Identification of odor-active trace compounds in blooming flower of Damask Rose (Rosa damascena)

Article abstract of DOI:10.1021/acs.jafc.9b03391

The flower scent of the damask rose (Rosa damascena) was studied. Two ultratrace components exhibiting high flavor dilution factors were detected as odor-active compounds via aroma extract dilution analysis (AEDA). One of these had a woody note and was identified as rotundone using multidimensional gas chromatography-mass spectrometry-olfactometry (MD-GC-MS-O), while the other had a citrus note and was identified as 4-(4-methylpent-3-en-1-yl)-2(5H)-furanone (MPF) by fractionation of a commercial rose absolute from R. damascena. To the best of our knowledge, this is the first study addressing the organoleptic importance of these two compounds in the rose scent. Sensory analysis was conducted to evaluate the effects of rotundone and MPF. Adding 50 μg/kg rotundone and 5 μg/kg MPF to the aroma reconstitute of R. damascena provided it with blooming and natural characteristics. Additionally, the existence of rotundone and MPF in five types of fragrant roses was investigated.

Full text of DOI:10.1021/acs.jafc.9b03391

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Chemistry and Biology of Aroma and Taste
Identification of Odor-active Trace Compounds in
Blooming Flower of Damask Rose (Rosa damascena)
Teruhisa Ohashi, Yamato Miyazawa, Susumu Ishizaki, Yoshiko Kurobayashi, and Tsukasa Saito
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03391 • Publication Date (Web): 12 Jun 2019
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Page 1 of 25
Journal of Agricultural and Food Chemistry
Identification of Odor-active Trace Compounds in
Blooming Flower of Damask Rose (Rosa
damascena)
Teruhisa Ohashi,* Yamato Miyazawa, Susumu Ishizaki, Yoshiko Kurobayashi, Tsukasa Saito
Research & Development Center, T. Hasegawa Co., Ltd., 29-7, Kariyado, Nakahara-ku,
Kawasaki-shi, 211-0022, Japan
AUTHOR ADDRESS: teruhisa_ohashi@t-hasegawa.co.jp
RUNNING HEAD
Odor-active Trace Compounds in Damask Rose (Rosa damascena)
*Teruhisa Ohashi FOOTNOTE.
TEL: +81-44-411-0298; FAX: +81-44-434-5257; E-mail: teruhisa_ohashi@t-hasegawa.co.jp
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ABSTRACT
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The flower scent of the damask rose (Rosa damascena) was studied. Two ultra-trace components
exhibiting high flavor dilution factors were detected as odor-active compounds via aroma extract
dilution analysis (AEDA). One of these had a woody note and was identified as rotundone using
multidimensional gas chromatography–mass spectrometry–olfactometry (MD-GC-MS-O), while
the other had a citrus note and was identified as 4-(4-methylpent-3-en-1-yl)-2(5H)-furanone
(MPF) by fractionation of a commercial rose absolute from R. damascena. To the best of our
knowledge, this is the first study addressing the organoleptic importance of these two compounds
in the rose scent. Sensory analysis was conducted to evaluate the effects of rotundone and MPF.
Adding 50 μg/kg rotundone and 5 μg/kg MPF to the aroma reconstitute of R. damascena
provided it with blooming and natural characteristics. Additionally, the existence of rotundone
and MPF in five types of fragrant roses was investigated.
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KEYWORDS: rose; Rosa damascena; odor-active compound; AEDA; isolation
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INTRODUCTION
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The rose scent is essential to flavors and fragrances. Particularly, the rose note is key to floral
perfume compositions. Typical aroma concentrates employed in fragrance products are rose oil,
rose water, rose absolute, and rose concrete. Nonetheless, the aromas of these processed products
differ from those of natural blooming rose flowers due to possible heating or oxidation during
the manufacturing processes. Furthermore, the scent of rose cannot be imitated by merely using
known chemicals, indicating that the remaining unknown components are key to the enigma of
the rose scent. Among the large rose varieties and forms, the damask rose (Rosa damascena) is
one of the main species cultivated for the fragrance industry1. Volatile compounds of natural rose
products from R. damascena have been comprehensively analyzed for many years.2,3 In these
studies, very small amount of constituents, such as rose oxides4 and rose ketones5,6, were
identified as key odorants of rose oil. Further, the rose oil aroma profile was recently
investigated using aroma extract dilution analysis (AEDA) and sensory analysis7. However, few
studies have reported the aroma of the blooming R. damascena flower. Thus, this study aims to
sensorially analyze and identify the main odorants in the aroma emitted from the blooming
flower of R. damascena and identify the compounds differentiating the natural rose scent from
that of artificial rose aroma reconstitutes. R. damascena has several cultivars. We planted R.
