Identification of rotundone as a potent odor-active compound of

May 18, 2017 - An investigation of the aromas of grapefruit, orange, apple, and mango revealed the presence of an odor-active compound that gave off a...
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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 R&D Center, T. Hasegawa Co., Ltd., 29-7 Kariyado, Nakahara-ku, Kawasaki-shi 211-0022, Japan S Supporting Information *

ABSTRACT: 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. KEYWORDS: rotundone, odor-active compound, woody odor, fruits, grapefruit, orange, apple, mango, isolation

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INTRODUCTION

have a woody odor, and the odor closely resembled the aroma of dried wood chips when assessed by GC-O; 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 odor-active 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 literature. However, the unique “peppery/spicy” odors of rotundone were never perceived from the sniffing port of our GC-O analyses of the aromas of the above fruits. From these facts, we could not 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 odor-active compound in fruit aromas by sensory analyses.

Owing to the development of analytical technology, most of key odorants, which exist even in trace amounts in foods, have been identified. Recently, our group reported that (6Z,8E)undeca-6,8,10-trien-3-one, (4Z,7Z)-trideca-4,7-dienal, and cis-3methyl-4-decanolide were identified as novel odor-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 Because these compounds were present in only trace amounts in the volatiles of each food, their structure elucidation was difficult; however, by application of repeated fractionations and/or multidimensional gas chromatography−mass spectrometry (MD-GC-MS), 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 present in the aromas of foods in a concentration that is too low to detect the mass spectra of the odorants but 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 (GCO) on the basis of the task in our laboratory, and, through these GC-O analyses, it was found that one odorant was commonly detected only by GC-O. 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 © 2017 American Chemical Society

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MATERIALS AND METHODS

Materials. The high-boiling fraction of orange essential oil was purchased from R. C. Treatt & Co. Ltd. (Suffolk, UK). Grapefruit (marsh seedless) and apple (Sun-Fuji) were purchased in a local Received: Revised: Accepted: Published: 4464

March 7, 2017 May 17, 2017 May 18, 2017 May 18, 2017 DOI: 10.1021/acs.jafc.7b00929 J. Agric. Food Chem. 2017, 65, 4464−4471

Article

Journal of Agricultural and Food Chemistry

Figure 1. Synthesis of authentic rotundone. supermarket in Kawasaki, Japan, and mango (Alphonso) puree was available commercially from O’will Corp. (Tokyo, Japan). Chemicals. The following chemicals were purchased commercially: (−)-guaiol, cobalt(II) 2-ethylhexanoate, cobalt(II) acetylacetonate, cobalt(II) naphthenate (Sigma-Aldrich, St. Louis, MO, USA); toluene, pyridine, N,N-dimethyl-4-aminopyridine, 4-methyl-2-pentanone (Junsei Chemical Co., Ltd., Tokyo, Japan); acetic anhydride (Daicel Corp., Tokyo, Japan), silica gel 60N (spherical neutral, particle size 63−210 μm, Kanto Chemical Co., Inc., Tokyo, Japan); and Wakogel C-100 (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Gaseous oxygen was generated from an oxygen concentrator (LFY-I-5F, Malus Corp., 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). Synthesis of Authentic Rotundone. Rotundone [(3S,5R,8S)-5isopropenyl-3,8-dimethyl-3,4,5,6,7,8-hexahydro-1(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). 2-[(3S,5R,8S)-3,8-Dimethyl-1,2,3,4,5,6,7,8-octahydroazulen-5-yl]propan-2-yl acetate (3, Guaiyl Acetate). A mixture of (−)-guaiol (2; 445 mg, 2.00 mmol), acetic anhydride (225 mg, 2.20 mmol), and N,Ndimethyl-4-aminopyridine (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 1.5 h of stirring at room temperature, acetic anhydride (5.17 g, 50.6 mmol), N,N-dimethyl-4-aminopyridine (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 2 N HCl and extracted with ethyl acetate. The organic layer was washed with saturated aqueous 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%). 2-[(3S,5R,8S)-3,8-Dimethyl-3,4,5,6,7,8-hexahydro-1(2H)-azulenon-5-yl]propan-2-yl acetate (4). Oxygen gas was bubbled into a mixture of guaiyl acetate (3; 353 mg, 1.33 mmol), cobalt(II) 2ethylhexanoate (22.9 mg), cobalt(II) acetylacetonate (20.0 mg), cobalt(II) naphthenate (48.5 mg), and 4-methyl-2-pentanone (10 mL) at 70 °C for 2.5 h. The reaction mixture was cooled to room temperature, poured into saturated aqueous 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 2-[(3S,5R,8S)-3,8-dimethyl3,4,5,6,7,8-hexahydro-1(2H)-azulenon-5-yl]propan-2-yl acetate (4; yellow oil, 45.2 mg, yield 12%). (3S,5R,8S)-5-Isopropenyl-3,8-dimethyl-3,4,5,6,7,8-hexahydro1(2H)-azulenone (1; Rotundone). 2-[(3S,5R,8S)-3,8-Dimethyl3,4,5,6,7,8-hexahydro-1(2H)-azulenon-5-yl]propan-2-yl acetate (4; 47.7 mg, 0.171 mmol) was agitated using a magnetic stirring bar of appropriately small dimension (2 mm diameter 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%). 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 GC-MS were calculated as 2300 and 1680, respectively. Elucidation of the Woody Odor Compound. Depending on the sample amount, fractionation was sequentially conducted on the highboiling fraction of orange essential oil by vacuum distillation, silica gel column chromatography, and preparative HPLC. Vacuum Distillation. The high-boiling fraction of orange essential oil (801.3 g) was distilled under reduced pressure to obtain distillate fractions (fraction i-1, 136 °C/1.0 kPa, 282.4 g; fraction i-2, 136 °C/ 1.0 kPa, 96.8 g; fraction i-3, 136−157 °C/0.9 kPa, 165.4 g; fraction i-4, 157−169 °C/0.9 kPa, 85.6 g; fraction i-5, 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 GC-O analysis. Accordingly, fractions i3−i-5 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 were fractionated by silica gel chromatography (silica gel 60N: 1800 g, hexane/ethyl acetate = 3:1, v/ v) into five fractions (fractions ii-1−ii-5), each of which was evaporated and subjected to GC-O. A 112.8 g sample of concentrated effluent was collected from fraction ii-3, which was confirmed to have the target aroma by GC-O. 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 (fractions iii1−iii-7), each of which was evaporated and subjected to GC-O. A 68.0 g sample of concentrated effluent was collected from fractions iii-3−iii5, 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 of concentrated sample was obtained from their fractions and confirmed to have the target aroma by GC-O. 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. GC-MS analysis of the HPLC fraction indicated that the main component was the target compound (36.7%). HRMS (EI) calculated for C15H22O was 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 4465

