Identification and Characterization of 3-epi-Rotundone, a Novel

Jun 9, 2017 - ... was identified in the aroma of grapefruit, orange, apple, and mango. ... organoleptic properties, which were odor description (woody...
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Identification and Characterization of 3-epi-Rotundone, a Novel Stereoisomer of Rotundone, in Several Kinds of Fruits Akira Nakanishi,* Makiko Ito, Keisuke Yoshikawa, Tomoko Maeda, Susumu Ishizaki, and Yoshiko Kurobayashi R&D Center, T. Hasegawa Co., Ltd., 29-7, Kariyado, Nakahara-ku, Kawasaki-shi, 211-0022, Japan S Supporting Information *

ABSTRACT: A novel stereoisomer of rotundone, 3-epi-rotundone, was identified in the aroma of grapefruit, orange, apple, and mango. 3-epi-Rotundone was prepared by the isomerization of rotundone, and its structural elucidation was confirmed by comparing the 1D and 2D nuclear magnetic resonance and nuclear Overhauser effect spectroscopy spectra with those of rotundone. The odor thresholds of rotundone and 3-epi-rotundone in water were determined by a triangle test as 5 and 19100 ng/kg, respectively. The odor of 3-epi-rotundone was evaluated as woody, spicy, peppery, citrus, grapefruit-like, powdery, and celery-like, which was a greater range of odor characteristics than that for rotundone. Results of odor evaluation of 3-epirotundone revealed that its unique organoleptic properties, which were odor description (woody, spicy, and peppery), anosmic properties in neat form, and strong adaptation, were similar to those of rotundone. 3-epi-Rotundone might be a valuable substance to apply new types of woody, peppery, and spicy notes. KEYWORDS: 3-epi-rotundone, rotundone, fruit, aroma, threshold

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INTRODUCTION Sesquiterpenes (including modified sesquiterpene such as sesquiterpenoid) are widely distributed in nature and are known to have various bioactivities, for example, pheromonal, antitumor, antiviral, antimicrobial, and anti-inflammatory effects.1 In particular, odorous sesquiterpenes are important constituents in most essential oils, and they are usually oxygenated compounds. Various odorous sesquiterpenes such as α- and β-sinenal (an orange-like odor), nootkatone (a grapefruit-like odor), and patchoulol (a patchouli-like odor) are well-known; however, novel sesquiterpenes most likely still await their discovery in nature. Because sesquiterpenes comprise three units of isoprene, they have a wide range of possible structural combinations. In addition, because sesquiterpenes are difficult to synthesize and isolate from nature, the structural elucidation of novel naturally occurring sesquiterpenes is difficult. However, as mentioned above, odorous sesquiterpenes often have a characteristic odor that is indispensable for reproducing the aroma of the natural product; the discovery of novel odorous sesquiterpenes is thus a challenging field of research for the flavor and fragrance industry. Rotundone (Figure 1), which is a oxygenated sesquiterpene composed of a guaiene carbon skeleton, has been identified as a natural sesquiterpenoid in the essential oils from nut grass weed (Cyperus rotundus),2 agarwood,3−5 the Aquilaris tree,6 the tubers of nagarmotha (cypriol, Cyperus scariosus).7 It has also been found as a potent odorant in patchouli oil,8 Shiraz wine (which is characterized by a peppery aroma), Shiraz grape (the Vitis vinifera cultivar used to make Shiraz wine), spices (white and black peppers, marjoram, oregano, etc.),9 and oak-aged spirits.10 Recently, investigation of the volatile fraction of Boswellia sacra gum resin by Niebler et al. led to the identification of rotundone and a novel oxygenated sesqui© XXXX American Chemical Society

Figure 1. Chemical structures of rotundone, rotundone-d5, and 3-epirotundone (numbers labeled in this chemical structure were used for signal assignments of 1H and 13C NMR and not for compound names).