damascena ‘trigintipetala,’ which is one of the cultivars grown for rose oil production in
Bulgaria,8 and examined its beautiful flower scent in detail. To define the odor-active
compounds in the volatiles of R. damascena flower, AEDA was conducted for the aroma
concentrate obtained by dynamic headspace sampling. Further, follow the structural elucidation
of the two odor-active unknowns present in trace. These two compounds were found to have
“woody” note or “citrus-like” note and have never been regarded as important in the rose scent.
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In addition, we discuss the effects of these compounds on the rose aroma reconstitutes of R.
damascena and the existence of these compounds in five types of fragrant rose cultivars.
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MATERIALS AND METHODS
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Materials. Seedlings of R. damascena ‘trigintipetala,’ Rosa centifolia ‘bullata,’ Rosa ‘Neige
Parfum,’ Rosa ‘Pope John Paul II,’ Rosa ‘Lady Hilingdon,’ and Rosa ‘Grand Mogul’ were
obtained from nurseries in Japan and planted in the garden of the T. Hasegawa R&D Center.
Absolute from R. damascena was obtained from Biolandes Co., (Le Sen, France).
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Chemicals. Wakogel C-100 was purchased from Wako Pure Chemical Industries, Ltd., Osaka,
Japan. All other reagents and solvents were obtained from commercial suppliers and were used
without further purification. The chemicals for triangle test were obtained from T. Hasegawa
(Kawasaki, Japan). Rotundone [(3S,5R,8S)-5-isopropenyl-3,8-dimethyl-3,4,5,6,7,8-hexahydro-
1(2H)-azulenone] (6) was synthesized from (−)-guaiol in our laboratory.9
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Synthesis of Authentic 4-(4-methylpent-3-en-1-yl)-2(5H)-furanone (MPF). Using a
modified literature procedure,10 4-(4-methylpent-3-en-1-yl)-2(5H)-furanone (1) was synthesized
from 3-(1-ethoxyethoxy)prop-1-yne (2) via ethoxycarbonylation, conjugate addition of the 4-
methylpent-3-en-1-yl moiety, deprotection, and lactonization (Figure 1). The description of the
reaction processes in Figure 1 is provided in the Supporting Information.
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Dynamic Headspace (DHS) Analysis. The petals of R. damascena ‘trigintipetala’ (20
flowers; 42.6 g) were hand-picked from the garden of the T. Hasegawa R&D Center in the
morning. Open flowers that emitted a strong scent were chosen. Immediately after picking, they
were placed in a glass chamber. A constant flow rate of 1.5 L/min was employed, with air
entering the chamber through a charcoal filter and leaving it via a passage through 2.0 g of
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Tenax TA 60/80 adsorbent (GL Sciences Co., Tokyo, Japan). The volatiles were collected for 6
h at 25°C before using 20 mL each of pentane and diethyl ether for elution. The eluent was
collected and concentrated to ca. 100 µL by solvent distillation using a Vigreux column at 43°C.
The concentrate was subjected to GC-MS/flame ionization detector (FID) using a polar column
(InertCap WAX).
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Aroma Extract Dilution Analysis (AEDA). The flavor dilution (FD) factors of the odor-
active compounds were determined by AEDA.11 The concentrated volatiles of R. damascena
were diluted stepwise with diethyl ether to obtain dilutions in the series 1:5, 1:25, 1:125, 1:625,
and each dilution was analyzed via GC-O with a polar column (InertCap WAX). Two
experienced assessors conducted AEDA.
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Determination of the Citrus-like Odor Compound. The absolute from R. damascena was
fractionated by distillation, silica gel column chromatography, and high-performance liquid
chromatography (HPLC).
