and flow rate were all the same as those used for the
GCꢀMS analysis as described above. To compare with authentic rotundone for identification, an
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InertCap 1MS capillary column (0.25 mm i.d. × 60 m, 0.25 ꢁm film thickness, GL Sciences Co.) was
also used. At that time, the oven temperature was held at 40 °C for the first 3 min and then increased to
250 °C at a rate of 5 °C/min.
Multidimensional Gas Chromatography–Mass Spectrometry/Olfactometry (MD-GC-MS/O).
MDꢀGCꢀMS/O analyses were performed using an Agilent 7890 GC combined with a Gerstel cooled
injection system CISꢀ4 and a FID, connected to an Agilent 7890 GC combined with a 5975 quadrupole
mass spectrometer and a Gerstel ODP3 sniffing port equipped with a Gerstel multicolumn switching
system. The effluent of the first column (InertCap WAX capillary column, 0.32 mm i.d. × 60 m, 0.25
ꢁm film thickness, GL Sciences Co.) at the end of the capillary was divided into two branches and
routed by deactivated fused silica capillaries to the FID and to the column switching device, where the
compounds eluted from the first column could be transferred directly into the second column (InertCap
1MS capillary column, 0.25 mm i.d. × 60 m, 0.25 ꢁm film thickness, GL Sciences Co.). The effluent of
the second column at the end of the capillary was divided into two branches and routed by deactivated
fused silica capillaries to the mass detector and sniffing port. The sample was injected in 1 ꢁL volumes
in splitless mode. The injection temperature was held at 10 °C for the first 0.5 min and then increased to
250 °C at a rate of 12 °C/s. The oven temperature of the first GC was maintained at 40 °C for the first 5
min and then increased to 190 °C at a rate of 10 °C/min and held for 20 min, with a constant helium
carrier gas flow of 7.5 mL/min. At a target retention time (24.1–24.5 min), the effluent was transferred
to a cold trap at −50 °C. After 0.5 min, the effluent was eliminated again, whereupon the trapped
material was heated to 250 °C and then directed to the second column. The oven temperature of the
second GC was maintained at 40 °C for the initial 3 min and then increased to 280 °C at a rate of 10
°C/min. The ionization mode was the same as in the GCꢀMS analyses as described above.
1
13
Nuclear Magnetic Resonance (NMR) Spectra. H and C NMR, heteronuclear multiple quantum
correlation (HMQC), and heteronuclear multiple bond correlation (HMBC) experiments were performed
using a JNMꢀECX400 spectrometer (JEOL Ltd., Tokyo, Japan). Using CDCl3 as the solvent, chemical
shifts (δ) were measured relative to tetramethylsilane, which was used as an internal standard (δ = 0.00
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ppm). The chemical shifts and coupling constants (J) are expressed in parts per million (ppm) and hertz
(Hz), respectively.
High Resolution Mass Spectra (HRMS). HRMS were recorded using an AccuTOF GCv 4G (JEOL
Ltd., Tokyo, Japan) equipped with an InertCap WAX capillary column (0.25 mm i.d. × 60 m, 0.25 ꢁm
film thickness, GL Sciences Co.). The injection conditions, oven temperature program, carrier gas, and
flow rate were all the same as in the GCꢀMS analyses as described above.
Sensory Analyses. The panel comprised 10–13 panelists, all of whom were employees of the R&D
Center of T. Hasegawa Co., Ltd. They were trained to recognize and rate intensities of aromas with
about 100 odorous chemicals and raw materials. The attributes (grapefruit: sweet, sour, bitter, fresh,
juicy, peely, complex, and discordant; orange: sweet, sour, floral, fresh, juicy, peely, complex, and
discordant; apple: sweet, sour, green, fruity, pulpy, complex, and discordant; mango: sweet, sulfurous,
green, fruity, metallic, ripe, complex, and discordant) were agreed upon in preliminary sessions where
the panelists evaluated natural fruits (grapefruit, orange, apple, and mango). In these attributes,
“complex” and “discordant” were defined as “complex flavor of natural fruits” and “unpleasant taste or
artificial flavor (unnatural)” by the panelists.
Two types of model beverages for each fruit aroma were evaluated: one comprised a taste solution
(6.5% of high fructose corn syrup and 0.1% of citric acid in water) with each fruit aroma reconstitute
prepared as described below, and the other comprised a taste solution with the same aroma reconstitute
and rotundone (5 ng/kg).