terpene, mustakone, as potent odorants contributing to the aroma of frankincense.11 In addition, investigation of the essential oil from Cyperus scariosus R.Br. by Clery et al. revealed that rotundone and a new sesquiterpenoid, cyperen-8-one, contributed to the odor of cypriol oil.12 In our series of studies on rotundone, we identified rotundone as a potent odor-active component in the aromas of grapefruit, orange, apple, and mango and quantitated its concentrations in grapefruit peel and juice.13,14 Because rotundone has three asymmetric centers, it has eight stereoisomers; however, no reports have described them. In our previous investigations of the aromas of these fruits, we noted an unknown compound that was assumed to be one of the stereoisomers of rotundone. In this study, we elucidated the structure and organoleptic properties of this compound. Received: Revised: Accepted: Published: A

April 12, 2017 June 8, 2017 June 9, 2017 June 9, 2017 DOI: 10.1021/acs.jafc.7b01696 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

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8.35 to 8.60 min (3-epi-rotundone), the eluents were collected and then concentrated in vacuo to obtain rotundone (24.3 mg, purity >99.9% by GC-FID) and 3-epi-rotundone (21.4 mg, purity >99.9% by GC-FID). 3-epi-Rotundone: 1H NMR: δ 4.74 (1H, q, J = 0.8, H12a), 4.71 (1H, quint, J = 1.6, H12b), 2.98 (1H, ddq, J = 11.2, 3.6, and 7.6, H10), 2.66 (1H, app quint, J = 6.8, H4), 2.59 (1H, ddd, J = 0.8, 6.4, and 18.8, H3α), 2.54 (1H, dd, J = 15.6 and 11.6, H6β), 2.41 (1H, dt, J = 15.2 and 2.4, H6α), 2.00 (1H, dd, J = 18.4 and 1.6, H3β), 1.92 (1H, m, H7), 1.87−1.74 (3H, overlapping m, H8,9α), 1.77 (3H, t, J = 0.8, H13), 1.52 (1H, m, H9β), 1.17 (3H, d, J = 6.8, H14), 1.03 (3H, d, J = 7.2, H15). 13C NMR: δ 208.1 (C2), 177.4 (C5), 150.8 (C11), 145.4 (C1), 109.0 (C12), 46.2 (C7), 43.1 (C3), 37.2 (C4), 36.1 (C6), 32.6 (C9), 30.9 (C8), 26.9 (C10), 20.4 (C13), 19.3 (C14), 16.7 (C15). GC−MS: 219 (16), 218 (M+, 100), 203 (42), 176 (9), 175 (28), 163 (14), 162 (18), 161 (28), 148 (13), 147 (38), 137 (25), 135 (16), 134 (21), 133 (24), 121 (15), 120 (18), 119 (30), 109 (11), 107 (19), 105 (31), 95 (12), 93 (20), 91 (38), 81(9), 79 (22), 77 (24), 69 (11), 67 (17), 65 (11), 55 (13), 53 (11), 41 (21), 39 (13). HRMS (EI) calculated for C15H22O 218.16706, found 218.16501. [α]20 D −65.8° (CHCl3; c 0.57). The retention indices of 3-epi-rotundone on a polar column (InertCap WAX) and a nonpolar column (InertCap 1MS) by GC−MS were calculated as 2305 and 1680, respectively. The analytical data of the isolated rotundone were identical to those reported previously,9 except the assignments of H6α and H6β in 1H NMR,which were inverse based on our NOESY experiment. Detection of an Unknown Compound in Several Kinds of Fruits. In our research on the identification of rotundone in orange, grapefruit, apple, and mango13 and in the quantitation of rotundone in grapefruit peel and juice,14 an unknown compound was always detected adjacent to rotundone. The procedures of sample preparation and analytical conditions were described in our previous reports.13,14 Sensory Analyses: Odor Thresholds. The odor thresholds of 3epi-rotundone and rotundone in water were determined with reference to the literature.9,15 The panelists [n = 32 (men = 22, women = 10) for 3-epi-rotundone, n = 25 (men = 16, women = 9) for rotundone] were employees of the R&D Center of T. Hasegawa Co., Ltd. They had been trained to recognize and quantify aromas using about 100 odorous chemicals and raw materials. The 3-epi-rotundone concentrations employed were 12.8, 64, 320, 1 600, 8 000, 40 000, 200 000, and 1 000 000 ng/kg. The rotundone concentrations employed were 0.00512, 0.0256, 0.128, 0.64, 3.2, 16, 80, 400, 2 000, and 10 000 ng/kg. The highest concentration samples employed were prepared by diluting an ethanol solution of 3-epi-rotundone and rotundone 10 000 times with water. An equal volume of ethanol was added to the corresponding blank samples. Two grams of each sample were administered into a closed sensory vial (total volume of 30 mL) and given a random three-digit numerical code. The samples were presented in the ascending order of sample concentration. Three samples in a set were presented in random order and identified only by three-digit random numbers. The sample that was different from the other two was always the spiked sample. Assessments were conducted orthonasally in a quiet room maintained at 23 °C. At the same time as the threshold assessment, panelists also evaluated the odor of the sample that they had successfully recognized. Each panelist was assigned a best estimate threshold value, which was the geometric mean of the highest concentration missed and the next higher concentration tested. For all panelists who successfully detected 3-epirotundone and rotundone below the highest concentration tested [n = 24 (men = 15, women = 9) for 3-epi-rotundone, n = 22 (men = 13, women = 9) for rotundone], the geometric mean of the individual best estimate thresholds was then calculated to give each subgroup threshold value.

