Article pubs.acs.org/JAFC
Constituents of Cypriol Oil (Cyperus scariosus R.Br.): N‑Containing Molecules and Key Aroma Components Robin A. Clery,* Julie R. L. Cason, and Veronika Zelenay Givaudan Schweiz AG, Ueberlandstrasse 138, CH-8600 Dübendorf, Switzerland S Supporting Information *
ABSTRACT: Cypriol oil, the essential oil from Cyperus scariosus R.Br., has been investigated to reveal minor nitrogen-containing molecules and minor components responsible for the odor. A total of 21 nitrogenous components are reported, of which epiguaipyridine (32 mg/kg), guaia-9,11-dienpyridine (9 mg/kg), and cananodine (10 mg/kg) were the most abundant. A new ketone, cyperen-8-one, with a significant woody, ambery odor could also be isolated and identified along with a novel lactone, cyperolactone, and an alcohol. Rotundone was found to have the highest odor-activity value of the measured compounds and, together with the other ketones, contributes to the woody−amber character of cypriol oil. KEYWORDS: Cyperus scariosus, Cyperaceae, cypriol, nagarmotha, volatiles, aroma, guaia-9,11-dienpyridine, cyperen-8-one, cyperolactone
■
INTRODUCTION Cypriol oil is the essential oil steam-distilled from the rhizomes of Cyperus scariosus, which is known in India as Nagarmotha, Nagar Mustaka, or nut grass and has a long history of traditional use in both medicine and perfumery.1 The essential oil is produced commercially from wild collected plants that grow abundantly in certain parts of India, especially in riverine environments in the states of Madhya Pradesh and Chhattisgarh. The plant regenerates and spreads rapidly by means of its rhizomes and is thus considered a weed when it invades cultivated land. The essential oil has long been used in perfumery and has a warm, woody, spicy, balsamic character with facets of cedarwood, vetiver, and sometimes a smoky character if the rhizomes have been burnt or dried over a fire during processing. C. scariosus also grows wild across the Indian subcontinent, in East Africa, around the Indian Ocean, in China and Australia, although the precise botanical assignment of some specimens has recently brought the correct botanical nomenclature for this species into question.2 Many other species in the genus Cyperus also produce aromatic essential oils, notably C. rotundus, C. maculatus, C. articulatus, and C. alopecuroides, some of which grow in the same range as C. scariosus. Some old publications,3,4 describe isolation of the main components from C. scariosus, but few recent publications deal adequately with the volatile composition of this essential oil. The major components of the closely related species C. rotundus have been widely reported with specific molecules identified by Ohira,5 while Sonwa and Koenig6 identified further minor components, and Jeong7 identified three nitrogenous rotundines. Reports of composition from the genus Cyperus have been reviewed by Gamal,8 who also reviewed the nitrogenous compounds9 but does not mention the guaipyridines we report here. As yet, no studies have established the link between the characteristic odor and the chemical composition. A recent review of C. scariosus by Srivastava10 cites various identifications for components found © XXXX American Chemical Society
in cypriol oil; unfortunately, several structures are incorrect, and we were not able to resolve many of the identifications given. Recently, the demand for cypriol in perfumery has increased because of its unique woody, ambery, and earthy character that is appreciated by perfumers as an alternative to the more expensive agarwood oil and which is difficult to reproduce synthetically. The odor quality can be very variable between sources and this lead us to investigate the composition in more detail, especially the nitrogenous components as previously described in ambrette oil.11 Thus, we aim to investigate minor and trace components that may have low odor-detection thresholds to identify previously unreported components that contribute to the desirable odor.
