Trace Components in Spearmint Oil and Their Sensory Evaluation

Chapter 11

Trace Components in Spearmint Oil and Their Sensory Evaluation 1

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Tomoyuki Tsuneya , Masakazu Ishihara , Minora Shiga , Shigeyasu Kawashima , Hiroshi Satoh , Fumio Yoshida , and Keiichi Yamagishi 1

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Shiono Koryo Kaisha, L t d , Niitaka 5-17-75, Osaka 532, Japan Lion Corporation, Honjo 1-3-7, Sumitaku, Tokyo 130, Japan

Downloaded by YORK UNIV on June 29, 2012 | http://pubs.acs.org Publication Date: April 6, 1993 | doi: 10.1021/bk-1993-0525.ch011

2

The basic component in Scotch spearmint oil (Mentha cardiaca, Gerard ex Baker) was investigated by means of GC, GC-MS and other analytical techniques. Thirty nine nitrogen compounds were identified as trace components in the oil. Among them, eleven pyridinecompounds, including 2-acetyl-4-isopropenylpyridine, were identified for the first time. The chemical structures of these compounds were elucidated by spectral data and by synthesis. The odor qualities of these compounds were evaluated, and a discussion is given of their contributions to the spearmint flavor. Spearmint oil, along with peppermint oil, is a popular flavoring material used extensively in chewing gums and toothpastes. Although spearmint oil alone is used in these finished products, the practice of mixing it with peppermint oil, known as mixed-mint or doublemint, has enjoyed increasing popularity because it enhances the flavor characteristics of spearmint oil. Taxonomically, spearmint is divided into two species: Mentha spicata Huds and Mentha gentilis f. cardiaca. Mentha spicata Huds is a native spearmint, and the latter is Scotch spearmint. Both oils are produced mainly in the USA - in the Northwestern states of Washington and Oregon, and in the Midwestern states of Michigan, Indiana and Wisconsin. The output of spearmint oil from 1989 to 1991 in the USA is shown in Table 1. Recent growing demands for spearmint flavor and the expansion of planting areas in these producing areas seem to have increased the output of spearmint oil. There is a definite difference in odors between the native spearmint and Scotch spearmint. The Scotch spearmint is generally recognized as superior to the native variety. China is beginning to produce a considerable amount of spearmint oil. Though firm data are not available, it has been predicted that the output of spearmint oil in China could reach about 500 tons. Canova (7) has conducted extensive studies on the flavor components of spearmint oil and has reported 194 compounds. Some characteristic compounds have been reported by Tsuneya et al. (2), Takahashi et al. (5), Ichimura et al. (4), 0097-6156/93/0525-0137$06.50A) © 1993 American Chemical Society

In Bioactive Volatile Compounds from Plants; Teranishi, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

138

BIOACnVE VOLATILE COMPOUNDS F R O M PLANTS

Sakurai et al. (5), Surburg et al. (6), and Shimizu et al. (7). Sakurai et al. (8) have reported three pyridine compounds in spearmint oil. Over 200 flavor components of spearmint oil have been identified (9,10). The authors present here some of the newly identified pyridine compounds characteristic in the oil and sensory evaluations of these compounds.