MATERIALS AND METHODS

Materials. The high-boiling fraction of orange essential oil, grapefruit, apple, and mango were used as described in our previous reports.13,14 Chemicals. Rotundone was prepared from guaiyl acetate, which was purchased from Charavot SA (Grasse Cedex, France), as described by Wood et al. 9 The following chemicals were purchased commercially: sodium ethoxide (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and 99.5% ethanol (Junsei Chemical Co., Ltd., Tokyo, Japan). All other reagents and solvents were of analytical grade. 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) 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., Tokyo, Japan). The effluent of 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 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 5 min, and then increased to 230 °C at a rate of 3 °C/min with a constant helium carrier gas flow of 1.8 mL/min. Mass spectra in the electron impact (EI) mode were recorded at 70 eV ionization energy. The linear retention indices (RIs) of the compounds were calculated in relation to the retention times of a homologuous series of n-alkanes (C5−C30). The purities of the synthesized compounds (rotundone and 3-epi-rotundone) were calculated by integration of the chromatogram obtained using the FID. High-Resolution Mass Spectra (HRMS). The HRMS were recorded on 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 condition, oven temperature program, carrier gas, and flow rate were all the same as the conditions of GC−MS described above. Nuclear Magnetic Resonance (NMR) Spectra. 1H and 13C NMR, heteronuclear multiple quantum correlation (HMQC), heteronuclear multiple bond correlation (HMBC), and nuclear Overhauser effect spectroscopy (NOESY) were performed on a JNM-ECX400 spectrometer (JEOL Ltd., Tokyo, Japan). Using CDCl3 as solvent, chemical shifts (δ) were measured from using tetramethylsilane 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. For signal assignments of 1 H and 13C NMR, the numbers labeled in the chemical structure of Figure 1 were used. The purities of the synthesized compounds (rotundone and 3-epi-rotundone) were confirmed by the integration of the NMR signal. Specific Rotation. The specific rotation was recorded on a P-2300 polarimeter (JASCO Corporation, Tokyo, Japan). Preparation of 3-epi-Rotundone. Rotundone (150 mg, 0.687 mmol) was added to a 1.0 M ethanol solution of sodium ethoxide (5 mL), and the mixture was stirred at room temperature for 2 days. The mixture was quenched by saturated aqueous NH4Cl and extracted with ethyl acetate. The organic layer was washed with water and brine, dried over MgSO4, and concentrated in vacuo. The residue (146 mg) was purified by preparative high-performance liquid chromatography (HPLC) separation. The HPLC instrument system consisted of a Shimadzu LC-20AT, Shimadzu SIL-20AC, Shimadzu CTO-20A, Shimadzu SPD-M20A, and Shimadzu CBM-20A (Shimadzu Corporation, Kyoto, Japan). Two TCI Chiral CH-S columns (5 μm particle size, 250 mm × 4.6 mm i.d., Tokyo Chemical Industry Co., Ltd.) and a CHIRALPAK IA (5 μm particle size, 250 mm × 4.6 mm i.d., Daicel Corporation, Osaka, Japan) were connected in the instrument system. The 5% n-hexane/ethanol = 95/5 solution of the sample was injected. The isocratic elution was set as follows: 0−10 min, n-hexane/ethanol = 99/1. The solvent flow rate was 2.0 mL/min, and the column temperature was 30 °C. The detection wavelength was set at 230 nm. In the retention times from 7.35 to 7.55 min (for rotundone) and from