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Molecular Distillation. Rose absolute from R. damascena (330.5 g) was distilled by
employing simple molecular distillation apparatus (MDU60-05J; Asahi Glassplant Inc., Japan)
under reduced pressure (90°C/2.5–0.01 kPa) to obtain a distillate (144.2 g) and residue (172.2 g).
The target citrus-like odor compound was detected by GC-O. The distillate was used in the next
step.
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Fractional Distillation. The distillate (137.5 g) from the molecular distillation was
fractionated by distillation to give distillate fractions (fraction ii-1, 85°C–90°C/0.23–0.27 kPa,
37.4 g; fraction ii-2, 99°C–104°C/0.23 kPa, 39.4 g; fraction ii-3, 110°C–115°C/0.23 kPa, 25.2 g)
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and a residue (33.4 g). The target compound was identified by GC-O. The residue was used in
the next step.
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Silica Gel Column Chromatography. The residue (32.4 g) from the fractional distillation
was further fractionated by column chromatography using silica gel (Wakogel C-100, 650 g)
with the following hexane/ethyl acetate mixtures: 100:0, 30:1, 10:1, 3:1, and 0:100 (3,900 mL
each). Each fraction was evaporated (fractions iii-1–iii-5) and analyzed by GC-O. A 11.1 g
sample of concentrated effluent was collected from fraction iii-4, which was confirmed to
contain the target aroma by GC-O.
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Preparative High-Performance Liquid Chromatography (HPLC). Two successive HPLC
fractionations were conducted on a concentrate of fraction iii-4 (10.7 g) obtained by silica gel
column chromatography. For each HPLC fractionation, the target compound was identified by
GC-O. Finally, the HPLC fraction (2.8 mg) was obtained. GC-MS-FID analysis of this fraction
suggested that the main component was the target compound (97%). High-resolution mass
spectrometry (HRMS) (field ionization mode) calculated for C10H14O2 was 166.09938 (found
166.10041). The chemical structure of the target compound was deduced from HRMS and
nuclear magnetic resonance (NMR) spectra. Identification was further confirmed by comparing
the analytical data and odor qualities of the isolated target compound with those of synthesized
authentic 4-(4-methylpent-3-en-1-yl)-2(5H)-furanone.
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High-Performance Liquid Chromatography Fractionation. The instrument system
comprised a Shimadzu LC-20AD pump, SIL-20ACHT autosampler, CTO-20AC column oven,
SPD-M20A detector, CBM-20A controller, and DGU-20A5 degasser (Shimadzu Corp., Kyoto,
Japan). Two successive HPLC fractionations were conducted. The initial HPLC conditions were
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as follows: an Inertsil ODS-3 column (5 μm particle size, 250 mm × 14 mm i.d., GL Sciences
Co., Tokyo, Japan) was connected to the instrument system. The gradient elution was as follows:
0–23 min, methanol/water = 40:60 → 63:37; 23–28 min, methanol/water = 100:0; 28–35 min,
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methanol/water = 40:60. The solvent flow rate was 9.3 mL/min, and the column temperature was
30°C. The effluent was monitored at λ = 220 nm, the eluent was collected between 25.0 and 25.6
min, and methanol was evaporated using a rotary evaporator from it. NaCl (25% w/w) was added
to the residue before extracting with diethyl ether, and the diethyl ether layer was dried over
NaSO4 and concentrated in vacuo. A second HPLC fractionation was conducted on the
concentrate, under the following conditions: an Inertsil ODS-3 column (5 μm particle size, 250
mm × 4.6 mm i.d., GL Sciences Co., Tokyo, Japan) was connected to the instrument system. The
gradient elution was as follows: 0–18 min, acetonitrile/water = 55:45 → 73:27; 18–28 min,
acetonitrile/water = 100:0; 28–40 min, acetonitrile/water = 55:45. The solvent flow rate was 1.0
mL/min, and the column temperature was 40°C. At retention times between 21.3 and 22.5 min,
the eluent was collected, then was concentrated using the procedure described above.
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Gas Chromatography–Mass Spectrometry–Flame Ionization Detection (GC-MS-FID).