■
MATERIALS AND METHODS Chemicals. A 1 kg sample of cypriol oil was taken from our existing stock of commercially available cypriol oil, previously purchased from the state of Chhattisgarh in India. Reagents, solvents and some reference materials were purchased from Fluka/Sigma-Aldrich (Buchs, Switzerland): HCl (≥32%), pentane (≥99% GC), hexane−HPLC plus (≥95% GC), ethyl acetate−Chromasolv (≥99.7% GC), ethanol−HPLC (≥99.8%), tert-butyl methyl ether (MTBE)−HPLC plus (≥99.8%), tetradecane (≥99%), methyl decanoate (≥99%), phosphomolybdic acid solution (20% in ethanol), and TLC plates (10 × 20 cm, silica gel + fluorescence indicator). Anhydrous salts were purchased from Acros Organics (Fisher Scientific); magnesium sulfate (MgSO4) (≥97%, anhydrous), and sodium carbonate (Na2CO3) (≥99%, anhydrous). C6D6 solution for nuclear magnetic resonance (NMR) was purchased from Euriso-top (St. Aubin, France). A reference standard of pure nootkatone had previously been produced in-house by Received: February 10, 2016 Revised: May 12, 2016 Accepted: May 15, 2016
A
DOI: 10.1021/acs.jafc.6b00680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
was 40 °C, 2 °C/min to 280 °C (4 min). The injector and detector temperatures were held constant at 200 and 300 °C, respectively. Data were acquired and processed using HP ChemStation software. Quantitation. The quantitation of components in the essential oil and basic fraction was achieved following the principles described by Bicchi16 using the FID signal from the 50m HP-5 column with external calibration. For the essential oil, tetradecane was used as reference standard for the hydrocarbon peaks and nootkatone as the reference standard for the sesquiterpene ketones and other oxygenated compounds. For the basic fraction, quinoline was used as the reference standard. A five-point calibration was made covering the concentration range observed in the sample and the linear equation for the line of best fit used to calculate the g/kg value from peak areas. Compounds identified and characterized in subfractions but below the detection limit in the calibrated chromatogram were recorded as “trace”. GC−FID and Olfactometry. Polar-phase analysis was carried out using an Thermo Trace1300 GC system modified with an in-house olfactory detection port and fitted with a ZBWax-plus (polyethylene glycol) 30 m × 0.32 mm 0.25 μm film thickness column with hydrogen carrier at 2 mL/min and a temperature program of 50 °C (2 min) and 5 °C/min to 250 °C (2 min). The effluent from the column was split equally between the FID and nose port by means of a deactivated glass Y splitter and equal lengths of 0.25 mm i.d. uncoated fused silica transfer lines. The olfactory detector and FID were held at 230 and 280 °C respectively. Data was collected using Chromeleon 7.0 software from Dionex and an in-house voice recording system to register odor descriptions. GC Odor-Detection Thresholds. The GC odor-detection thresholds were measured on this system and recorded as the lowest perceived total mass (ng) of odorant delivered to the smelling port as calculated from FID peak area using external calibration with methyl decanoate. Although these data may not be directly comparable with literature values measured by other methods they are, in our experience, approximately equivalent to values of ng/L in air measured with a conventional olfactometer. The advantage of this technique is the ability to evaluate minute samples of material and to evaluate compounds without the need to produce olfactorily pure