Each panel was presented with a set of test samples coded by a random threeꢀdigit number with
instructions to taste each sample and rate the intensity according to each attribute using a fiveꢀpoint
linear scale from 1 (none) to 5 (very strong). The results were averaged for each attribute and plotted on
a spider chart. The evaluation was conducted in a quiet room maintained at 25 °C.
The differences among the average scores of the evaluated samples were compared using tꢀtests. On
the Tukey’s multiple comparison tests, the standard error measurement and least significant differences
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for each attribute were calculated (p < 0.05, 0.01) and compared with the differences between the
evaluated samples.11–16
Aroma reconstitutes. The fruit aroma reconstitutes were prepared based on quantitative data reported
in the literature.17–20 The components of each reconstitute and their concentrations in model beverages
are shown in Table 1.
RESULTS AND DISCUSSION
Elucidation of the Woody Odor Compound. To elucidate the “woody” compound detected in the
aroma of grapefruit, orange, apple, and mango, we aimed to obtain the mass spectrum of the compound
and isolate sufficient amounts of the compound for NMR experiments if possible. Because the target
“woody” compound existed at very low concentrations even in the aroma concentrates of these fruits, a
huge amount of these fruits would be necessary to isolate this compound. Therefore, we searched for
substitute resources that were readily available. As the target has relatively higher retention indices,
2300 in the polar column and 1680 in the nonpolar column, we thought it should be a sesquiterpenoid,
which usually has a higher boiling point. Hence, we noticed a highꢀboiling fraction of an orange
essential oil that was abundant in sesquiterpenoids and commercially available. Unfortunately, the target
woody odor could not be detected in the highꢀboiling fraction by mass spectrometry, but it was strongly
detectable in olfactometry, so we decided to concentrate it from the material. Despite the application of
fractionation by vacuum distillation and repeated silica gel column chromatography to the material, even
a clear mass spectrum of the target compound could not be obtained. However, the application of
fractionation by preparative HPLC resulted in the target compound being successfully purified to show a
clear mass spectrum and the final purity was improved to 36.7%, as determined by GCꢀFID (Figure 2).
Fortunately, despite this relatively low purity, sufficient analytical data for structural analysis could be
obtained because no single impurity exceeding 10% contaminated the target compound. Based on
HRMS and NMR data, the isolated compound was assumed to be rotundone. It was possible to
synthesize authentic rotundone from commercially available guaiol, although the yield of the oxidation
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step was very low (Figure 1). Finally, the target “woody” compound was definitively identified as
rotundone by matching its mass spectrum, NMR data, retention index, and odor quality with those of
authentic rotundone (Figure 3).
Rotundone has been identified as a natural sesquiterpenoid in essential oils of nut grass weed
(Cyperus rotundus),21 agarwood,22–24 Aquilaris tree,25 and tubers of nagarmotha (cypriol, Cyperus
scariosus),26 besides its identification as an odorꢀactive component.5–9 Based on these reports, rotundone
could be isolated and its NMR data could be obtained from nut grass weed and agarwood in which
rotundone was relatively abundant,21,22 however it was difficult to obtain even a clear mass spectrum in
natural products such as Shiraz wine, black peppercorn, oak aged spirit, and frankincense, in which
rotundone was previously identified as an odorꢀactive component.5–9 The first complete identification of
rotundone in fruits, including its mass spectrum, retention index, odor quality, and NMR data (which
were more difficult to obtain than a mass spectrum), was performed in this study.
Identification of Rotundone in Grapefruit, Apple, and Mango. The next question was whether
this compound was actually present in grapefruit, apple, and mango. To confirm this, it is necessary to
obtain mass spectrum of rotundone from each fruit. In the above study, it was found that preparative
HPLC is a useful tool for purification, so we decided to apply this method this time as well. In the case
of grapefruit and mango, clear mass spectra of rotundone were obtained simply by using GCꢀMS with a
polar column after HPLC fractionation. On the other hand, a clear mass spectrum was not obtained for
apple aroma concentrate even after HPLC fractionation, because the target compound spectrum
completely overlapped with the spectra of other compounds in GCꢀMS with both polar and nonpolar
columns. A clear mass spectrum was obtained successfully without further concentration by using MDꢀ
GCꢀMS/O. Finally, by matching the mass spectra, retention indices on two columns of different polarity,
and odor qualities, the unknown with a woody note in grapefruit, apple, and mango was determined to
be rotundone.
The observed woody odor in this study differed from what was known so far, by means of odor
quality. Hinoki (Japanese cypress, Chamaecyparis obtusa), which is a very highꢀquality timber and has
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been used as a building material in Japan, was the right fit to adequately demonstrate this difference.