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RESULTS AND DISCUSSION Detection of an Unknown Compound in Several Kinds of Fruits. In our series of analyses on rotundone in the aroma of grapefruit, orange, apple, and mango,13,14 an unknown compound that had a mass spectrum similar to that of B

DOI: 10.1021/acs.jafc.7b01696 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. Mass spectra of rotundone and the unknown compound in the final isolate when rotundone was purified from the high boiling point fraction of orange essential oil.

Figure 3. Ion chromatograms (scan mode, m/z 218) of rotundone and the unknown compound in (I) white grapefruit peel, (II) apple, and (III) mango, and selected ion chromatograms (selected ion monitoring mode, m/z 218.2) of rotundone and the unknown compound in (IV) white grapefruit juice [oven temperatures: (I and III) 40 °C for 5 min, 3 °C/min to 230 °C; (II and IV) 40 °C for 3 min, 10 °C/min to 180 °C].

rotundone was always confirmed to be present accompanying rotundone. The RI on an InertCap WAX column of unknown compound was slightly larger than that of rotundone and its mass spectrum was exactly the same as that of rotundone except for the difference in ion intensity of m/z = 203. Even when rotundone was purified from the high boiling point fraction of orange essential oil, the compound was detected in the final isolate (Figure 2). Furthermore, in the course of the identification of rotundone in grapefruit, apple, and mango and the quantitation of rotundone in grapefruit peel and juice, this compound was always detected adjacent to rotundone,

although its ratio relative to rotundone differed among these different types of fruit (Figure 3). This compound did not separate with rotundone during gas chromatography using a nonpolar column (InertCap 1MS) and was detected right after rotundone which had a strong woody odor; therefore, the odor properties of this compound were difficult to evaluate by gas chromatography−olfactometry. On the basis of the RIs and mass spectrum of this compound, it was assumed that the unknown compound was a stereoisomer of rotundone. However, because the target component seemed to be less C

DOI: 10.1021/acs.jafc.7b01696 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 4. Mass spectra of rotundone-d5 and the byproduct obtained when rotundone-d5 was synthesized.

Figure 5. Mass spectra of rotundone and 3-epi-rotundone obtained when rotundone was treated with NaOEt/EtOH.

byproduct was hypothesized to be 3-epi-rotundone-d5. The difference between the MS spectra of rotundone-d5 and the byproduct was similar to the difference between the MS spectra of rotundone and the unknown compound (Figure 4). From these points, it was suggested that the target unknown compound would be 3-epi-rotundone (Figure 1). To verify this assumption, rotundone was treated with 1 M ethanol solution of NaOEt based on the method of preparation of rotundone-d5. As a result, a 1:1 mixture of rotundone and a compound that had the same mass spectrum as the unknown compound in the above-mentioned types of fruits was obtained

abundant in nature than rotundone, we considered that it would be difficult to isolate. Structural Elucidation of the Unknown Compound. When rotundone-d5 (Figure 1), which was used as an internal standard for the stable isotope dilution assay, was synthesized in our laboratory from rotundone, as described by Siebert et al.,16 a byproduct having a mass spectrum similar to that of rotundone-d5 was generated (Figure 4). However, previous studies using rotundone-d5 did not report formation of any byproduct.16−29 Considering the reaction mechanism of deuteration that the H4 of rotundone was deuterated, the D