samples for assessment. Fractionation by Flash Chromatography: Basic Fraction. The basic extract was fractionated by flash chromatography using an Armen pump (Interchim, France) and a LS5600 fraction collector. A total of 400 mg of basic extract was dissolved in 1 mL of hexane, loaded onto a preconditioned 25 g silica cartridge (PF15SiHP Interchim, France), and eluted at 20 mL/min with a step gradient composed of 200 mL of hexane− ethyl acetate (40:60); 90 mL of ethyl acetate; and 70 mL of ethanol. Fractions of 10 mL were collected and analyzed by TLC, GC−FID, and GC−MS. Fractionation by Flash Chromatography: Neutral Fraction. The neutral fraction was separated by flash chromatography using an Armen pump (Interchim, France) and an LS-5600 fraction collector. A total of 4 g was dissolved in 4 mL of hexane and loaded onto a preconditioned 120 g silica cartridge and eluted at 80 mL/min with a step-gradient composed of 800 mL of hexane; 1200 mL of hexane−ethyl acetate (95:5); 600 mL of ethyl acetate ; 200 mL of ethanol. Fractions of 25 mL were collected and analyzed by TLC, GC− FID, and GC−MS. Fractions were olfactorily assessed and
recrystallization of a synthetic sample from hexane to a purity of >99% by GC. Calibration mixtures for RI calculations were prepared from individual components purchased from Fluka/ Sigma-Aldrich. Extraction. Cypriol oil (800 g) was washed with 2 M HCl solution (4 × 300 mL), rinsed with water (3 × 300 mL), then dried with anhydrous MgSO4 and filtered to give the neutral fraction. This was stored in a fridge at 4 °C. The acidic washes (pH 1−2) were combined and back-washed with pentane (3 × 300 mL) and then basified with Na2CO3 to pH 8. This neutralized aqueous phase was extracted with methyl tert-butyl ether (MTBE) (3 × 300 mL) and the MTBE extract dried with anhydrous MgSO4, filtered and concentrated on a rotary evaporator at 40 °C under vacuum to give 650 mg of the basic fraction. The original essential oil and the basic fraction containing the nitrogenous components were submitted to gas chromatography−mass spectrometry (GC−MS) and gas chromatography−olfactometry (GC−O). The GC−MS analyses indicated a significant number of peaks for which no adequate match could be found in the MS libraries. Further flash fractionation and purification was followed to isolate these compounds for NMR analysis. Gas Chromatography−Mass Spectrometry and Olfactometry (GC−MS/O). Identification and odor analysis of components was carried out using an Agilent 6890N GC, fitted with a DB-5 (5% diphenyl polysiloxane) capillary column (50 m × 0.32 mm i.d., 0.52 μm film thickness), with helium carrier gas at 2 mL/min. Injection was 1 μL splitless (0.75 min). The column effluent was split between an Agilent 5975 inert MSD (EI 70 eV, mass range 25−350) and a specially modified Gerstel odor detection port (ODP2) via a Capillary Flow Technology splitter plate. The injector and MSD transfer line were held constant at 250 °C. In general, GC−O experiments were carried out with an oven program: 35 °C (1 min), 15 °C/ min ramp to 50 °C, then 5 °C/min ramp to 280 °C (hold for 5 min), and the analytical data and retention indices were calculated from a program with a slower ramp of 2 °C/min. Data was acquired and processed using MSD ChemStation (Rev. D.02.00.275) and Gerstel ODP Recorder software (version 2.7.5.2). Gas Chromatography−Mass Spectrometry (GC−MS) Polar Phase. Identification of components by MS and retention index (RI) measurements on a polar phase were carried out using an Agilent 7890 GC with 