The hinokiꢀlike woody odor could be smelled definitely with the peppery/spicy odor from authentic
rotundone using paper smelling strips. The various odor descriptions of rotundone such as peppery/spicy
and woody are caused by differences in rotundone concentration as mentioned in the literatures.6,8 The
odor of rotundone is difficult to detect at higher concentrations, but the peppery/spicy and woody notes
are detected at low to medium concentrations. In this study, the sole woody note that resembles hinoki
aroma could be detected at very low concentrations, particularly when perceived at the sniffing port on
GCꢀO.
Sensory Analyses. Rotundone has been reported to be a key peppery odorant in the Shiraz grape, the
aroma of which is characterized as peppery,6 however, until now the effect of rotundone on the aroma of
other fruits was not known. It was reported that rotundone has a quite low odor threshold (8 ng/kg in
water);5 however the hinokiꢀlike woody odor of rotundone has never been directly detected in these
fruits aromas. Therefore, we supposed that the concentrations of rotundone in these fruits were near the
threshold. We have previously reported that odor components with relatively low odor thresholds can
exhibit flavorꢀmodifying effects at around or below their odor threshold.2 Sensory analyses were
therefore performed to assess the effects of rotundone on the aromas of grapefruit, orange, apple, and
mango at a subꢀthreshold level by tasting model beverages (Figure 4). Two types of model beverage
were prepared for each fruit aroma. One comprised a taste solution (6.5% high fructose corn syrup and
0.1% citric acid in water) with each fruit aroma reconstitute, and the other comprised a taste solution
with the same aroma reconstitute and rotundone (5 ng/kg). Panelists evaluated these model beverages
using the paired comparison method. It was found that even when added at a subꢀthreshold level,
rotundone didn’t confer directly its characteristic woody odor, but had significant effects on several
flavors in the model beverages containing fruit aroma reconstitutes. In particular, it was found that after
addition of rotundone, the overall flavors approximated the natural flavors of the fruits owing to
decreases of “discordant” defined as “unpleasant taste or artificial flavor (unnatural)” and increases of
“complex” defined as “complex flavor of natural fruits”. From these sensory analyses results, it is
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expected that by adding a small amount of rotundone, it is possible to obtain more preferable beverages,
which have flavors similar to those of natural fruits.
In conclusion, rotundone was identified as a potent aroma compound in grapefruit, orange, apple, and
mango. Rotundone added at even subꢀthreshold levels to model beverages comprising reconstituted fruit
aromas approximated the whole natural flavors of these fruits. This is the first study to identify
rotundone as an odorꢀactive component in popular fruits. Furthermore, although identification has not
yet been confirmed, rotundone was tentatively identified by matching the retention indices and odor
qualities in many other fruits such as lemon, lime, blood orange, mandarin orange, banana, strawberry,
Muscat grape, melon, watermelon, and Japanese pear (data not shown). Our findings demonstrate that
rotundone is widely distributed in various kinds of fruits, and it contributes significantly to their aromas.
ABBREVIATIONS USED
VSC, volatile sulfur compound; GCꢀO, gas chromatography–olfactometry; GCꢀMS, gas
chromatography–mass spectrometry; EI, electron impact; FID, flame ionization detector; MDꢀGCꢀMS/O,
multidimensional gas chromatography–mass spectrometry/olfactometry; NMR, nuclear magnetic
resonance; HMQC, heteronuclear multiple quantum correlation; HMBC, heteronuclear multiple bond
correlation; HRMS, high resolution mass spectra; HPLC, high performance liquid chromatography.
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REFERENCES
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(1) Miyazawa, N.; Tomita, N.; Kurobayashi, Y.; Nakanishi, A.; Ohkubo, Y.; Maeda, T.; Fujita, A. Novel
character impact compounds in yuzu (Citrus junos Sieb. ex Tanaka) peel oil. J. Agric. Food Chem. 2009,
57, 1990–1996.
(2) Saito, T.; Shiibashi, H.; Myoga, H.; Haraguchi, K.; Masuda, Y.; Kurobayashi, Y.; Nammoku, T.;
Yamazaki, H.; Nakamura, M.; Fushiki, T. Aroma compounds contributing to dried bonito. Nippon
Shokuhin Kagaku Kogaku Kaishi 2014, 61, 519–527.
(3) Nakanishi, A.; Miyazawa, N.; Haraguchi, K.; Watanabe, H.; Kurobayashi, Y.; Nammoku, T.