EXPERIMENTAL PROCEDURE Sample Preparation: As shown in Figure 1, a 49 kg sample of Midwest Scotch spearmint oil (1983 crop) was extracted three times with 7.5 kg of one normal aqueous solution of hydrochloric acid. The acidic extract was processed to yield 2.8 g of basic components. The GC analysis by flame thermoionic detector (FTD) of the extracted so-called components showed that only 18% of this material were truly composed of basic compounds. Upon further work, it was found that the yield of basic components was 0.00102% (10.2 ppm) of the original oil. Samples were prepared from 400 g amounts of other varieties of spearmint oils and peppermint Mitcham oils (Williamette). The yields of the basic components of Farwest Scotch, Farwest native and peppermint Mitcham oils (Williamette) were 8 ppm, 10 ppm and 5.5 ppm respectively. Gas Chromatography (GC): A 0.28 mm (i.d.) x 40 m SF-96 fused silica capillary column and 0.25 mm(i.d.) x 60 m DB-1 fused silica capillary column were used in a Hitachi K-163 equipped with flame ionization detector (FID), flame thermoionic detector (FTD) and flame photometric detector (FPD). The oven temperature was programmed from 50° (5 min isothermal) to 240°C at 3°C/min. Similar conditions were used for a DB-1 column. The temperature of the injector was set up at 260°C. Fig. 2 shows the gas chromatogram (FTD) assigned by the peaks of the newly identified basic components. Other peaks assigned are shown in Table 3. Column Chromatography and Medium Pressure Liquid Chromatography (MPLC): For the purpose of the isolation of unknown compounds, fractionation by column chromatography is shown in Table 2. Further separation of the basic component was undertaken by use of MPLC (Merck Lobar Column:Lichroprep Si-60, 40 - 60 ^m, 24 cm x 1 cm i.d.). Gas Chromatography-Mass Spectrometry (GC-MS): A Hitachi 663 GC was combined with a Hitachi M-80 A mass spectrometer (EI mode) with a M-0101 data processor. A 0.25 mm (i.d.) x 60 m DB-1 fused silica capillary column was used. The oven temperature was programmed from 75°C (5 min isothermal) to 240°C. The temperature of the injector was 250°C. The mass spectra were recorded at an ionization voltage 20 eV at an ion source temperature of 200°C. For high resolution mass spectra (HR-MS), the same instrument was used at an ionization energy of 70 eV. Infrared Spectrometry (IR): Measurement of the sample was with an IR Jasco IRA-1 instrument. Proton Nuclear Magnetic Resonance ( H-NMR): Measurement of sample was taken on a Hitachi R-24 B instrument. !


11. TSUNEYA ET AL.

Trace Components in Spearmint OU

Table 1. Output of Spearmint Oil in the USA Varieties 1 2 3 4

Farwest Scotch Midwest Scotch Farwest Native Midwest Native Total

1991

1990

1989 552,000 420,000 845,000 108,000

900,000 378,000 1,080,000 86,000

1075,500 680,000 1,196,000 180,000

1,925,000

2,444,000

3,131,500


(Courtesy of LP. Callison & Sons Incorporated)

Spearmint MWS Oil (49 Kg) Extraction withlNHCI

Organic Laytr

Aquaous Laytr Extraction with Toluana

Organic Layer

Aquaous Laytr NautralizatJon with 2N NaOH (pH 11) Exraction with Et 0 2

Organic Laytr Washing with Brint Conctntration Basic Fraction (2.8 g) Si0 Column Chromatography 2

[CC-1

I

~

ICC-14 I

Figure 1. Scheme 1. Sample preparation o f the basic fraction.


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4- Acetyl-2-isopropenylpyridine 5- [(Z)-l -Butcn-1 -yl]-2-propylpyridine 3-[(Z)-1 -Buten-1 -yl]-4-propylpyridine 5-[(E)-l-Buten-l-yl]-2-propylpyridine 3-[(£)-l-Buten-l-ylj-4-propylpyridine

40

Figure 2. Gas chromatogram (FTD) of the basic fraction of Scotch spearmint oil (see text for GC conditions).

2-Isopropyl-4-methylpyridine 4-Isopropenyl-2-methy]pyridine 2-Ethyl-4-isopropenylpyridine 2-Acetyl-4-isopropylpyridine 2,4-Diisopropenylpyridine 2-Acetyl-4-isopropenylpyridine

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TSUNEYA ET AL.

Trace Components in Spearmint Oil

Table 2. Column Chromatography of the Basic Fraction Fr. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Eluting Solvent hexane 2.5% ether/hexane 5% 5% 10% 10% 20% 20% 30% 30% 50% 70% ether methanol

Volume (ml) 250 200 75 75 75 75 75 75 80 70 100 100 150 200

Yield (mg) 0 0 6 18 60 57 249 235 227 225 291 322 53Θ 521


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BIOACTIVE VOLATILE COMPOUNDS F R O M PLANTS

Table 3. Compounds Identified from the Basic Fraction in Midwest Scotch Spearmint Oil Peak No

8

Compound

b

Known Unknown Means of identification c

1323 770

1323 772

M S , RI, IR, NMR, Syn M S , RI

12 36 22 24 28 4 7 17 39 34 1

(Greater than 10% ) 2-Acetyl-4-isopropenylpyridine (X)* 2-Methylpyridine (1.0-10%) 2-Acetylpyridine 3-[(£)-l-Buten-l-yl]-4-propypyridine(XV)* 3-[(Z)-l-Buten-l-yl]pyridine (V) 3-[(£)-l-Buten-l-yl]pyridine (VI)** 2,4-Diisopropenylpyridine (DC)* 2,6-Dimethylpyridine 3-Ethylpyridine 4-Isopropyl-2-methylpyridine (ΠΙ)** 5-Phenyl-2-propylpyridine (XVII) 3-Phenylpyridine Pyridine

