Characterization of the Major Odor-Active Compounds in the Leaves

Apr 9, 2015 - Confirmation of 1-Phenylethane-1-thiol as the Character Impact Aroma Compound in Curry Leaves and Its Behavior during Tissue Disruption,...
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Characterization of the Major Odor-Active Compounds in the Leaves of the Curry Tree Bergera koenigii L. by Aroma Extract Dilution Analysis Martin Steinhaus* Deutsche Forschungsanstalt für Lebensmittelchemie (German Research Center for Food Chemistry), Lise-Meitner-Straße 34, 85354 Freising, Germany ABSTRACT: Curry leaves are a popular seasoning herb with a pronounced sulfury and burnt odor, the molecular background of which was yet unclear. Application of an aroma extract dilution analysis to the volatile fraction of curry leaves isolated by solvent extraction and solvent-assisted flavor evaporation afforded 23 odor-active compounds with flavor dilution (FD) factors ranging from 1 to 8192. On the basis of the comparison of their retention indices, mass spectra, and odor properties with data of reference compounds, the structures of 22 odorants could be assigned, 15 of which had not been reported in curry leaves before. Odorants with high FD factors included 1-phenylethanethiol (FD factor 8192), linalool (4096), α-pinene (2048), 1,8-cineole (1024), (3Z)-hex-3-enal (256), 3-(methylsulfanyl)propanal (128), myrcene (64), (3Z)-hex-3-en-1-ol (32), and (2E,6Z)-nona2,6-dienal (32). The unique sulfury and burnt odor exhibited by 1-phenylethanethiol in combination with its high FD factor suggested that it constitutes the character impact compound of fresh curry leaf aroma. KEYWORDS: Bergera koenigii, Murraya koenigii, curry tree, curry leaves, 1-phenylethanethiol

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INTRODUCTION The curry tree Bergera koenigii L., previously known as Murraya koenigii (L.) Spreng. but now grouped into the genus Bergera,1 is a small evergreen tropical tree in the rue family (Rutaceae) and native to South and Southeast Asia. Its imparipinnate compound leaves are up to 35 cm in length and consist of up to 25 leaflets. The individual leaflets, commonly known as “curry leaves”, grow up to a size of 6 cm × 2 cm and show an asymmetrically lanceolate shape. Curry leaves are a popular seasoning herb, particularly in southern Indian and Sri Lankan cuisine, where they are widely used in curries and chutneys, thus substantially contributing to the characteristic local food flavor. In these tropical regions, the curry trees are usually grown as home garden plants, because only the freshly picked leaves exhibit the desired aroma. In the temperate zones of the United States and Europe, where the popularity of curry leaves has immensely increased in recent years, potted indoor plants provide steady access to fresh leaves with an authentic aroma. The characteristic aroma of curry leaves combines green and terpeny notes with a pronounced sulfury and burnt odor note. Numerous reports on curry leaf volatiles have been published to date.2−18 In the pioneering study,2 27 compounds were identified in the essential oil obtained from Sri Lankan curry leaves by hydrodistillation; most of them were terpene and sesquiterpene hydrocarbons. Further studies utilized plant material grown in Bangladesh,10 China,3 India,5,6,8,9,11,13,15−17 Malaysia,4,14 Nigeria,7 and Thailand18 and led to a total number of structurally characterized curry leaf oil constituents of >100. Among the major oil components reported for different curry leaf oils were β-caryophyllene, α-pinene, β-phellandrene, terpinen-4-ol, sabinene, (E)-β-ocimene, (Z)-β-ocimene, αphellandrene, α-copaene, 3-carene, and α-humulene. On the basis of the analysis of major leaf oil constituents, curry trees can be grouped into distinct chemotypes.12,14,16 A very recent © XXXX American Chemical Society

study is so far the only one that aimed at identifying the volatiles not only in the hydrodistilled essential oil but also in the fresh leaves.18 Using solid phase microextraction, 51 compounds, almost exclusively (sesqui)terpenes and (sesqui)terpenoids, were detected in the headspace above freshly picked leaf material. Despite the large number of investigations on curry leaf volatiles, no systematic study has yet aimed at evaluating the contribution of individual compounds to the aroma of curry leaves. In particular, none of the substances so far reported as volatile constituent was able to plausibly explain the sulfury and burnt note, which is highly characteristic for curry leaf aroma independent of the plant origin. To approach the molecular background of fresh curry leaf aroma, the aims of the present study were to isolate the volatiles of freshly picked curry leaves using a mild workup procedure based on solvent extraction and solvent-assisted flavor evaporation (SAFE)19 and to screen the volatile fraction for aroma-active compounds by application of an aroma extract dilution analysis (AEDA).20 To cover very highly volatile odorants not accessible by AEDA, a static headspace GC-O (SH-GC-O) analysis was additionally performed.