DOI: 10.1021/acs.jafc.7b00929 J. Agric. Food Chem. 2017, 65, 4464−4471

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Journal of Agricultural and Food Chemistry

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 μm particle size, 250 mm × 4.6 mm i.d., GL Sciences Co.) and a Sunrise C28 column (5 μm 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. Identification of Rotundone in Grapefruit, Mango, and Apple. The first HPLC conditions were as follows: an Inertsil ODS-3 column (5 μm 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. The second HPLC conditions were as follows: a Sunrise C28 column (5 μm 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). GC-MS analyses were performed using an Agilent 7890 gas chromatograph combined with an Agilent MSD5975 quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) and a flame ionization detector (FID) equipped with an InertCap WAX capillary column (0.25 mm i.d. × 60 m, 0.25 μm 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 FID. The injection port was held at 250 °C. The split ratio was 50:1, and 1 μL 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 n-alkanes. The purities of the synthesized compounds were calculated by integration of the chromatogram obtained using the FID. Gas Chromatography−Olfactometry (GC-O). GC-O 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 μm 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 GC-MS analysis as described above. To compare with authentic rotundone for identification, an InertCap 1MS capillary column (0.25 mm i.d. × 60 m, 0.25 μm 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). MD-GC-MS/O analyses were

pieces and extracted with pentane (2.4 L) for 30 min under vigorous stirring. After the pentane solution had been separated by decantation, the residue was extracted again with pentane (1.2 L). After removal of 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 GC-FID). 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 C-100) 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 GC-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 GC-O 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 it had been left 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 MD-GCMS/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 GC-O 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 sufficient mixing, 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 GC-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 GC-O 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 Corp., Milford, MA, USA). Instrument system B comprised a Shimadzu LC-20AT, a Shimadzu SIL-20AC, a Shimadzu CTO-20A, a Shimadzu SPD-M20A, and a Shimadzu CBM-20A (Shimadzu Corp., 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. Elucidation of the Woody Odor Compound. The first HPLC conditions were as follows: an Inertsil ODS-3 column (5 μm particle size, 250 mm × 4.6 mm i.d., GL Sciences Co., Tokyo, Japan) 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 4466

DOI: 10.1021/acs.jafc.7b00929 J. Agric. Food Chem. 2017, 65, 4464−4471

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Journal of Agricultural and Food Chemistry Table 1. Concentration of Components Used for Fruit Aroma Reconstitutes concentration (μg/kg) no.