991 1438 1131 1176 1301 846 923 1059 1684 1426 702

993 1442 1131 1177 1293 844 921 1056 1685 1420 694

MS,RI M S , RI, Syn M S , RI, Syn M S , RI, Syn M S , RI, IR, NMR, Syn M S , RI M S , RI M S , RI, Syn M S , RI, Syn MS.RI M S , RI

27 31 33 35 32 22 18 20 21 6 25 9 11 5 19 16 23 38 13 14 26

(0.1 - 1.0%) 2-Acetyl-4-isopropylpyridine (VITJ)* 4-Acetyl-2-isopropenylpyridine (XI)* 3-[(Z;- 1-Buten-1 -yl]-4-propylpyridine (XUJ)* 5-[(E)-1 -Buten-1 -yl]-2-propylpyridine (XIV)* 5-[(Z)-l-Buten-l-yl]-2-propylpyridine (XII)* 3-[(Z)-l-Buten-l-yl]pyridine (V) 2-Butylpyridine 3-Butylpyridine 4-Butylpyridine 2,5-Dimethylpyrazine 2-Ethyl-4-isopropenylpyridine (VII)* 2-Ethyl-6-methylpyrazine 5-Ethyl-2-methylpyridine 2-Ethylpyridine 4-Isopropenyl-2-methylpyridine (IV)* 2-IsopropyM-methylpyridine (Π)* 2-Pentylpyridine 3-Phenyl-4-propylpyridine (XVI) 3-Propylpyridine 4-Propylpyridine Quinoline

1260 1333 1383 1431 1378 1131 1065 1108 1124 881 1189 960 982 863 1100 1035 1174 1615 1019 1024 1206

1261 1331 1388 1432 1379 1131 1067 1105 1123 873 1183 959 986 866 1097 1033 1167 1616 1018 1023 1201

M S , RI, Syn MS, RI, Syn M S , RI, Syn M S , RI, Syn M S , RI, Syn M S , RI, Syn M S , RI MS, RI M S , RI M S , RI M S , RI, Syn M S , RI M S , RI MS, RI M S , RI MS, RI, Syn MS, RI M S , RI, Syn MS, RI MS, RI MS, RI

37 15 29 3 10 8

(Less than 0.1%) 3-Benzylpyridine 4-Isopropenylpyridine (I)** Methyl anthranilate 3-Methylpyridine 2-Propylpyridine 3-Vinylpyridine

1500 1034 1311 819 960 932

1497 1031 1312 820 961 928

MS, MS, MS, MS, MS, MS,


30 2

RI RI, Syn RI RI RI RI

* newly identified, ** identified first from nature, Known* ; Kovats Indices (DB-1) of known compound, Unknown ; Kovats Indices (DB-1) of unknown compound, (Greater than 10%) ; G C area %. b


c

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TSUNEYA ET AL.