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MATERIALS AND METHODS

Curry Leaves. Seven-year-old trees of B. koenigii L. were purchased in a local garden center near Freising, Germany, in 2012 and kept indoors at temperatures between 22 and 25 °C. For analyses, young, but fully developed, healthy leaves were picked, and the leaflets were stripped off by hand. To confirm that they exhibited the typical aroma Received: March 6, 2015 Revised: April 9, 2015 Accepted: April 9, 2015

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DOI: 10.1021/acs.jafc.5b01174 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry profile, some leaflets were crushed between fingertips and then orthonasally evaluated by experienced panelists familiar with authentic Sri Lankan cuisine. Reference Odorants. 1, 2, (R)-2, (S)-2, 4−7, 10, 14−19, (R)-19, 20, 21, and 23−27 were obtained from Sigma-Aldrich (Taufkirchen, Germany); 3, 8, 11−13, (R)-21, and (S)-26 were synthesized as detailed below. Miscellaneous Chemicals and Reagents. 6-Methylheptanoic acid was purchased from Alfa Aesar (Karlsruhe, Germany). 7Methyloctan-1-ol was from C/D/N Isotopes (Pointe-Claire, QC, Canada). Dess−Martin periodinane and (2S)-2-methylbutan-1-ol were from Sigma-Aldrich. Dichloromethane, diethyl ether, and pentane were freshly distilled before use. Silica gel 60 (0.040−0.063 mm) was purchased from VWR (Darmstadt, Germany) and purified as follows: After extraction with hydrochloric acid (32%; 3 h), the gel was washed with water until the eluate was acid free, dried at 120 °C until constant weight, and adjusted to 7% water content. Mercurated agarose gel was prepared from Affi-Gel 10 (Bio-Rad, Munich, Germany).21 Syntheses. The following reference odorants were synthesized using procedures detailed in the literature: 3,22 11,23 13,24 and (S)21.25 (S)-26 was obtained from (2S)-2-methylbutan-1-ol by oxidation with Dess−Martin periodinane,26 as detailed below for the synthesis of 6-methylheptanal. 6-Methylheptan-1-ol. In an argon atmosphere a solution of 6methylheptanoic acid (288 mg, 2 mmol) in anhydrous diethyl ether (5 mL) was added to a suspension of lithium aluminum hydride (152 mg, 4 mmol) in anhydrous diethyl ether (10 mL) under stirring. The mixture was heated at reflux for 1 h. Under cooling (0 °C) a saturated aqueous ammonium chloride solution (10 mL) was added, followed by hydrochloric acid (2 mol/L; 10 mL). The phases were separated, and the aqueous phase was extracted with diethyl ether (10 mL). The combined organic phases were washed with saturated aqueous sodium hydrogen carbonate solution (5 mL) and dried over anhydrous sodium sulfate. The solvent was removed in vacuo to give 6-methylheptan-1-ol as a colorless oil (226 mg = 87%). RI FFAP, 1510; MS (EI, 70 eV), m/ z (%) 39 (19), 40 (3), 41 (75), 42 (32), 43 (74), 44 (4), 45 (3), 53 (5), 54 (3), 55 (89), 56 (100), 57 (33), 67 (6), 68 (21), 69 (81), 70 (30), 71 (4), 83 (6), 84 (19), 97 (20), 98 (2). 6-Methylheptanal (8). 6-Methylheptan-1-ol (130 mg, 1 mmol) in dichloromethane (5 mL) was added to Dess−Martin periodinane (530 mg, 1.25 mmol) in dichloromethane (5 mL), and the mixture was stirred for 1 h at ambient temperature. Diethyl ether (80 mL) and a solution of sodium thiosulfate (1 mol/L; 100 mL) in saturated aqueous sodium hydrogen carbonate solution were added, and the mixture was intensely shaken until the initial opacity disappeared. The organic phase was washed with aqueous sodium hydrogen carbonate solution (0.5 mol/L; 100 mL) and dried over anhydrous sodium sulfate. The solvent was removed, and the raw product was purified by column chromatography with silica gel (6 g). After a washing with pentane (50 mL), the target compound was eluted with pentane/ diethyl ether (45 mL + 5 mL) and its exact concentration in the eluate (50 mL) was determined by GC-FID using octanal as internal standard. Yield, 74.2 mg = 58%; purity, 90% (GC-FID); MS (EI, 70 eV), m/z (%) 39 (16), 41 (79), 42 (13), 43 (100), 44 (30), 45 (9), 53 (4), 55 (17), 56 (30), 57 (44), 67 (12), 68 (6), 69 (36), 71 (4), 82 (31), 83 (4), 84 (11), 85 (21), 93 (2), 95 (18), 100 (4), 109 (1), 110 (2); 1H NMR (400 MHz, CDCl3), δ 0.87 (d, 6H, J = 6.6 Hz, CH(CH3)2), 1.15−1.26 (m, 2H, CH2CH2CH(CH3)2), 1.28−1.37 (m, 2H, CH2CH2CH2CH2CHO), 1.54 (nonet, 1H, J = 6.6 Hz, CH2CH(CH3)2), 1.61 (quint, 2H, J = 7.4 Hz, CH2CH2CH2CHO, 2.42 (td, 2H, J = 7.4, 1.9 Hz, CH2CH2CHO), 9.76 (t, 1H, J = 1.9 Hz, CH2CHO); 13C NMR (100 MHz, CDCl3), δ 22.3 (CH2CH2CHO), 22.5 (CH(CH3)2), 26.9 (CH2(CH2)2CHO), 27.8 (CH(CH3)2), 38.6 (CH2CH(CH3)2), 43.9 (CH2CHO), 203.0 (CHO). 7-Methyloctanal (12). Using the procedure detailed for 8, 12 was obtained by oxidation of 7-methyloctan-1-ol (144 mg, 1 mmol) and purification by silica gel chromatography. The exact concentration of 12 in the eluate (50 mL) was determined by GC-FID using nonanal as internal standard. Yield, 64.4 mg = 45%; purity, 96% (GC-FID); MS (EI, 70 eV), m/z (%) 39 (16), 41 (73), 42 (16), 43 (78), 44 (32), 45