compound

grapefruit

orange

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

acetaldehyde limonene ethyl 3-hydroxyhexanoate (Z)-hex-3-enal myrcene decanal linalool ethyl butanoate vanillin α-pinene hexanal octanal nonanal ethyl 2-methylpropanoate ethyl hexanoate ethyl 2-methylbutanoate trans-4,5-epoxy-(E)-dec-2-enal 3,6-dimethyl-3a,4,5,7a-tetrahydro-1-benzofuran-2(3H)-one (E,E)-deca-2,4-dienal oct-1-en-3-one 4-sulfanyl-4-methylpentan-2-one (E)-non-2-enal 3-methylsulfanylpropanal 1-p-menthene-8-thiol 3-methylbutanol 2-methylbutanol butanoic acid hexan-1-ol butan-1-ol (E)-hex-2-enal β-damascenone dimethyl sulfide terpinolene linalool oxide 2,5-dimethyl-4-methoxy-3(2H)-furanone δ-3-carene ethyl octanoate γ-terpinene ethyl decanoate p-methylacetophenone β-ionone (E,Z)-nona-2,6-dienal

6150 2308 117 108 94 89 76 70 69 42 33 32 9.3 5.8 4.3 3.9 3.1 1.1 1.0 0.8 0.8 0.5 0.2 0.01

8305 85598 1136 187 594 45 81 1192 67 308 197 25 13 8.8 63 48 4.3 0.8 1.2 4.1

apple

mango 940

11

3.4

160 11 1500

4305

280 33

0.27

0.6 0.4 639 270 74

12330 1450 900 306 4.6 1.3

7.9 23560 1710 1660 1260 610 530 140 140 27 19

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 GC-MS analyses as described above. Nuclear Magnetic Resonance (NMR) Spectra. 1H and 13C NMR, heteronuclear multiple quantum correlation (HMQC), and heteronuclear multiple bond correlation (HMBC) experiments were performed using a JNM-ECX400 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 ppm). The chemical shifts and coupling constants (J) are expressed in parts per million (ppm) and hertz (Hz), respectively.

performed using an Agilent 7890 GC combined with a Gerstel cooled injection system CIS-4 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 μm 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 μm 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 μL 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 4467

DOI: 10.1021/acs.jafc.7b00929 J. Agric. Food Chem. 2017, 65, 4464−4471

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Journal of Agricultural and Food Chemistry

Figure 2. FID chromatogram of the isolated “woody” compound from the high-boiling fraction of an orange oil.

Figure 3. Comparison of mass spectra and 1H NMR spectra of the isolated “woody” compound and those of authentic rotundone. 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 a set of test samples coded by a random three-digit number with instructions to taste each sample and rate the intensity according to each attribute using a five-point 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 t tests. On the Tukey’s multiple-comparison

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 μm film thickness, GL Sciences Co.). The injection conditions, oven temperature program, carrier gas, and flow rate were all the same as in the GC-MS analyses 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 (for grapefruit, sweet, sour, bitter, fresh, juicy, peely, complex, and discordant; for orange, sweet, sour, floral, fresh, juicy, peely, complex, and discordant; for apple, sweet, sour, green, fruity, pulpy, complex, and discordant; for mango, sweet, sulfurous, green, fruity, metallic, ripe, complex, and discordant) were agreed upon in preliminary sessions during which the panelists evaluated natural fruits (grapefruit, 4468

DOI: 10.1021/acs.jafc.7b00929 J. Agric. Food Chem. 2017, 65, 4464−4471

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Journal of Agricultural and Food Chemistry

Figure 4. Spider charts of sensory analyses of rotundone in fruit model beverages. tests, the standard error measurement and least significant differences 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 on the basis of quantitative data reported in the literature.17−20 The components of each reconstitute and their concentrations in model beverages are shown in Table 1.

nated the target compound. On the basis of 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 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 odor-active component.5−9 On the basis of 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 odor-active 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 the 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 GC-MS 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 GC-

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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 highboiling 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 high-boiling 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 GC-FID (Figure 2). Fortunately, despite this relatively low purity, sufficient analytical data for structural analysis could be obtained because no single impurity exceeding 10% contami4469

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Journal of Agricultural and Food Chemistry

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.

MS with both polar and nonpolar columns. A clear mass spectrum was obtained successfully without further concentration by using MD-GC-MS/O. Finally, by matching the mass spectra, retention indices on two columns of different polarities, 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 high-quality timber and has been used as a building material in Japan, was the right fit to adequately demonstrate this difference. The hinoki-like 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 literature.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 GC-O. 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 hinoki-like 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 flavor-modifying 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 subthreshold 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 subthreshold level, rotundone did not 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 the 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 analysis results, it is 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 subthreshold 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 odor-active component in popular fruits. Furthermore, although identification has not yet been confirmed, rotundone was tentatively identified by matching

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00929. 1 H and 13C NMR, COSY, HMQC, and HMBC spectra of isolated rotundone and authentic rotundone; comparison of mass spectra of rotundone in grapefruit, apple, and mango with that of authentic rotundone (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(A.N.) Phone: +81-44-411-0298. Fax: +81-44-434-5257. Email: [email protected]. ORCID

Akira Nakanishi: 0000-0003-4764-7744 Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED VSC, volatile sulfur compound; GC-O, gas chromatography− olfactometry; GC-MS, gas chromatography−mass spectrometry; EI, electron impact; FID, flame ionization detector; MDGC-MS/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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