Determination of the absolute configuration of a novel odourꢀactive lactone, cisꢀ3ꢀmethylꢀ4ꢀdecenolide,
in wasabi (Wasabia japonica Matsum.). Flavour Frag. J. 2014, 29, 220–227.
(4) Dunkel, A.; Steinhaus, M.; Kotthoff, M.; Nowak, B.; Krautwurst, D.; Schieberle, P.; Hofmann, T.
Nature’s chemical signatures in human olfaction: A foodborne perspective for future biotechnology.
Angew. Chem., Int. Ed. 2014, 53, 7124–7143.
(5) Kaiser, R. Agarwood – the wood of the God. In Meaningful Scents around the World. Olfactory,
Chemical, Biological and Cultural Considerations; Kisakürek, M. V., Eds.; Verlag Helvetica Chimica
Acta: Zürich, WileyꢀVCH: Weinheim, 2006; pp 59–64.
(6) Wood, C.; Siebert, T. E.; Parker, M.; Capone, D. L.; Elsey, G. M.; Pollnitz, A. P.; Eggers, M.; Meier,
M.; Vössing, T.; Widder, S.; Krammer, G.; Sefton, M. A.; Herderich, M. J. From wine to pepper:
rotundone, an obscure sesquiterpene, is a potent spicy aroma compound. J. Agric. Food Chem. 2008, 56,
3738–3744.
(7) Genthner, E. R. Identification of rotundone as an important contributor to the flavor of oak aged
spirits. Ph.D. Dissertation, University of Illinois at UrbanaꢀChampaign, 2014.
(8) Niebler, J.; Zhuravlova, K.; Minceva, M.; Buettner, A. Fragrant sesquiterpene ketones as trace
constituents in frankincense volatile oil of Boswellia sacra. J. Nat. Prod. 2016, 79, 1160–1164.
17
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397
398
399
400
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405
406
407
408
409
410
411
412
413
414
415
416
417
418
(9) Clery, R. A.; Cason, J. R. L.; Zelenay, V. Constituents of cypriol oil (Cyperus scariosus R. Br.): Nꢀ
containing molecules and key aroma components. J. Agric. Food Chem. 2016, 64, 4566–4573.
(10) 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.
(11) Aishima, T. Laboratory methods for sensory analysis of food: part 1. J. Jpn. Soc. Food Sci. Technol.
2001, 48, 311–320.
(12) Aishima, T. Laboratory methods for sensory analysis of food: part 2. J. Jpn. Soc. Food Sci. Technol.
2001, 48, 378–392.
(13) Aishima, T. Laboratory methods for sensory analysis of food: part 3. J. Jpn. Soc. Food Sci. Technol.
2001, 48, 453–466.
(14) Aishima, T. Laboratory methods for sensory analysis of food: part 4. J. Jpn. Soc. Food Sci. Technol.
2001, 48, 539–548.
(15) Aishima, T. Laboratory methods for sensory analysis of food: part 5. J. Jpn. Soc. Food Sci. Technol.
2001, 48, 637–642.
(16) Aishima, T. Laboratory methods for sensory analysis of food: part 6. J. Jpn. Soc. Food Sci. Technol.
2001, 48, 697–703.
(17) Buettner, A.; Schieberle, P. Evaluation of key aroma compounds in handꢀsqueezed grapefruit juice
(Citrus paradise Macfayden) by quantitation and flavor reconstitution experiments. J. Agric. Food
Chem. 2001, 49, 1358–1363.
(18) Buettner, A.; Schieberle, P. Evaluation of aroma difference between handꢀsqueezed juice from
valencia late and navel oranges by quantitation of key odorants and flavor reconstitution experiments. J.
Agric. Food Chem. 2001, 49, 2387–2394.
(19) Steinhaus, M.; Bogen, J.; Schieberle, P. Key aroma compounds in apple juice – changes during
juice concentration. In Flavour science: Recent Advances and Trends; Bredie, W. L. P., Peterson, M. A.,
Eds.; Elsevier: Amsterdam, The Netherlands, 2006, pp189–192.
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(20) Boonbumrung, S.; Tamura, H.; Mookdasanit, J.; Nakamoto, H.; Ishihara, M.; Yoshizawa, T.;
Varanyanond, W. Characteristic aroma components of the volatile oil of yellow keaw mango fruits
determined by limited odor unit method. Food Sci. Technol. Res. 2001, 7, 200–206.