(7), 53 (4), 54 (6), 55 (63), 56 (68), 57 (100), 58 (5), 67 (24), 68 (18), 69 (24), 70 (15), 71 (9), 72 (3), 81 (27), 82 (16), 83 (9), 95 (7), 96 (11), 98 (5), 99 (4), 109 (19), 114 (3), 124 (3); 1H NMR (400 MHz, CDCl3), δ 0.87 (d, 6H, J = 6.6 Hz, CH(CH3)2), 1.13−1.34 (m, 6H, CH2CH2CH2CH(CH3)2), 1.52 (nonet, 1H, J = 6.6 Hz, CH2CH(CH3)2), 1.58−1.67 (m, 2H, CH2CH2CH2CHO, 2.41 (td, 2H, J = 7.4, 1.9 Hz, CH2CH2CHO), 9.75 (t, 1H, J = 1.9 Hz, CH2CHO); 13C NMR (100 MHz, CDCl3), δ 22.1 (CH2CH2CHO), 22.5 (CH(CH3)2), 27.1 (CH2(CH2)3CHO), 27.9 (CH(CH3)2), 29.4 (CH2(CH2)2CHO), 38.7 (CH2CH(CH3)2), 43.9 (CH2CHO), 202.9 (CHO). Isolation of Curry Leaf Volatiles. Curry leaves (10 g) and anhydrous sodium sulfate (50 g) were cryomilled at the temperature of liquid nitrogen. The obtained homogenate was added to dichloromethane (150 mL) and stirred for 30 min at ambient temperature. After filtration, nonvolatiles were removed by SAFE during 45 min at 40 °C. The distillate was concentrated to a final volume of 1 mL, first using a Vigreux column (50 × 1 cm) and subsequently a Bemelmans microdistillation device.27 Separate curry leaf volatile isolates were prepared for screening by AEDA, fractionation by silica gel chromatography, and isolation of volatile curry leaf thiols. Fractionation of Curry Leaf Volatiles. A curry leaf volatiles isolate prepared as detailed above was applied onto a slurry of purified silica gel (8 g) in pentane in a water-cooled (12 °C) glass column (1 cm i.d.). Elution was performed by pentane/diethyl ether mixtures of 100 + 0, 90 + 10, 70 + 30, 50 + 50, and 0 + 100 (v + v; 50 mL each), and the eluate was collected in 15 10 mL portions followed by two 50 mL portions. Each fraction was concentrated to 0.5 mL. Isolation of Volatile Curry Leaf Thiols. From a curry leaf volatiles isolate prepared as detailed above, the thiols were isolated using a procedure published earlier28 with some modifications. In detail, the isolate was initially applied onto mercurated agarose gel (1 g) in a glass column (0.5 cm i.d.). After the column had been rinsed with dichloromethane (50 mL), the thiols were eluted with dithiothreitol (10 mmol/L) in dichloromethane (50 mL). The excess of dithiothreitol was removed by SAFE distillation, and the distillate was concentrated to 0.5 mL. GC-O and GC-FID Analyses. A Trace GC Ultra gas chromatograph (Thermo Scientific, Dreieich, Germany) was equipped with a cold-on-column injector, an FID, and a tailor-made sniffing port. The following fused silica columns were used: (1) DB-FFAP, 30 m × 0.32 mm i.d., 0.25 μm film; (2) DB-5, 30 m × 0.32 mm i.d., 0.25 μm film (both Agilent, Waldbronn, Germany); (3) BGB-176, 30 m × 0.25 mm i.d., 0.25 μm film; (4) BGB-174E, 30 m × 0.25 mm i.d., 0.25 μm film (both BGB Analytik, Rheinfelden, Germany). The carrier gas was helium at 70 kPa (DB-FFAP, DB-5) and 120 kPa (BGB-176, BGB174E), respectively. The initial oven temperature was 40 °C (2 min). Gradients typically were 6 °C/min for DB-FFAP and DB-5 and 2 °C/ min for BGB-176 and BGB-174E. For GC-O analyses, the column effluents were divided 1:1 using a deactivated Y-shaped glass splitter and two deactivated fused silica capillaries (50 cm × 0.25 mm i.d.) connecting the splitter to the FID and the sniffing port, respectively. The sniffing port consisted of a cylindrically shaped aluminum device (105 mm × 24 mm o.d.) with a beveled top and central drill hole (2 mm) housing the capillary. It was mounted on a heated (250 °C) detector base of the GC. During a GC-O run (35 min), the panelist placed her/his nose closely above the top of the device and sniffed the effluent. Whenever an odor was perceived, the retention time and the odor quality were noted in the FID chromatogram printed by a recorder. Linear retention indices (RI) of the odor-active compounds were calculated from their retention times and the retention times of adjacent n-alkanes by linear interpolation. GC-O analyses of the concentrated curry leaf volatiles isolate were performed by six trained panelists, and results were combined. AEDA. The concentrated curry leaf volatiles isolate (1 mL) was stepwise diluted 1:2 with dichloromethane to obtain dilutions of 1:2, 1:4, 1:8, 1:16, etc. Each diluted sample was then analyzed by GC-O using the FFAP column. Odor-active compounds were assigned a flavor dilution (FD) factor, representing the dilution factor of the B