GC-MS-FID was conducted with an Agilent 7890 gas chromatograph coupled with an Agilent
MSD5975 quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) and an
FID fitted with an InertCap WAX capillary column (0.25 mm i.d. × 60 m, 0.25 μm film
thickness, GL Sciences Co.). The effluent from the column at the end of the capillary was
divided into two branches and routed via deactivated fused silica capillaries to the mass
spectrometer and FID. The injection port was maintained at 250°C. The split ratio was 10:1, and
1 μL of sample was injected. The oven temperature was maintained at 40°C for the first 2 min
then increased to 230°C at 3°C/min, with a constant helium carrier gas flow of 1.8 mL/min. Mass
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spectra in the electron impact (EI) mode were recorded at 70 eV in scan mode (m/z of 29–550).
The temperature of the transfer line was 250°C. The linear retention indices (RIs) of the
compounds were calculated with respect to the retention times of a homologous series of n-
alkanes (C5–C30). The purities of the isolated or synthesized compounds [4-(4-methylpent-3-en-
1-yl)-2(5H)-furanone] were calculated by integrating the chromatogram obtained with the FID.
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Gas Chromatography–Olfactometry (GC-O). GC-O was conducted with an Agilent 7890
GC coupled with an Agilent MSD5975 quadrupole mass spectrometer and a Gerstel ODP3
sniffing port (Gerstel GmbH & Co. KG, Mülheim an der Ruhr, Germany) fitted with an InertCap
WAX capillary column (0.25 mm i.d. × 60 m, 0.25 μm film thickness, GL Sciences Co.). The
effluent from the column at the end of the capillary was divided into two branches and routed via
deactivated fused silica capillaries to the mass spectrometer and sniffing port. The sample
volume, split ratio, injection temperature, oven temperature program, carrier gas, and flow rate
were the same as those employed for performing the aforementioned GC-MS-FID analysis. The
linear RIs of the compounds were calculated with respect to the retention times of the
homologous series of n-alkanes (C5–C30). The GC-O analysis of the fractions from HPLC was
performed by a trained panelist who was experienced in the detection of odorants using the
aforementioned AEDA analysis.
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Multidimensional Gas Chromatography–Mass Spectrometry–Olfactometry (MD-GC-
MS-O). MD-GC-MS-O was conducted using a selectable 1D/2D GC-MS system with an Agilent
7890A GC fitted with a low thermal mass (LTM) system.12 The GC was coupled with a Gerstel
cooled injection system CIS-4 and a FID (250ºC), a 5975 mass selective detector, and a sniffing
port fitted with a Gerstel multi-column switching system. The effluent from the first column
(DB-WAX capillary column; 0.25 mm i.d. × 30 m; film thickness 0.25 μm, LTM; Agilent
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Technologies, Inc.) at the end of the capillary was divided into three branches and routed via
deactivated fused silica capillaries to the FID, mass detector, and sniffing port. The branch
routing could be switched with the column switching device to eliminate the compounds eluted
from the first column or transfer them directly to the second column (DB-1 capillary column;
0.25 mm i.d. × 30 m; film thickness 0.25 μm; Agilent Technologies, Inc.). The effluent from the
second column at the end of the capillary was also divided into three branches and routed via
deactivated fused silica capillaries to the FID, mass detector, and sniffing port. Each sample (the
concentrate of DHS sampling) was injected in 1 μL volumes in splitless mode. The injection
temperature was maintained at 10°C for the first 0.5 min then increased to 240°C at 12°C/s. The
oven temperature of the first column was kept at 40ºC then increased to 230ºC at 10ºC/min,
maintaining a constant pressure of 362 kPa. The effluent was eliminated again when the target
retention time (rotundone: 20.2–20.6 min, MPF: 21.8–22.6 min) was reached, whereupon the
trapped material was heated to 250ºC and directed to the second column. The oven temperature
of the second column was maintained at 40ºC for the initial time (rotundone: 21.0 min, MPF:
23.0 min), and then increased to 230ºC at 20ºC/min, maintaining a constant pressure of 362 kPa.
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High-Resolution Mass Spectrometry (HRMS). HRMS was performed on an AccuTOF GCv
4G instrument (JEOL Ltd., Tokyo, Japan) fitted with an InertCap WAX capillary column (0.25
mm i.d. × 60 m, 0.25 μm film thickness; GL Sciences Co.). The injection conditions, oven
temperature program, carrier gas, and flow rate were the same as those described above for GC-
MS/FID.