5977A MSD fitted with an Innowax (polyethylene glycol) capillary column (30 m × 0.25 mm i.d. × 0.25 μm film thickness) with helium carrier gas at 1.2 mL/min constant flow. Injection was 1 μL, split (1:50). The temperature program was 35 °C (2 min), 10 °C/ min to 50 °C, and 2.5 °C/min to 240 °C (5 min). The injector temperature was 230 °C, MS: EI 70 eV, mass range 25−350. Peak identification was primarily achieved using AMDIS software in retention indices (RI) calibration mode with RI calibrated in-house MS libraries and using commercially available MS libraries12−14 with the NIST MS Search software. RI values were calculated according to van den Dool15 using nalkanes for the DB5 phase and both n-alkanes and methyl esters of linear aliphatic acids for the polar wax phase. Gas Chromatography−Flame Ionization Detector (GC−FID). Analysis was carried out using a Hewlett-Packard 6890 gas chromatograph, fitted with a HP-5 (5% diphenyl polysiloxane) capillary column (50 m × 0.2 mm i.d., 0.33 μm film thickness). Injection was 1 μL split (1:10) with helium carrier gas at 56 psi (2.5 mL/min). The temperature program B
DOI: 10.1021/acs.jafc.6b00680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Guaipyridine (2) C15H21N. GC−MS (EI) m/z (%): 215 (59, M+), 214 (55), 200 (35), 186 (69), 174 (29), 172 (35), 158 (100), 146 (27), 144 (45), 132 (38). RIDB5(Alk) 1660, RIWAX(ME) 1504. 1H NMR (C6D6, 600 MHz): δH 6.96 (1H, d, J = 7.7 Hz, H2), 6.63 (1H, d, J = 7.7 Hz, H3), 4.82 (1H, s, H13b), 4.75 (1H, br s, H13a), 3.41 (1H, br d, J = 14.3 Hz, H6b), 3.27 (1H, dd, J = 14.3, 9.8 Hz, H6a), 2.72−2.66 (1H, m, H10), 2.45 (3H, s, H15), 2.19 (1H, br t, J = 10.0 Hz, H7), 1.83−1.76 (1H, m, H8b), 1.64−1.60 (1H, m, H8a), 1.59−1.55 (2H, m, H9), 1.73 (3H, s, H12), 1.12 (3H, d, J = 7.2 Hz, H14), 13C NMR (C6D6, 150 MHz): δC 159.7 (C5), 154.8 (C4), 150.9 (C11), 137.3 (C1), 135.8 (C2), 120.5 (C3), 109 (C13), 44.6 (C7), 44.4 (C6), 37.8 (C10), 33.4 C9), 31.4 (C8), 24 (C15), 20.8 (C12), 18.4 (C14). Guai-9,11-dienpyridine (3) C15H19N. LC−HRMS m/z: [M + H]+ calc, 214.1590; found, 214.1590. GC−MS (EI) m/z (%): 213 (38, M+), 212 (49), 198 (47), 182 (15), 172 (100), 170 (17), 158 (43), 156 (25), 144 (26). RIDB5(Alk) 1664, RIWAX(ME) 1532. 1H NMR (C6D6, 600 MHz): δH 7.11 (1H, d, J = 7.9 Hz, H2), 6.70 (1H, d, J = 7.5 Hz, H3), 5.84 (1H, tq, J = 7.2, 1.5 Hz, H9), 5.03 (1H, s, H13b), 4.77 (1H, quint, J = 1.5 Hz, H13a), 3.16 (1H, dd, J = 11.7, 6.4 Hz, H6b), 3.08 (1H, quint, J = 6.8 Hz, H7), 3.03 (1H, dd, J = 11.7, 7.5 Hz, H6a), 2.48 (3H, s, H15), 2.02−1.97 (1H, m, H8b), 1.95−1.90 (1H, m, H8a), 1.88 (3H, s, H14), 1.70 (3H, s, H12), 13C NMR (C6D6, 150 MHz): δC 159.3 (C5), 155.9 (C4), 149.3 (C11), 135.1 (C1), 133.3 (C2), 133.3 (C10), 127 (C9), 120.5 (C3), 109.7 (C12), 54.7 (C7), 41.3 (C6), 31.1 (C8), 24.4 (C15), 22.2 (C14), 21.3 (C13). Cananodine (4) C15H23NO. GC−MS (EI) m/z (%): 175 (73), 174 (90), 172 (20), 160 (100), 158 (39), 147 (27), 146 (50), 144 (34), 132 (42), 59 (47). RIDB5(Alk) 1833, RIWAX(ME) 1998. 1H NMR (C6D6, 600 MHz): δH 7.1 (1H, d, J = 7.9 Hz, H2), 6.7 (1H, d, J = 7.9 Hz, H3), 3.53 (1H, d, J = 13.2 Hz, H6a), 2.73 (1H, dd, J = 13.2, 9.4 Hz, H6b), 2.67−2.62 (1H, m, H10), 2.48 (3H, s, H15), 1.96−1.90 (1H, m, H8a), 1.64−1.60 (1H, m, H9a), 1.32−1.28 (2H, m, H8b, H7), 1.12 (3H, s, H12), 1.1−1.04 (1H, m, H9b), 1.09 (3H, d, J = 7.2 Hz, H14), 1.04 (3H, s, H13). 