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Identification of Components: Identification of components was accomplished by comparison of known retention indices and mass spectra with those of authentic reference standards. In the absence of published information, authentic samples for mass spectra were purchased. However, the identity of most compounds was confirmed by matching their mass spectra and retention indices with those of synthesized compounds. Isolation of 2-Acetyl-4-isopropenylpyridine (X): Compound X was isolated by the method mentioned earlier to give 77 mg of a colorless oil: RI 1323; IR (film) 3100, 3070, 1700, 1598, 1250, 910, 853 cm ; Ή-NMR (CC1 ) δ 2.17 (br, s, 3H, CH ) 2.65 (s, 3H, CH ), 5.28(m, 1H, CH =), 5.61(m, 1H, CH =), 7.43(dd, J = 1.6 and 5 Hz, 1H, ArH), 7.97(d,J = 1.6 Hz, ArH) 8.54(d, J=5 Hz, 1H, ArH); EI-MS, m/z(rel. int.) 161(M , 86), 133(28), 119(100), 118(65), 91(31), 43(30). Isolation of 2,4-Diisopropenylpyridine (IX): Compound IX was isolated by the method mentioned earlier to give 13 mg as a colorless oil: RI 1293; IR (film) 3100, 1598, 900, 843 cm ; Ή-NMR (CC1 ) δ 2.07 (m, 3H, CH ), 5.16(m, 2H, CH =) 5.45(m, 1H, CH ), 5.74(m, 1H, CH ), 7.04(dd, J = 1.6 and 5 Hz, 1H, ArH), 7.35(d, J = 1.6 Hz, ArH), 8.36(d,J = 5 Hz, 1H, ArH); EI-MS, m/z(rel. int.)159(M , 100), 158(86), 144(18), 119(25), 91(13). Preparation of 2-Isopropyl-4-methylpyridine (II): Compound II was prepared by regioselective addition of isopropylmagnesium bromide to 1phenoxycarbonyl salt of 4-picoline by applying the method of Comins and Abdulla (77) in 44% yield after purification through silica gel chromatography (hexane/ether = 8:2) to give 6.53 g (44%) of II, a colorless oil: RI = 1036; IR(film) 3080, 1603, 1562, 820 cm ; Ή-NMR (CC1 ) δ 1.25(d,J=7 Hz, 6H, two CH ), 2.25(s, 3H, CH ), 2.94(dq, J=7 and 7 Hz, 2H, two CH), 6.76(d, J=5 Hz, 1H, ArH), 6.81(s, 1H, ArH), 8.26(d, J=5 Hz, 1H, ArH); EI-MS, m/z (rel. int.) 135(M , 40), 134(42) 120(100), 107(28), 93(23), 77(3), 65(5). Preparation of 4-Isopropenyl-2-methylpyridine (IV) and 2-Ethyl-4isopropenylpyridine (VII): Each regioselective alkylation of methyl and ethyl radicals generated from the corresponding carboxylic acid to the 2 position of methyl isonicotinate was performed according to the method of Minisci et al. (72). Each of the resulting 2-alkylated esters was treated with methylmagnesium bromide followed by dehydration procedure to give compounds IV and VII in 11 % and 18% yields, respectively, after column chromatography on silica gel (ether/hexane = 3:7). Compound IV: RI = 1100; IR (film) 1600, 1534, 900, 835 cm ; Ή-NMR (CDC1 ) δ 2.14(d, J=5 Hz, 1H, ArH), 7.16(s, 1H, ArH), 8.43(d,J=5 Hz, 1H, ArH); EI-MS, m/z(rel. int.) 133(M , 100), 132(30), 118(19), 117(18), 91(33), 65(11). Compound VII: RI = 1189; IR (film): 1600, 1543, 900, 840 cm ; Ή-NMR (CDC1 ): δ 1.31(t,J=7 Hz, 3H, CH ), 2.11(d,J=1.6 Hz, 3H, CH ), 2.83(q, J=7 Hz, 2H, CH ), 5.18(m, 1H, CH =), 5.49(m, 1H, CH =), 7.08(d, J=7 Hz, 5H, ArH), 7.12(s, 1H, ArH), 8.43(d, J=5 Hz, 1H, ArH); EI-MS, m/z(rel. int.) 147(M , 70), 146(100), 130(5), 119(21), 91(7). Preparation of 2-Acetyl-4-isopropylpyridine (VIII): Compound VIII was prepared from 2-acetylpyridine and isobutyric acid by applying the method of 1