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Nuclear Magnetic Resonance (NMR) Spectroscopy. The 1H NMR (400 MHz, CDCl3,
residual CHCl3 at δ = 7.26 as internal standard) and 13C NMR (100 MHz, CDCl3, CDCl3 at δ =
77.0 as internal standard) experiments were conducted using a JNM-ECX 400 spectrometer
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(JEOL Ltd., Tokyo, Japan). The chemical shifts and coupling constants (J) are expressed in parts
per million (ppm) and hertz (Hz), respectively.
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Odor Threshold Definition of MPF. The odor threshold of MPF in water was evaluated
using a previously reported method.13 Panelists [n = 23 (16 males and 7 females; age 20–60
years)] were employees of the R&D Center of T. Hasegawa Co., Ltd. and were trained to
recognize and quantify aromas using approximately 100 odorous chemicals and raw materials.
The MPF concentrations used were 1.3, 6.4, 32, 160, 800, 4,000, 20,000, and 100,000 ng/kg. The
most concentrated samples were prepared by diluting an ethanol solution of MPF 10,000 times
with water. Equal volumes of ethanol were added to the corresponding blank samples. Two
grams of each sample were added to a closed sensory vial (total volume 30 mL) and allocated a
random three-digit numerical code. The samples were presented in ascending order of sample
concentration. Three samples in a series were presented in random order and identified only by
their three-digit random numbers. The spiked sample always differed from the other two.
Orthonasal assessments were performed in a quiet room maintained at 23°C. Along with the
threshold assessment, panelists assessed the odor of the sample they had successfully recognized.
All panelists were assigned a best estimate threshold value, which was the geometric mean of the
highest concentration missed and next highest concentration tested. The geometric mean of the
individual best estimate threshold was then calculated to afford the threshold value.
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Triangle Test. The triangle test was conducted to evaluate the effects of rotundone and MPF
on the rose aroma reconstitutes of R. damascena. The panel comprised 52 panelists (30 males
and 22 females; age 20–60 years), who were employees of the R&D Center of T. Hasegawa Co.,
Ltd. and were trained to recognize and rate intensities using approximately 100 odorous
chemicals and raw materials. Four aroma reconstitutes (samples A–D) were assessed. The aroma
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reconstitutes were produced by mixing dipropylene glycol with the odorants showing a high FD
factor by AEDA at concentrations based on the GC peak area percentages of the DHS analysis
described previously. Aroma reconstitute A comprised seven compounds [2-phenylethanol (20
g/kg), citronellol (12 g/kg), geraniol (10 g/kg), nerol (7.0 g/kg), citral (mixture of neral and
geranial; 0.75 g/kg), rose oxide (0.13 g/kg), and linalool (0.075 g/kg)]. Aroma reconstitutes B, C,
and D were produced by adding rotundone (50 µg/kg), MPF (5 µg/kg), and rotundone (50 µg/kg)
and MPF (5 µg/kg) to aroma reconstitute A, respectively. A 2 g portion of each sample was
placed in a closed sensory vial (total volume 10 mL) and allocated a random three-digit number
as a code. Each panelist was presented with a series of test samples (AAB, ABB, AAC, ACC,
AAD, or ADD) with instructions to sniff each sample and identify samples B, C, or D against A.
Simultaneously, the panelists assessed the odor of the sample they had successfully recognized.
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Identification of Rotundone and MPF in Various Types of Roses. The headspace gases of
the living flowers of R. centifolia ‘Bullata,’ R. ‘Neige Parfum,’ R. ‘Pope John Paul II,’ R. ‘Lady
Hilingdon,’ and R. ‘Grand Mogul’ were pumped through 0.4 g of Tenax TA (0.5 L/min, 3 h) at
22°C–25°C. The adsorbents were eluted with 4 mL each of pentane and diethyl ether. The
eluents were concentrated and subjected to MD-GC-MS-O to tentatively identify rotundone and
MPF using the procedure described above.