DOI: 10.1021/acs.jafc.5b01174 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Odorants in the SAFE Distillate Obtained from Fresh Curry Leaves no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

odoranta f

butane-2,3-dione α-pinene 3-methylbut-2-ene-1-thiolf (3Z)-hex-3-enal myrcene limonene 1,8-cineole 6-methylheptanal unknown octanal 2-acetyl-1-pyrroline 7-methyloctanal (5Z)-octa-1,5-dien-3-onef (3Z)-hex-3-en-1-ol nonanal (2E)-oct-2-enal 3-(methylsulfanyl)propanal 3-isobutyl-2-methoxypyrazinf linalool (2E,6Z)-nona-2,6-dienal 1-phenylethanethiol calameneneg γ-heptalactonef

odorb

RIc FFAP

RIc DB-5

FD factord

first reporte

buttery resinous, terpeny skunky green, grassy terpeny, geranium leaf-like terpeny, citrusy terpeny, eucalyptus-like citrusy, soapy mushroom-like citrusy, soapy roasty, popcorn-like citrusy, soapy metallic, geranium leaf-like green, grassy citrusy, soapy fatty, nutty cooked potato-like bell pepper-like citrusy cucumber-like sulfury, burnt clove-like coconut-like

980 1031 1112 1151 1167 1181 1222 1232 1252 1278 1335 1340 1369 1378 1385 1425 1458 1517 1546 1580 1601 1818 1822

960 931 820 800 992 1028 1031 964 − 1004 915 1068 984 854 1104 1057 917 1185 1100 1159 1120 1544 1165