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difference in threshold resulting simply from reversing the configuration at one stereogenic center is an interesting finding. The odor of 3-epi-rotundone was described by panelists who participated in the determination of its threshold as woody, spicy, peppery, citrus, grapefruit-like, powdery, and celery-like, which is a greater range of characteristics than that for rotundone evaluated as woody, peppery, and spicy. Interestingly, these odor descriptions could not be obtained unless 3epi-rotundone was diluted to some extent; it was almost odorless in neat form. Furthermore, most panelists experienced a strong adaptation to 3-epi-rotundone; once the odor of 3-epirotundone had been detected, detecting it again became difficult or impossible the next time. The organoleptic properties of 3-epi-rotundone are similar to those of rotundone9 and mustakone,11 namely, the odor description, anosmic properties in neat form, and strong adaptation, with the exception being the very low odor thresholds of these latter compounds (mustakone, 0.069 ng/L in air).11 This is the first study on the identification of 3-epi-rotundone in natural products and its organoleptic properties. To confirm its contribution to fruit aromas, quantitation studies are required. Our experience says that adaptation and anosmia in neat form are not a problem when mixed with other ingredients as flavorings and perfumes. Therefore, in the flavor and fragrance industry, 3-epi-rotundone might be valuable to apply a new type of woody, peppery, and spicy note, being compatible with the aroma of fruits, particularly citrus fruits, owing to its citrus and grapefruit-like odor and comparatively low odor threshold.

(Figure 5). To obtain structural data on this unknown compound, it was isolated from the mixture using preparative HPLC. Because rotundone has a very low odor threshold, even a trace contamination of rotundone could affect the organoleptic properties of the target compound; therefore, complete separation was required. Because of the high similarity in polarity, chiral HPLC columns were chosen. To further optimize the separation process, three chiral HPLC columns (two columns of Chiral CH-S and one column of CHIRALPAK IA) were used in conjunction, which resulted in the pure target compound being obtained successfully. The 1D and 2D NMR data of the target compound were comparable to those of rotundone and supported the assertion that it is a stereoisomer of rotundone. To confirm its stereochemistry, NOESY experiments were performed on rotundone and the stereoisomer (Figure 6). When comparing

Figure 6. Selected NOE correlations observed in the NOESY of rotundone and 3-epi-rotundone.

both correlations in H14, rotundone had correlations between H14 and H3β, H4, H6α, and H6β whereas the stereoisomer had correlations between H14 and H3α, H3β, H4, H6α, and H7. These findings revealed that the stereoisomer generated by base treatment of rotundone was 3-epi-rotundone [(3R,5R,8S)-5isopropenyl-3,8-dimethyl-3,4,5,6,7,8-hexahydro-1(2H)-azulenone]. The mass spectrum and RI of the unknown compound in the different types of fruits were identical to those of 3-epirotundone; therefore, it was confirmed that 3-epi-rotundone is present in nature. Organoleptic Properties of 3-epi-Rotundone. Rotundone has a very low olfactory threshold in water (8 ng/L),9 whereas the odor of isolated 3-epi-rotundone was relatively weak. Therefore, the odor thresholds of these compounds in water were determined by triangle testing (Table 1). Wood et al. reported that ∼20% of the panelists could not detect rotundone at the highest concentration tested in the odor threshold experiment.9 In our odor threshold tests, 8 out of 32 panelists for 3-epi-rotundone and 3 out of 25 panelists for rotundone could not be detectable at the highest concentration tested as well; therefore, we calculated the odor thresholds for the subgroup of the panelists who succeeded in detection. The subgroup odor threshold of rotundone (5 ng/L) determined in this study showed good similarity to the value reported previously in the literature (8 ng/L).9 The results revealed that the subgroup odor threshold of 3-epi-rotundone (19100 ng/L) is relatively low in the class of food-related odorants, but it was ∼4000 times higher than that of rotundone. This marked

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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.7b01696. NMR spectra of rotundone and 3-epi-rotundone (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-44-411-0298. Fax: +81-44-434-5257. E-mail: [email protected]. ORCID

Akira Nakanishi: 0000-0003-4764-7744 Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED GC−MS, gas chromatography−mass spectrometry; FID, flame ionization detector; EI, electron impact; RI, retention index; NMR, nuclear magnetic resonance; HMQC, heteronuclear multiple quantum correlation; HMBC, heteronuclear multiple bond correlation; NOESY, nuclear Overhauser effect spectroscopy; HRMS, high-resolution mass spectra; HPLC, highperformance liquid chromatography