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RESULTS AND DISCUSSION
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DHS Analysis and AEDA. DHS sampling was used to prepare the headspace aroma
concentrate of R. damascena ‘trigintipetala.’ If the flowers are left unpicked until daytime, their
scent weakens and deteriorates. Thus, the flower petals were hand-picked in the morning. When
using only the petals, the flower scent remained constant during a six-hour sampling. Moreover,
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sampling from the petals of 20 flowers reduced the influence of individual differences between
flowers. The olfactory properties of the aroma concentrate represented those of the living flower.
We conducted AEDA to define odor-active compounds in the aroma concentrate of R.
damascena. Consequently, along with major compounds including 2-phenylethanol, geraniol,
citronellol, and nerol, two ultra-trace components (unknowns A and B) with a high FD factor
(FD 625) were detected as odor-active compounds (Table 1). These results were different from
those of the reported rose oil7; rose oxide, trans-nerolidol, and geranyl butyrate showed the
highest FD factor in AEDA analysis, whereas the compounds corresponding to unknown A and
B were not detected.
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Elucidation of Unknown Compounds. Unknown A was identified as rotundone, with a
woody note, by MD-GC-MS-O analysis using the same retention time, MS, and comparing the
odor qualities with those of the authentic synthesized rotundone (Figure 2). Rotundone was
identified as an odor-active component in patchouli oil,14 frankincense oil,15 Shiraz wine,16
peppers,16 and several fruits (grapefruit, orange, apple, and mango).9 To the best of our
knowledge, rotundone has not been reported in roses.
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However, a clear mass spectrum of unknown B (citrus note) was obtained by MD-GC-MS-O,
although its structure could not be identified by MS library matching. Therefore, we attempted to
obtain sufficient quantities of the compound for NMR experiments. The target citrus-like
compound was detected in commercial rose absolute from R. damascena. Thus, it was isolated
from rose absolute by several fractionation steps. First, molecular distillation was performed to
remove nonvolatile components from the absolute. Then, fractional distillation was conducted to
separate the target compound from 2-phenethyl alcohol, which is a major constituent
(approximately 60%) of the volatile fraction. Subsequent fractionation via silica gel column
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chromatography and two-step preparative HPLC successfully purified the target compound. As
confirmed by GC-FID, the final purity of the isolated compound (2.6 mg) was 97%. This was
assumed to be 4-(4-methylpent-3-en-1-yl)-2(5H)-furanone (MPF; 1) from high-resolution mass
and NMR spectra. Identification was verified further by matching the analytical data and odor
qualities of the isolated compound with those of the synthesized MPF (Figure 3). In a preamble
to a report on rose oil analysis by Kovats,6 Ohloff and Demole reported MPF in rose oil.5
However, details (quantity, character, and odor) of MPF were unmentioned. MPF was also
identified in the secretions of acarid mites and tentatively named as α,β-acariolide,17 but its
biological function remains unclear. Although headspace analysis of the flower of several types
of hybrid rose cultivars were reported18, 19, 20, rotundone and MPF were not identified. To the best
of our knowledge, this is the first study that reports the organoleptic properties of MPF. In the
threshold measurement of MPF, panelists reported that this emitted a citrus-like (lemon, orange,
and grapefruit) and floral odor (muguet and jasmine) with a moderately low threshold of 3.6
µg/kg in water.
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Evaluation of the Effects of Rotundone and MPF. A triangle test was conducted to
investigate the effects of rotundone and MPF on rose aroma reconstitutes of R. damascena. The
concentrations of rotundone and MPF were initially settled at levels where the characteristic odor
(the woody odor of rotundone and the citrus-like odor of MPF) was not sensorially distinctly
recognized. Figure 421 shows the results of this test. Aroma reconstitutes B and C were not
significantly discriminated from aroma reconstitute A. Only aroma reconstitute D was
significantly discriminated from aroma reconstitute A. Furthermore, panelists who could
discriminate between aroma reconstitutes D and A assessed the aroma of D as “more blooming
than A” or “more natural than A.” The panelists did not distinguish aroma reconstitutes A and D
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as “woody” or “citrus-like.” The effects of these two added compounds are expected to
differentiate the natural rose aroma from that of artificial rose aroma reconstitutes.