8 2048 16 256 64 2 1024 4 8 4 8 4 8 32 1 8 128 4 4096 32 8192 8 4

− 3 − − 4 2 7 − − − − − 4 8 − − − 4 − − − −

a

Each odorant was identified by comparing its retention indices on two GC capillaries of different polarity (FFAP, DB-5) and its mass spectrum obtained by GC-MS, as well as its odor quality as perceived at the sniffing port during GC-O with data obtained from authentic reference compounds analyzed in parallel. bOdor quality as perceived at the sniffing port during GC-O. cRetention index; calculated from the retention time of the compound and the retention times of adjacent n-alkanes by linear interpolation. dFlavor dilution factor;20 dilution factor of the highest dilution of the concentrated SAFE distillate in which the odorant was detected during GC-O by any of three panelists. eReference reporting the compound for the first time as a curry leaf volatile. The dash (−) indicates compounds that have not been reported in curry leaves before. fA clear mass spectrum of the compound could not be obtained in the curry leaf isolates, identification was based on the remaining criteria detailed in footnote a. gNo reference compound was available. The compound was tentatively identified on the basis of MS database30 spectra and RI data in the literature.34 Stereochemistry was not assigned. Two-Dimensional Heart-Cut Gas Chromatography−Mass Spectrometry (GC-GC-MS). A Combi PAL autosampler (CTC) was mounted on a Trace GC Ultra equipped with a cold-on-column injector (Thermo) and an FFAP capillary, 30 m × 0.32 mm i.d., 0.25 μm film, or a DB-5 capillary, 30 m × 0.25 mm i.d., 1 μm film (Agilent). The column end was connected to a moving column stream switching system (MCSS) (Thermo), conveying the eluate time-programmed through deactivated fused silica capillaries (0.32 mm i.d.) either simultaneously to an FID and a sniffing port or via a heated (250 °C) transfer line to a cold trap localized in the oven of a CP 3800 GC (Varian). The tailor-made trap consisted of a piece of steel tubing housing the capillary and could be cooled by means of liquid nitrogen. Downstream, the capillary was connected to the BGB-176 or BGB174E column described above. The end of this column was connected to a Saturn 2200 mass spectrometer (Varian) operated in the CI mode with methanol as reagent gas. For the determination of the enantiomeric distribution of 2, 19, and 21, start temperatures were 40 °C (2 min) and gradients were 6 °C/min in the first dimension (FFAP) and 3 °C/min in the second dimension (BGB-176). For the determination of the enantiomeric distribution of 26, start temperatures were 40 °C (2 min) and 35 °C (2 min) and gradients were 1 and 2 °C/min, in the first (DB-5) and second dimensions (BGB174E), respectively. A heart-cut (0.5 min) of the eluate of the first column containing the respective target compound was transferred via the MCSS and the transfer line to the precooled trap. Then the trap cooling was turned off, and the second oven was started. The retention times of the target compounds in the first and second dimensions were previously determined using reference compounds. NMR Spectroscopy. 1H and 13C NMR spectra were recorded at 25 °C using an Avance 400 NMR spectrometer (Bruker, Rheinstetten, Germany) and tetramethylsilane as the internal standard (δ = 0.00

highest dilution in which the respective odorant was detected by any of three trained panelists (two females, one male). Gas Chromatography−Mass Spectrometry (GC-MS). Mass spectra were generated in the electron impact (EI) mode at 70 eV and a scan range of m/z 35−300 using a HP 5890 gas chromatograph (Hewlett-Packard, Heilbronn, Germany) connected to an MAT 95 sector field mass spectrometer (Finnigan, Bremen, Germany). Columns and further GC conditions were equivalent to those used in the GC-O analyses. Static Headspace Gas Chromatography (SH-GC). Curry leaves (1 g) and anhydrous sodium sulfate (5 g) were cryomilled, and the homogenate was placed into a 20 mL septum-sealed vial. After 5 min at ambient temperature, 5 mL of the headspace was withdrawn using a gastight syringe and injected by means of a Combi PAL autosampler (CTC Analytics, Zwingen, Switzerland) via a cold-on-column injector (helium, 110 kPa) onto a deactivated fused silica precolumn (0.2 m × 0.53 mm i.d.) installed in a Trace GC Ultra gas chromatograph (Thermo). Volatiles were trapped on the precolumn using a cold trap 915 at −150 °C. The precolumn was connected to the main column and an outlet solenoid valve via a three-way connector. During trapping, the solenoid valve was open to maintain a flow of 20 mL/min through the precolumn. After injection had been finished, the solenoid valve was closed, and the trap was heated to 250 °C to transfer the trapped volatiles onto the main column, a DB-5, 30 m × 0.25 mm i.d., 1 μm film (Agilent). The oven start temperature was 0 °C maintained by liquid nitrogen oven cooling. After 2 min, the temperature was raised at 6 °C/min. The end of the main column was connected to a pressure-controlled stream switching system (S+H Analytik, Mönchengladbach, Germany), which transferred the eluate to an FID and a sniffing port and/or a Saturn 2100 mass spectrometer (Varian, Darmstadt, Germany). C

DOI: 10.1021/acs.jafc.5b01174 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry ppm). Correct signal assignment was confirmed by 2D NMR spectroscopy using COSY, HMQC, and HMBC experiments (data not shown).