Table 1. Odor Thresholds in Water and Odor Descriptions of 3-epi-Rotundone and Rotundone 3-epi-rotundone rotundone a

RI on a polar columna

threshold (ng/L) in waterb

odor descriptionc

2305 2300

19100 5

woody, spicy, peppery, citrus, grapefruit-like, powdery, celery-like woody, spicy, peppery

InertCap WAX. bCalculated as recognition threshold. cEvaluated at the concentrations around each threshold. E

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rotundone concentration in red wine made from Vitis vinifera L. cv. Duras. Aust. J. Grape Wine Res. 2014, 20, 401−408. (22) Mayr, C. M.; Geue, J. P.; Holt, H. E.; Pearson, W. P.; Jeffery, D. W.; Francis, I. L. Characterization of the key aroma compounds in Shiraz wine by quantitation, aroma reconstitution, and omission studies. J. Agric. Food Chem. 2014, 62, 4528−4536. (23) Takase, H.; Sasaki, K.; Shinmori, H.; Shinohara, A.; Mochizuki, C.; Kobayashi, H.; Saito, H.; Matsuo, H.; Suzuki, S.; Takata, R. Analysis of rotundone in Japanese Syrah grapes and wines using SBSE with heart-cutting two-dimensional GC/MS. Am. J. Enol. Vitic. 2015, 66, 398−402. (24) Huang, A.-C.; Sefton, M. A.; Taylor, D. K. Comparison of the formation of peppery and woody sesquiterpenes derived from αguaiene and α-bulnesene under aerial oxidative conditions. J. Agric. Food Chem. 2015, 63, 1932−1938. (25) Davies, C.; Nicholson, E. L.; Böttcher, C.; Burbidge, C. A.; Bastian, S. E. P.; Harvey, K. E.; Huang, A.-C.; Taylor, D. K.; Boss, P. K. Shiraz wines made from grape berries (Vitis vinifera) delayed in ripening by plant growth regulator treatment have elevated rotundone concentrations and “pepper” flavor and aroma. J. Agric. Food Chem. 2015, 63, 2137−2144. (26) Zhang, P.; Barlow, S.; Krstic, M.; Herderich, M.; Fuentes, S.; Howell, K. Within-vineyard, within-vine, and within-bunch variability of the rotundone concentration in berries of Vitis vinifera L. cv. Shiraz. J. Agric. Food Chem. 2015, 63, 4276−4283. (27) Zhang, P.; Howell, K.; Krstic, M.; Herderich, M.; Barlow, E. W. R.; Fuentes, S. Environmental factors and seasonality affect the concentration of rotundone in Vitis vinifera L. cv. Shiraz wine. PLoS One 2015, 10, e0133137. (28) Takase, H.; Sasaki, K.; Shinmori, H.; Shinohara, A.; Mochizuki, C.; Kobayashi, H.; Ikoma, G.; Saito, H.; Matsuo, H.; Suzuki, S.; Takata, R. Cytochrome P450 CYP71BE5 in grapevine (Vitis vinifera) catalyzes the formation of the spicy aroma compound (−)-rotundone. J. Exp. Bot. 2016, 67, 787−798. (29) Mattivi, F.; Caputi, L.; Carlin, S.; Lanza, T.; Minozzi, M.; Nanni, D.; Valenti, L.; Vrhovsek, U. Effective analysis of rotundone at belowthreshold levels in red and white wines using solid-phase microextraction gas chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2011, 25, 483−488.