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268
269
270
271
272
273
Identification of Rotundone and MPF in Various Types of Roses. A further question was
whether these two compounds existed in other roses. To verify this, MD-GC-MS-O analysis was
used to investigate the existence of rotundone and MPF in five types of fragrant roses. R.
centifolia is one of the two main species with R. damascena cultivated for the perfume industry1.
R. centifolia ‘Bullata’ is one of the cultivars of R. centifolia. Other four types of roses are the
hybrid tea rose cultivars that emit a strong and rich fragrance. Rotundone was detected in all five
types, whereas MPF was detected in three types (i.e., R. centifolia ‘Bullata,’ R. ‘Neige Parfum,’
and R. ‘Pope John Paul II’). These results indicate that rotundone and MPF are widely
distributed not only in R. damascena but also in several types of rose cultivars and contribute to
their beautiful aroma.
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In conclusion, this is the first study to identify rotundone and MPF as potent aroma compounds
that are emitted from the blooming flower of R. damascena. The addition of rotundone and MPF
to the aroma reconstitute of R. damascena provided it with blooming and natural characteristics.
Furthermore, rotundone and MPF were tentatively identified by matching the RIs and odor qualities in
several fragrant roses, including the hybrid tea rose cultivars. To confirm the contributions of
rotundone and MPF to the aromas of the natural rose flowers, quantitative studies must be
conducted with respect to the headspace volatile composition of the flowers.
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ABBREVIATIONS USED
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285
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288
289
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DHS, dynamic headspace; AEDA, aroma extract dilution analysis; GC-O, gas
chromatography–olfactometry; GC-MS-FID, gas chromatography–mass spectrometry–flame
ionization detection; EI, electron impact; FID, flame ionization detector; RI, retention indices;
MD-GC-MS-O, multidimensional gas chromatography–mass spectrometry–olfactometry; NMR,
nuclear magnetic resonance; HRMS, high-resolution mass spectra; HPLC, high-performance
liquid chromatography; MPF, 4-(4-methylpent-3-en-1-yl)-2(5H)-furanone.
ETHICS & CONFLICT OF INTEREST
The authors declare no competing financial interest.
REFERENCES
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Figure Captions
Figure 1. Synthesis of 4-(4-methylpent-3-en-1-yl)-2(5H)-furanone.
Figure 2. Overlay of the total ion chromatograms of rotundone reference material (in blue) and
Rosa damascena aroma concentrate (in black) with the mass spectra of each.
Figure 3. Comparison of mass spectra and 1H NMR spectra of the isolated target citrus-like compound
and those of authentic MPF.
Figure 4. Accuracy rate of the triangle test (Identification of sample B, C, or D against A). A:
control (-rotundone, -MPF), B: +rotundone (50 μg/kg), C: +MPF (5 μg/kg), D: +rotundone (50
μg/kg), +MPF (5 μg/kg) n = 52, *Binominal test, p < 0.05
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Table 1. Odor-active compounds (FD ≥ 25) in the Rosa damascena aroma concentrate
RI on
FD
GC peak
identification
modec
odor
InertCap
WAX
no.
odorant
factor area, %b
qualitya
1
2
3
4
5
6
7
8
9
(Z)-rose oxide
linalool
rose, green
floral, fresh
rose, fresh
rose, fresh
rose, fresh
rose, fresh
rose, honey
clove-like
lactone
1361
1549
1752
1770
1810
1853
1930
2188
2288
2300
2466
3125
125
0.21
0.06
MS, RI, GC-O
MS, RI, GC-O
MS, RI, GC-O
MS, RI, GC-O
MS, RI, GC-O
MS, RI, GC-O
MS, RI, GC-O
MS, RI, GC-O
RI, GC-O
geranial
25
0.87
citronellol
nerol
3125
25
20.38
11.83
15.80
32.64
trace
trace
trace
trace
geraniol
3125
15625
3125
625
2-phenylethanol
eugenol
γ-undecalactone
10 unknown A
11 unknown B
woody
625
citrus-like
625
a Odor quality perceived at the sniffing port. b trace, trace amount (<0.001%).
c MS, reference mass spectrum; RI, retention indices; GC-O, gas chromatography–
olfactometry.
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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/Abstract Graphic
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