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RESULTS AND DISCUSSION Screening the Curry Leaf Volatiles for Odorants by AEDA. Freshly picked curry leaflets were cryomilled under addition of anhydrous sodium sulfate. The obtained powder was extracted by dichloromethane. Then, SAFE was applied on the extract to remove any nonvolatile material. The SAFE distillate, when placed on a fragrance test strip and sniffed after evaporation of the solvent, still fully reflected the characteristic aroma of fresh curry leaves. The SAFE distillate was gently concentrated, and the concentrate was submitted to AEDA. AEDA resulted in 23 odorants in the FD factor range of 1− 8192 (Table 1). The highest FD factor (8192) was found for sulfury- and burnt-smelling odorant 21, clearly resembling the dominating aroma note in the overall sensory profile of the curry leaves. High FD factors were further determined for citrusy-smelling odorant 19 (FD 4096), resinous-smelling odorant 2 (FD 2048), eucalyptus-like-smelling odorant 7 (FD 1024), grassy-smelling odorant 4 (FD 256), and cooked potatolike-smelling odorant 17 (FD 128). Structural Assignment of Odorants. As a first step, the RIs and odor descriptors of the odorants detected during AEDA were compared to previously published data of roughly 1600 odor-active compounds compiled in an in-house database. In the case of matching data, authentic reference compounds were obtained by purchase or synthesis and analyzed in an appropriate dilution by GC-O in parallel (i.e., same instrument, same day) to the curry leaf volatiles isolate. Because retention time and odor characteristics of the compound in the curry leaf volatiles isolate were in agreement with the retention time and odor characteristics of the reference compound on two GC-O systems of different separation phase polarities (DB-5, FFAP), the structures of 19 of the 23 curry leaf odorants (1−7, 10, 11, 13−21, 23) could be assigned. Final confirmation of these assignments was made by comparing the mass spectra of the curry leaf odorants and the reference compounds as obtained by GC-MS analysis. To avoid any overlay of mass spectra due to coelution, a particular problem with odor-active trace compounds, the curry leaf volatiles isolate was further fractionated before GC-MS analysis. Using silica gel as stationary phase and pentane/diethyl ether mixtures as eluents, the curry leaf volatiles were separated into 17 fractions according to their polarity. In a separate experiment, the thiols among the curry leaf volatiles were selectively isolated using mercurated agarose gel. Each fraction was then analyzed by GC-O to localize the odorants. Fractions containing any odorant detected during AEDA were subjected to GC-MS analysis (DB-5 and FFAP) in parallel with the reference compounds. Using this approach, 14 of the 19 previously assigned structures were confirmed by mass spectral data, among them all odorants with high (≥32) FD factors (Figure 1). Sulfuryand burnt-smelling odorant 21, exhibiting the highest FD factor (8192) in the AEDA, was recovered in the thiol fraction and finally identified as 1-phenylethanethiol. 1-Phenylethanethiol has not yet been reported in curry leaves and has rarely been found in nature before. Its only mention as an odor-active compound refers to Pontianak orange peel oil,25 where it significantly contributes to the specific odor.29 In that study, it was observed that 1-phenylethanethiol, when exposed to

Figure 1. Most potent odorants (FD factor ≥ 32) in the SAFE distillate obtained from fresh curry leaves. FD factors are given in parentheses.