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Minisci et al. (72), and showed the following spectral data: RI=1260; IR (film) 3080, 1700, 1599, 1358, 1202, 843 cm ; Ή-NMR (CDC1 ) δ 1.25(d, J=7 Hz, 6H, two CH ), 2.67(s 3H, CH , 2.96(dq, J=7 and 7 Hz, 1H, CH), 7.26(dd, J=1.5 and 5 Hz, 1H, ArH), 7.87(d, J = 1.5 Hz, 1H, ArH), 8.50(d, J=5 Hz, 1H, ArH); ΕΙ-MS m/z(rel.int.) 163(M , 84), 148(11), 135(28), 121(100), 106(25), 79(10), 43(16). Preparation of 2,4-Diisopropenylpyridine (IX): An ethereal solution of 3M methylmagnesium bromide was added dropwise to dimethyl 2,4-lutidinate (39 g, 0.2 mol) in ether (400 ml) cooled in an ice water bath. The reaction mixture was stirred for lh at room temperature and then was refluxed for another lh. The reaction mixture was poured into a cold sat. NH4CI. The aqueous layer was extracted three times with a mixed solvent (ether/ethyl acetate = 1:2). The combined organic layer was washed with brine and dried with anhydrous MgS0 . After removal of the solvent, the brownish residue (39.2 g) was chromatographed over silica gel(ether/hexane = 8:2) to give 22.7 g (58%) of the compound (1) as colorless crystals and 11.7 g(33%) of a 1:1 mixture of the compound (2) and (3) às a colorless oil. {Spectral data of compound (1)}:IR (KBr): 3280, 2960, 1600, 1180, 1102, 955 cm^H-NMR (CDC1 ), δ 1.48(s, 6H, two CH ), 1.51(s, 6H, two CH ), 3.69(br.s, 1H, OH), 5.12(br, s, 1H, OH), 7.39(dd, J=1.6 and 5 Hz, 1H, ArH), 7.42(d, J=1.6 Hz, 1H, ArH), 8.25(d, J = 5.0 Hz, 1H); EI-MS, m/z(rel. int.): 195(M 0.8), 180(100), 165(12), 137(22). {MS data of compound (2)}: EI-MS, m/z, (rel. int.): 179(M , 100), 164(58), 137(72), 78(35), 59(43), 43(63). {MS data of compound(3)}: EI-MS, m/z (rel. int.): 179(M , 5), 164(100), 121(33), 59(13), 43(5). The Diol (1) (20g, 0.1 mol) was refluxed in cone. H SO (50g) and acetic acid (118g) for 1 h. The reaction mixture was cooled and poured into an ice water bath and washed with toluene. The aqueous layer was made basic with 20% NaOH and extracted three times with a mixed solvent (ether/ethyl acetate = 1:1). The organic layer was washed with brine and dried with anhydrous MgS0 . After removal of the solvent, the residue was chromatographed over silica gel (ether/hexane = 1:5) to give 12.5 g compound IX as a colorless oil(yield:78%). The compound IX showed the following spectral data: RI 1301; IR(film):3100, 1598,900,843cm ; Ή-NMR (CDC1 ), δ 2.07(m,3H), 2.17(m,3H), 5.16(m,lH), 5.45(m,lH), 7.04(dd, J = 1.6 & 5 Hz, 1H), 7.35(d, J = 1.6 Hz, 1H), 8.36(d, J=5 Hz, 1H);EI-MS, m/z(rel. int.):159(M ,100), 158(86), 144(18), 119(25), 91(13). 2-Acetyl-4-isopropenylpyridine (X) and 4-Acetyl-2-isopropenylpyridine (XI): The mixtures of compound (2)/(3) = l:l(4g,20 mmol), H S0 (13g) and glac. acetic acid (30g) were refluxed for 1.5 hr. The reaction mixture was poured into ice water (50 ml) and was extracted with ether/toluene (1:1). The aqueous layer was neutralized with 20% aq. NaOH and was extracted three times with ether. The ether solution was washed with brine and dried (MgS0 ). After removal of the solvent, the residue was chromatographed over silica gel (ether/hexane =15:85) to give 1.33 g of compound XI as a colorless oil (yield:37%) and 1.22 g of compound X as colorless oil(yield:34%). Compound X showed the following spectral data: RI 1323: IR (film) 3100, 3070, 1700, 1598, 1250, 910, 853 cm ; Ή-NMR (CDC1 ), δ 2.17(br.s, 3H, CH ), 2.65(s, 3H, CH ), 5.28(m, 1H), 7.43(dd, J = 1.6 & 5 Hz, 1H), 7.97(d, J = 1.6 Hz, 1H) 1