REFERENCES

(1) Li, D.; Wang, K.-W. Natural new sesquiterpenes: structural diversity and bioactivity. Curr. Org. Chem. 2016, 20, 994−1042. (2) Kapadia, V. H.; Naik, V. G.; Wadia, M. S.; Dev, S. Sesquiterpenoids from the essential oil of Cyperus rotundus. Tetrahedron Lett. 1967, 8, 4661−4667. (3) Ishihara, M.; Tsuneya, T.; Uneyama, K. Guaiane sesquiterpenes from agarwood. Phytochemistry 1991, 30, 3343−3347. (4) Ishihara, M.; Tsuneya, T.; Uneyama, K. Components of the volatile concentrate of agarwood. J. Essent. Oil Res. 1993, 5, 283−289. (5) Naef, R. The volatile and semi-volatile constituents of agarwood, the infected heartwood of Aquilaria species. Flavour Fragrance J. 2011, 26, 73−89. (6) Tajuddin, S. N.; Yusoff, M. M. Chemical composition of volatile oils of Aquilaria malaccensis (Thymelaeaceae) from Malaysia. Nat. Prod. Commun. 2010, 5, 1965−1968. (7) Pandey, A. K.; Chowdhury, A. R. Essential oil of Cyperus scariosus R. Br. tubers from Central India. Indian Perfum. 2002, 46, 325−328. (8) Kaiser, R. Agarwood − the wood of the gods. In Meaningful Scents around the World: Olfactory, Chemical, Biological and Cultural Considerations; Kisakürek, M. V., Ed.; Verlag Helvetica Chimica Acta: Zürich, Switzerland, 2006; pp 59−64. (9) 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. (10) Genthner, E. R. Identification of rotundone as an important contributor to the flavor of oak aged spirits. Ph.D. Dissertation, University of Illinois at Urbana-Champaign, Champaign, IL, 2014. (11) 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. (12) Clery, R. A.; Cason, J. R. L.; Zelenay, V. Constituents of cypriol oil (Cyperus scariosus R. Br.): N-containing molecules and key aroma components. J. Agric. Food Chem. 2016, 64, 4566−4573. (13) Nakanishi, A.; Miyazawa, N.; Fukushima, Y.; Yoshikawa, K.; Maeda, T.; Kurobayashi, Y. Identification of rotundone as a potent odor-active compound of several kinds of fruits. J. Agric. Food Chem. 2017, 65, 4464. (14) Nakanishi, A.; Miyazawa, N.; Fukushima, Y.; Yoshikawa, K.; Maeda, T.; Kurobayashi, Y. Quantitaion of rotundone in grapefruit (Citrus paradisi) peel and juice by stable isotope dilution assay. J. Agric. Food Chem. 2017, DOI: 10.1021/acs.jafc.7b01319. (15) Meilgaard, M.; Civille, G. V.; Carr, B. T. Sensory Evaluation Techniques, 5th ed.; CRC Press: Boca Raton, FL, 2016; pp 153−163. (16) Siebert, T. E.; Wood, C.; Elsey, G. M.; Pollnitz, A. P. Determination of rotundone, the pepper aroma impact compound, in grapes and wine. J. Agric. Food Chem. 2008, 56, 3745−3748. (17) Caputi, L.; Carlin, S.; Ghiglieno, I.; Stefanini, M.; Valenti, L.; Vrhovsek, U.; Mattivi, F. Relationship of changes in rotundone content during grape ripening and winemaking to manipulation of the ‘peppery’ character of wine. J. Agric. Food Chem. 2011, 59, 5565−5571. (18) Capone, D. L.; Jeffery, D. W.; Sefton, M. A. Vineyard and fermentation studies to elucidate the origin of 1,8-cineole in Australian red wine. J. Agric. Food Chem. 2012, 60, 2281−2287. (19) Herderich, M. J.; Siebert, T. E.; Parker, M.; Capone, D. L.; Jeffery, D. W.; Osidacz, P.; Francis, I. L. Spice up your life: Analysis of key aroma compounds in Shiraz. In Flavor Chemistry of Wine and Other Alcoholic Beverages; Qian, M. C., Shellhammer, T. H., Eds.; ACS Symposium Series 1104; American Chemical Society: Washington, DC, 2012; pp 3−13. (20) Scarlett, N. J.; Bramley, R. G. V.; Siebert, T. E. Within-vineyard variation in the ‘pepper compound’ rotundone is spatially structured and related to variation in the land underlying the vineyard. Aust. J. Grape Wine Res. 2014, 20, 214−222. (21) Geffroy, O.; Dufourcq, T.; Carcenac, D.; Siebert, T.; Herderich, M.; Serrano, E. Effect of ripeness and viticultural techniques on the F

DOI: 10.1021/acs.jafc.7b01696 J. Agric. Food Chem. XXXX, XXX, XXX−XXX