13C NMR (C6D6, 150 MHz): δC 161.4 (C5), 154.3 (C4), 137.7 (C1), 131.7 (C2), 120.2 (C3), 92.5 (C11), 48.2 (C7), 40.1 (C6), 36.5 (C9), 35.2 (C10), 32.9 (C8), 27.4 (C12), 26.3 (C13), 23.9 (C15), 20.6 (C14). Cyperen-8-one (5) C15H22O. LC−HRMS m/z: [M + H]+ calc, 219.1743; found, 219.1742. GC−MS (EI) m/z (%): 218 (50, M+), 175 (40), 159 (20), 147 (60), 133 (100), 119 (23), 105 (46), 93 (20), 91 (32), 77 (19), RIDB5(Alk) 1626, RIWAX(ME) 1454. 1H NMR (C6D6, 600 MHz): δH 2.59−2.53 (1H, m, H3b), 2.46 (1H, d, J = 6.4 Hz, H7), 2.17 (1H, dd, J = 16.2, 6.8 Hz, H9b), 2.14 (1H, dd, J = 16.2, 10.5 Hz, H3a), 2.02−1.97 (1H, m, H2b), 1.87 (1H, dquint, J = 10.9, 6.8 Hz, H10), 1.75 (1H, ddd, J = 16.6, 10.9, 1.5 Hz, H9a), 1.69−1.65 (2H, m, H6), 1.46 (3H, s, H15), 1.24 (1H, ddd, J = 13.2, 7.9, 1.1 Hz, H2a), 0.78 (3H, s, H13), 0.74 (3H, d, J = 6.4 Hz, H14), 0.66 (3H, s, H12). 13C NMR (C6D6, 150 MHz): δC 210.8 (C8), 140.4 (C5), 130.5 (C4),66 (C1), 64.9 (C7), 44 (C9), 43.1 (C11), 42.6 (C3), 36.4 (C10), 26.4 (C6), 26.1 (C2), 25.1 (C12), 20 (C13), 17.4 (C14), 14.1 (C15). 3,4,8,8-Tetramethyl-4,5,6,7,8,8a-hexahydro-1H-3a,7methanoazulen-4-ol (6) C15H24O. LC−HRMS m/z: [M-H2O +H]+ calc, 203.1794; found, 203.1794. GC−MS (EI) m/z (%): 220 (28, M+), 205 (30), 147 (100), 145 (32), 121 (35), 120 (27), 119 (30), 107 (27), 105 (33), 91 (35). RIDB5(Alk) 1573, RIWAX(ME) 1432. 1H NMR (C6D6, 600 MHz): δH 5.2−5.18
bulked and concentrated according to the TLC, GC, and olfactory results. The fractions containing the target compounds were further fractionated using a 25 g silica cartridge (PF15SiHP Interchim, France) eluted at 20 mL/min with 320 mL of hexane−ethyl acetate (98:2). Fractions of 10 mL were collected and analyzed by TLC, GC−FID, and GC−MS. The fractions containing the target compounds were bulked, concentrated, and submitted to preparative gas chromatography (prep-GC). Prep-GC. Isolation and purification of compounds in quantities of 10−100 μg was achieved by preparative gas chromatography using either a DB-5 or Supelcowax column of dimensions 30 m × 0.75 mm i.d. × 1 μm film thickness. The effluent from the column was divided by a deactivated glass Y splitter to the FID and a trapping port to which is fitted a glass tube (0.5 mm i.d.) containing 5 mg Porapak Q as adsorbent, in turn connected to a valve and slight vacuum. At the point of elution of the peak from the column, as monitored by the FID, the valve is manually opened for the duration of the peak and the effluent from the column is drawn through the trap by the vacuum. The oven is typically held isothermal at a preoptimized temperature, such that repeated injections can be made and the target peaks trapped efficiently. The sample is desorbed from the glass trap using C6D6 and collected directly in a microNMR tube. Nuclear Magnetic Resonance Spectroscopy. NMR spectra were recorded on a Bruker Avance 600 spectrometer at 600 MHz (1H NMR) and 150 MHz (13C NMR) with a TCI cryoprobe probehead (Bruker) using C6D6. Chemical shift values (δ) were calibrated against a residual benzene signal set at 7.16 ppm (1H NMR) and 128.0 ppm (13C NMR). Assignments by heteronuclear single-quantum correlation (HSQC), heteronuclear multiple-bond correlation (HMBC), correlated spectroscopy (COSY), and nuclear Overhauser effect spectroscopy (NOESY) experiments were performed with TopSpin software from Bruker. The mixing time in the NOESY experiment was 2 s. High-Resolution Mass Spectroscopy. HRMS spectra were acquired on a Q-Exactive Orbi-trap LCMS (Thermo) system by direct injection of the diluted NMR solution. Electrospray ionization in positive (ESI+) and negative (ESI−) mode. MS acquisition in full-scan mode from 50 to 750 m/z with high mass resolution (140 000,