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8.54(d, J=5 Hz, 1H); EI-MS, m/z(rel. int.):161(M , 86), 133(28), 119(100), 118(65), 91(31), 43(30). Compound XI showed the following spectral data: RI 1333; IR (film): 3100, 1700, 1590, 1365, 1250, 910, 850 cm" ; Ή-NMR (CDC1 ), δ 2.24(m, 3H, CH ), 2.60(s, 3H, CH ), 5.36(m, 1H), 5.94(m, 1H), 7.56(dd, J=1.6 & 5 Hz, 1H), 7.88(d, J = 1.6 Hz, 1H), 8.74(d, J=5 Hz, 1H): EI-MS, m/z(rel. int.):161(M , 100), 160(70), 146(12), 118(30), 117(20), 91(18), 43(25). To determine the position of the acetyl group on the pyridine ring, compound X was also prepared by the following selective procedure (S 3 Fig 7). The regioselective acetylation of methyl isonicotinate with paraldehyde was performed by applying the method of Giordano et al. (13). The resulting methyl 2-acetylisonicotinate was transformed to the compound X by Grignard reaction after protecting the acetyl carbonyl group as its ethylenedioxy acetal, followed by dehydration procedure in 23% overall yield. The analytical data of compound X obtained by the above manner were identical with those of the natural one. 5-[(Z)-1 -Buten-1 -yl)]-2-propylpyridine (XII) and 5-[(E)-1 -Butenyl- l-yl)]-2propylpyridine (XIV): The alkylation of propyl radical to the 6 position of nicotinaldehyde was performed by applying the method of Minisci et al (12). The subsequent Grignard reaction of 6-propylnicotinaldehyde followed by dehydration gave a mixture (3:97) of compound XII and XIV as a colorless oil in 5.8% overall yield after purification through silica gel column chromatography (hexane/ether= 7:3). The minor isomer XII showed the following data: RI 1378; EI-MS m/z(rel. int.)175(M 5), 174(9), 160(23), 147(100), 132(12), 106(6), 91(3). The major isomer XIV showed the following data: RI 1431; IR(film) 1595, 1560,1485,960, 910 cm ; Ή-NMR (CDC1 ) δ 0.95(t,J=7 Hz, 3H, CH ), 1.09(t, J=7 Hz, 3H, CH ), 1.45-2.50(m, 4H, two CH ), 2,73(dd, J=7 and 8 Hz, 2H, CH ), 6.29(m, 2H, CH=CH), 7.05(d, J=7.5 Hz), 1H, ArH), 7.58(dd, J = 1.6 and 7.5 Hz), 1H, ArH), 8.45(d, J=1.6 Hz, 1H, ArH); EI-MS m/z(rel.int.) 175(M , 20), 174(15, 160(45), 147(100), 132(40), 92(5). Preparation of 3-[(Z)-l-Buten-l-yl)]-4-propylpyridine (XIII) and 3-[(E)-lButenyl-l-yl)]-4-propyl pyridine (XV): 4-propylnicotinaldehyde was synthesized according to the method of Comins et al. (14). The subsequent Grignard reaction of 4-propylnicotinaldehyde followed by dehydration gave a mixture (3:97) of compound XIII and XV in 23% overall yield after purification through silica gel column chromatography. The minor isomer XIII showed the following data: RI 1383; EI-MS m/z(rel. int.) 175(M , 100) 160(54), 146(33), 132(88), 118(40), 117(35). The major isomer XV showed the following data: RI 1438; IR (film) 3050, 1595, 1465, 1416, 970 cm ; Ή-NMR (CDC1 ) δ 0.93(t, J=7 Hz, 3H, CH ), 1.07(t, J=7 Hz, 3H, CH ), 1.60(m, 2H, CH ), 2.17(m, 2H, CHj), 2.56(dd, J=7 and 8 Hz, 2H, CHJ, 6.04(dt, J=5.5 and 16 Hz, 1H, CH=), 6.45(d, J=16 Hz, 1H, CH=), 6.88(d, J=5 Hz, 1H, ArH), 8.17(d, J=5 Hz, 1H, ArH), 8.43(s, 1H, ArH); EI-MS m/z(rel. int.) 175(M , 83), 160(45), 146(35), 132(100), 131(31), 118(41), 117(39). Preparation of Other Reference Compounds: 4-Isopropenylpyridine I was prepared by Grignard reaction of methyl isonicotinate followed by dehydration in 63% yield. Spectral data obtained were as follows: RI 1038; IR(film) 3100, 1595, 1402, 905, 830, cm ; ^ - N M R i C C l J δ 2.08(m, 3H, CH ), 5.14(m, 1H, 1