(21) Kapadia, V. H.; Naik V. G.; Wadia, M. S.; Dev, S. Sesquiterpenoids from the essential oil of
Cyperus rotundus. Tetrahedron Lett. 1967, 47, 4661–4667.
(22) Ishihara, M.; Tsuneya, T.; Uneyama, K. Guaiane sesquiterpenes from agarwood. Phytochemistry
1991, 30, 3343–3347.
(23) Ishihara, M.; Tsuneya, T.; Uneyama, K. Components of the volatile concentrate of agarwood. J.
Essent. Oil Res. 1993, 5, 283–289.
(24) Naef, R. The volatile and semiꢀvolatile constituents of agarwood, the infected heartwood of
Aquilaria species. Flavour Frag. J. 2011, 26, 73–89.
(25) Tajuddin, S. N.; Yusoff, M. M. Chemical composition of volatile oils of Aquilaria malaccensis
(Thymelaeaceae) from Malaysia. Nat. Prod. Commun. 2010, 5, 1965–1968.
(26) Pandey, A. K.; Chowdhury, A. R. Essential oil of Cyperus scariosus R. Br. tubers from Central
India. Indian Perfum. 2002, 46, 325–328.
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Figure legends
Figure 1. Synthesis of authentic rotundone.
Figure 2. FID chromatogram of the isolated “woody” compound from the highꢀboiling fraction of an
orange oil.
Figure 3. Comparison of mass spectra and 1H NMR spectra of the isolated “woody” compound and that
of authentic rotundone.
Figure 4. Spider charts of sensory analyses of rotundone in fruit model beverages.
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Table 1. Concentration of components used fruit aroma reconstitutes
concentration (ꢁg/kg)
No. compound
grapefruit
orange
apple
mango
940
1
2
3
4
5
6
7
8
9
acetaldehyde
limonene
ethyl 3ꢀhydroxyhexanoate
(Z)ꢀhexꢀ3ꢀenal
myrcene
decanal
linalool
ethyl butanoate
vanillin
6150
2308
117
108
94
8305
85598
1136
187
594
45
81
1192
67
11
89
76
70
69
160
11
3.4
10 αꢀpinene
11 hexanal
12 octanal
42
33
32
308
197
25
1500
4305
13 nonanal
9.3
13
14 ethyl 2ꢀmethylpropanoate
15 ethyl hexanoate
16 ethyl 2ꢀmethylbutanoate
17 transꢀ4,5ꢀepoxyꢀ(E)ꢀdecꢀ2ꢀenal
5.8
4.3
3.9
3.1
8.8
63
48
4.3
280
33
3,6ꢀdimethylꢀ3a,4,5,7aꢀtetrahydroꢀ1ꢀbenzofuranꢀ
2(3H)ꢀone
18
1.1
0.8
19 (E,E)ꢀdecaꢀ2,4ꢀdienal
20 octꢀ1ꢀenꢀ3ꢀone
21 4ꢀsulfanylꢀ4ꢀmethylpentanꢀ2ꢀone
22 (E)ꢀnonꢀ2ꢀenal
23 3ꢀmethylsulfanylpropanal
24 1ꢀpꢀmentheneꢀ8ꢀthiol
25 3ꢀmethylbutanol
26 2ꢀmethylbutanol
27 butanoic acid
1.0
0.8
0.8
0.5
0.2
0.01
1.2
4.1
0.27
0.6
0.4
639
270
74
12330
7.9
28 hexanꢀ1ꢀol
29 butanꢀ1ꢀol
30 (E)ꢀhexꢀ2ꢀenal
31 βꢀdamascenone
32 dimethyl sulfide
33 terpinolene
34 linalool oxide
35 2,5ꢀdimethylꢀ4ꢀmethoxyꢀ3(2H)ꢀfuranone
36 δꢀ3ꢀcarene
37 ethyl octanoate
38 γꢀterpinene
39 ethyl decanoate
40 pꢀmethylacetophenone
41 βꢀionone
1450
900
306
4.6
1.3
23560
1710
1660
1260
610
530
140
140
27
42 (E,Z)ꢀnonaꢀ2,6ꢀdienal
19
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Figure 1.
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Figure 2.
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Figure 3.
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Figure 4.
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TOC Graphic.
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Synthesis of authentic rotundone.
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FID chromatogram of the isolated “woody” compound from the high-boiling fraction of an orange oil.
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Comparison of mass spectra and 1H NMR spectra of the isolated “woody” compound and that of authentic
rotundone.
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