increased temperatures, readily undergoes 1,2-elimination to form H2S and styrene.25 This might explain why the compound has escaped discovery in curry leaves so far. The compound might have been destroyed by the high temperatures applied during hydrodistillation or in the hot GC injection port. This finding exemplifies the importance of avoiding elevated temperatures in aroma research, for example, by using mild isolation techniques such as SAFE and by using the cold-oncolumn injection technique in GC. Odorants 19, 2, and 7, assigned the second (4096), third (2048), and fourth highest FD factors (1024) in the AEDA, were identified as citrusy-smelling linalool, resinous-smelling αpinene, and eucalyptus-like-smelling 1,8-cineole. All three have been identified in curry leaves before, but their high aroma potency has not been recognized yet. Further odorants with comparatively high FD factors were identified as (3Z)-hex-3enal (green, grassy; FD 256; 4), 3-(methylsulfanyl)propanal (cooked potato-like; FD 128; 17), myrcene (terpeny, geranium leaf-like; FD 64; 5), (3Z)-hex-3-en-1-ol (green, grassy; FD 32; 14), and (2E,6Z)-nona-2,6-dienal (cucumber-like; FD 32; 20). Of these, only myrcene has been reported as curry leaf constituent before. Mass spectral confirmation of the structural assignment was furthermore achieved for 2-acetyl-1-pyrroline (roasty, popcorn-like; FD 8; 11), (2E)-oct-2-enal (fatty, nutty; FD 8; 16), octanal (citrusy, soapy; FD 4; 10), limonene (terpeny, citrusy; FD 2; 6), and nonanal (citrusy, soapy; FD 1; 15). No clear mass spectra were obtained for five trace odorants with low FD factors (1, 3, 13, 18, 23). However, their retention times and odor properties on the two GC-O systems (DB-5, FFAP) were identical to those of butane-2,3-dione (1), 3methylbut-2-ene-1-thiol (3), (5Z)-octa-1,5-dien-3-one (13), 3isobutyl-2-methoxypyrazin (18), and γ-heptalactone (23). The retention indices and odor qualities of four odorants (8, 9, 12, 22) did not match with any available database entries, but GC-MS analyses of the curry leaf fractions resulted in D

DOI: 10.1021/acs.jafc.5b01174 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

reoxidation of the obtained alcohol to the aldehyde using Dess−Martin periodinane. Analysis of the synthesized compound by GC-O and GC-MS on the two separation systems (DB-5, FFAP) resulted in the same data as obtained for odorant 8, which thus was finally identified as 6-methylheptanal. 6-Methylheptanal has not been reported from curry leaves yet, but has been found among the volatiles of other plants in the rue family.31−33 In particular, it has been found as an aroma-active compound in mandarin peel oils32 and orange essence oil.31 As odorant 12 exhibited RIs (DB-5, 1068; FFAP, 1340) that were approximately 100 units higher than those of 6methylheptanal (DB-5, 964; FFAP, 1232), it was assumed to be its homologue, 7-methyloctanal. Synthesis of the compound by oxidation of the corresponding alcohol 7-methyloctan-1-ol confirmed this structural assignment, and odorant 12 was finally identified as 7-methyloctanal. This compound has rarely been reported from nature yet, and was also previously unknown as curry leaf constituent. Clove-like-smelling odorant 22, after silica gel fractionation, was recovered in the hydrocarbon fraction. Its mass spectrum matched the MS database30 entry of calamenene, an aromatized sesquiterpene hydrocarbon, and its RI was in agreement with data from the literature.34 However, no reference compound was available. For that reason, the identification of calamenene remained tentative. No mass spectrum could be obtained for mushroom-likesmelling odorant 9. Its odor quality, however, was clearly reminiscent of the odor of the button mushroom impact compound 1-octene-3-one. Taking into account that the RI of odorant 9 (FFAP, 1252) was lower than that of 1-octene-3-one (1293), and the difference was similar to that observed for the methyl-branched aldehydes 8 and 12 and their respective straight-chain isomers, it might be speculated that compound 9 could be a branched isomer of 1-octene-3-one such as 6methyl-1-hepten-3-one. However, no attempt was made to prove this assumption, and odorant 9, therefore, remained unidentified. Screening for Highly Volatile Odorants by SH-GC-O. Very highly volatile odorants with boiling points below that of the extraction solvent are not covered by AEDA, because they are lost during the concentration of the SAFE distillate, so a static headspace GC-O analysis was performed as a complementary screening method. Four additional odorants were detected in the headspace above freshly cut curry leaves and structurally assigned by comparing their GC-O and GCMS data with those of reference compounds. These odorants were fresh-, green-, and fruity-smelling acetaldehyde (24) and the malty-smelling compounds 2-methylpropanal (25), 2methylbutanal (26), and 3-methylbutanal (27) (Table 2). Enantiomeric Distribution of Chiral Curry Leaf Odorants. As enantiomers of chiral odorants may exhibit different odor thresholds or even different odor qualities, proper structure assignment of odor-active compounds must include the determination of the enantiomeric ratio. Seven of the total of 26 structurally assigned curry leaf odorants in this study were chiral. Among these were the three compounds with the highest FD factors in the AEDA, namely, 1-phenylethanethiol (21), linalool (19), and α-pinene (2), as well as highly volatile 2methylbutanal (26) detected by SH-GC-O. To determine their enantiomeric ratios, a heart-cut GC-GC-MS system was equipped with a chiral cyclodextrin phase in the second dimension. Baseline separation of the enantiomers was achieved

congruent mass spectra on both separation systems (DB-5, FFAP) for odorants 8, 12, and 22. An MS database30 library search applied on the mass spectra of odorants 8 and 12 (Figure 2) returned octanal and nonanal,