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CH =), 5.45(m, 1H, CH =), 7.14(m, 2H, ArH), 8.35(m, 2H, ArH),; EI-MS m/z(rel. int.) 119(M , 100), 118(60), 104(17), 91(33), 79(11). The mixture (7:93) of Ζ and Ε isomers of 3-(l-buten-l-ylpyridine V and VI were prepared by Grignard reaction of nicotinaldehyde followed by dehydration in 63% yield. Spectral data obtained were as follows: Compound V: RI 1131; EI-MS m/z(rel. int.) 133(M , 100), 132(43), 118(85), 117(43), 91(23), 65(13). Compound VI: RI 1176; IR(film)3050,1570, 1420, 1025, 970, 800, 710cm- ; Ή NMR (CC1 ) δ 1.07(t, J=7 Hz, 3H, CH ), 2.21(m, 2H, CH ), 6.17(m, 2H, CH=CH), 6.98(dd, J=5 and 8 Hz, 1H, ArH), 7.44(dt, J = 1.6 and 8Hz, 1H, ArH), 8.21(dd, J = 1.6 and 5 Hz, 1H, ArH); EI-MS m/z(rel. int) 133(M , 85), 132(45), 118(100), 117(47), 91(24), 65(11). 4- Isopropyl-2-methylpyridine (III) was prepared according to the method of Comins et al. (77) in 52% yield. Spectral data obtained were as follows: RI 1060; IR (film) 3080, 3030, 2975, 1603, 1560, 920, 830 cm ; 'Η-NMR (CDC1 ) δ 1.20(d, J=7 Hz, 6H, two CH ), 2.47(s, 3H, CH ), 2.79(dq, J=7 and 7 Hz, 1H, CH), 6.84(d, J=5 Hz, 1H, ArH), 6.87(s, 1H, ArH), 8.25(d, J=5 Hz, 1H, ArH); EI-MS m/z(rel. int.) 135(M , 64), 120(100), 106(4), 93(6), 77(13), 65(5), 42(6), 41(6). 3-Phenyl-4-propylpyridine(XVI) was prepared from 3-phenylpyridine and butyric acid by applying the method of Minisci et al. (72) and it showed the same physiochemical properties of those reported by Sakurai et al. (8). 5- Phenyl-2-propylpyridine(XVII) was prepared according to the method of Sakurai et al. (8) and it showed the same physiochemical properties as those reported. 2

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3

+

Results and Discussion Identification of Pyridine Compounds: Fig. 2 shows the gas chromatogram of the basic part in Midwest Scotch spearmint oil, and also shows the peaks of the eleven new pyridine compounds identified in this study. As can be seen in Figure 2, the existence of more than 100 nitrogen compounds was detected first with FTD and subsequently they were analyzed by GC-MS. It had been predicted that many of these compounds might be unknown or unfamiliar compounds. This fact led to much interest in and elucidation of the structures. In particular, the main component which showed a unique mass spectrum was especially interesting. The main component had a molecular weight of 161 m/z. The GC-MS (Fig. 3) and high resolution mass spectrum showed the molecular formula, C,oH NO; [M ] 161.0834. Compound X isolated by chromatography was submitted to the following instrumental analyses. IR spectrum (Fig. 4) shows the presence of an acetyl carbonyl group at 1700 and 1250 cm . The H-NMR signals (Fig. 5) at δ 2.17 (br, s, 3H), 5.28(m,lH) and 5.61(m,lH) suggested the existence of an isopropenyl group. The signals at δ 7.43(dd, J = 1.6 and 5 Hz, 1H), 7.97(d, J=1.6 Hz, 1H) and 8.54(d, J=5 Hz, 1H) suggested that the acetyl and the isopropenyl groups are attached to the 2- and 4-positions or the reverse positions on the pyridine ring, respectively. These spectral data supported two assigned structures of 2-acetyl-4-isopropenyl pyridine (X) or 4-acetyl-2-isopropenylpyridine (XI). The structure of compound X was determined first by synthesis as shown +

n

1

!


11.

TSUNEYA ET AL.


147

HR-MS Found [M*] m/z 161.0834 C,oH O,Ni RI=1323 n


100

200 mix

Figure 3. Mass spectrum of compound X (isolated).

3000

2000

1500

1000 „cm- i

Figure 4. IR spectrum of compound X (isolated).

American Chemical Society Library 1155 16th St., N.W. Washington, O.C. 20036 In Bioactive Volatile Compounds from Plants; Teranishi, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