Figure 2. Mass spectra (EI) obtained for the citrusy-, soapy-smelling odorants 8 (A) and 12 (B) isolated from curry leaves.

respectively, with high matching factors. Octanal and nonanal, which had also been detected by AEDA of the curry leaf volatiles (odorants 10 and 15), however, showed clearly different RIs. On the other hand, the similarities in the mass spectra were obvious. Similar to octanal (MS not shown), odorant 8 showed characteristic fragments of m/z 110, corresponding to a loss of water from the molecular ion m/z 128, and m/z 110, corresponding to a loss of carbon monoxide, thus suggesting the presence of an aldehyde group and a molecular mass of 128 u in analogy to octanal. Accordingly, odorant 12 showed fragments of m/z 124 and 114 in analogy to nonanal. Odorants 8 and 12 not only shared similar mass spectra with octanal and nonanal but also appeared in the same fraction after silica gel chromatography and exhibited virtually the same citrusy, soapy odor quality. In summary, the data suggested that odorants 8 and 12 were chain isomers of octanal and nonanal. A branched carbon skeleton would also correspond to the clearly lower RIs of odorants 8 (DB-5, 964; FFAP, 1232) and 12 (DB-5, 1068; FFAP, 1340) in comparison to octanal (DB-5, 1004; FFAP, 1278) and nonanal (DB-5, 1104; FFAP, 1385), respectively. Branched isomers typically show lower RIs than the corresponding straight-chain molecules due to their smaller surface and thus lower ability to interact with the GC stationary phase. Type and position of the branching, however, were difficult to predict. The similarity in the odor qualities at least suggested that the branching was rather distant from the odotope, that is, rather not too close to the aldehyde group. Following the latter idea, we hypothesized that odorant 8 could be 6-methylheptanal. The compound was synthesized from commercially available 6-methylheptanoic acid by reduction with lithium aluminum hydride and subsequent E

DOI: 10.1021/acs.jafc.5b01174 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Pontianak oranges belong to the genus Citrus and thus also to the Rutaceae family, the similarity in the enantiomeric ratio may reflect a common biosynthetic pathway. The odor thresholds of (1R)- and (1S)-1-phenylethanethiol were reported to be in the same order of magnitude.25 In summary, gentle isolation of the volatile fraction from fresh curry leaves followed by systematic and nontargeted screening for odor-active compounds by application of an AEDA and a SH-GC-O analysis resulted in a total number of 27 odorants, among which, on the basis of their high FD factors, sulfury-smelling 1-phenylethanethiol, citrusy-smelling linalool, resinous-smelling α-pinene, eucalyptus-like-smelling 1,8-cineole, and grassy-smelling (3Z)-hex-3-enal most likely show a vital contribution to the overall aroma. To confirm this assumption and to unequivocally identify the key aroma compounds of fresh curry leaves, further experiments are currently under way. These will include exact quantitation of the most potent odorants detected in the screening experiments, the sensory evaluation of aroma reconstitution models based on the obtained concentration data as proof of success, and finally omission tests to assess the contribution of the individual odorants to the overall aroma of curry leaves.37 The omission tests will particularly elucidate whether 1-phenylethanethiol can be considered as the character impact compound of fresh curry leaves. Its exceedingly high FD factor, its characteristic sulfury and burnt odor quality, and the absence of any other potent odorant with a sulfury odor note, however, support this assumption.

Table 2. Odorants (RI DB-5 < 1000) in the Headspace above Fresh Curry Leaves no.

odoranta

odorb

RIc DB-5

first reportd

24 25 1 26 27 4 2 5

acetaldehyde 2-methylpropanal butane-2,3-dione 2-methylbutanale 3-methylbutanale (3Z)-hex-3-enal α-pinene myrcene

fresh, green, fruity malty buttery malty malty green, grassy resinous, terpeny terpeny, geranium leaf-like