m

148


in Scheme 2, (Figure 6), and the position of the substituted group was confirmed by selective synthesis as shown in Scheme 3 (Figure 7). The spectral properties of the synthetic product were identical with those of one isolated from spearmint oil. Small amounts of 4-acetyl-2-isopropenylpyridine (XI) were also confirmed by comparing its mass spectrum and RI data with those found in nature. On the other hand, compound IX (Peak No. 28 in Fig. 2) which appeared just ahead of the main component in GC showed a molecular weight of 159 m/z from GC-MS and the molecular formula C„H N from the HR-MS (Fig. 8). The IR spectrum (Fig. 9) showed no absorption of the carbonyl group. The H-NMR spectrum (Fig. 10) showed the signals at 2.07 (m, 3H, CH ), 2.17 (m, 3H, CH ), 5.16 (m, 2H, CH =), 5.45 (m, 1H, CH =) and 5.74 (m, 1H, CH =), which suggest the existence of two isopropenyl groups. The coupling patterns of three proton signals of the pyridine ring were quite similar to those of compound X. These spectral data supported the assigned structure of 2,4-diisopropenylpyridine (IX), which was confirmed by the synthesis drawn in scheme 2. The spectral data of the synthesized compound were absolutely identical with those of the natural one. Besides these compounds, unknown compounds were further studied to elucidate their chemical structures. To determine the structures of other compounds for which authentic MS data were not available, many pyridine compounds had to be synthesized to elucidate their structures by examining their mass spectra. By synthesizing the unknown compounds and by analyzing their mass spectra and by matching the RI with those of the natural ones, thirty nine pyridine compounds were identified as shown in Table 3. In this table, the compounds with an asterisk marks are newly identified ones. Double asterisk marks indicate that the compounds which were identified for the first time in nature but had been previously synthesized. Figures 11 to 17 show mass spectra of new pyridine compounds, except for compounds IX and X. The mass spectra of XIV and XV are similar to those of compounds XII and XIII because they are the trans and cis forms, respectively, see Figures 16 and 17. 13

!

3


2

2

3

2

Existence of Pyridine Compounds in Nature Maga (15) and Vernin (76) have reviewed in detail pyridine compounds identified in nature, in various natural products such as fish, meat, poultry, vegetables, cereals, nuts, dairy products, fruits, spices, non-alcoholic beverages like tea and coffee, alcoholic beverages, tobacco, and essential oils. Approximately 100 pyridine derivatives with various ring substitutions have been identified. In essential oils, some characteristic pyridine compounds have been reported in jasmin (77), orange flower (78), and peppermint (8). Toyoda (77) reported 14 unique pyridine compounds such as esters of nicotinic acid from jasmine absolute. Sakurai (8) identified in peppermint and spearmint oils several pyridine derivatives substituted at 3,4- and 2,5-positions with phenyl groups. It is also noticed that 16 and 30 nitrogen compounds have been identified in black tea (19) and Burley tobacco (76), respectively. One of the interesting features of this work is that 8 pyridine compounds have been reported which have isopropenyl, isopropyl, and acetyl groups substituted in the 2,4-positions (Figure 18). As for the few 2,4substituted pyridines found in nature so far, 2,4-dimethylpyridine in Burley


TSUNEYA ET A L .


149


11.

(3) Figure 6. Scheme 2. Synthesis of compounds IX, X, and XI. In Bioactive Volatile Compounds from Plants; Teranishi, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

XI

150


C0 Mo 2

t-BuOOH CF C0 H

A

3

COjMe HOfCHsfoOH

2

F*S0

p-TsOH

4

OH

C0 M 2

ltoMgBr/Et 0 2

H2SO4/ACOH


fee Ο "Ο Figure 7. Scheme 3. Synthesis of compound X .

HR-MS Found [M*] m/z 159.1004 CnH^Nj RI=1293 100

BOO

Figure 8.

Figure 9.

Mass spectrum of compound IX (isolated).

IR spectrum of compound IX (isolated).



11.

9

151


TSUNEYA ET AL.

8

7

6

5

4

3

2

1

0

Figure 10. 'Η-NMR spectrum of compound IX (isolated). 100

Figure 11. Mass spectrum of compound II (synthesized). 100'

C9H11N1

133

-

MW-133 RI-1100

30

9Î I 63 1M

7

117

I

7

Ι , , „ . Λ , . . Μ Μ " Μ ' 50

100

130

200

Figure 12. Mass spectrum of compound IV (synthesized). In Bioactive Volatile Compounds from Plants; Teranishi, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

m/z

152 100

BIOACTIVE VOLATILE COMPOUNDS F R O M PLANTS C10H13N1

146

1

MW-147 RI-1189

30

ν * "1

1 • "'"M • •

1 50

119

1 1

104

ι fr" 111 » H

ι

11

W

130 h •• •

Η

"

800

150

100

mh

Figure 13. Mass spectrum of compound VII (synthesized). Downloaded by YORK UNIV on June 29, 2012 | http://pubs.acs.org Publication Date: April 6, 1993 | doi: 10.1021/bk-1993-0525.ch011

100

C10H13N1O1

MW-163 RU1260

eoo Figure 14. Mass spectrum of compound VIII (synthesized). C10H11N1O1

100

MW-161 RI-1333

£00

Figure 15. Mass spectrum of compound XI (synthesized). 100'

C12H17N1

147

MW-175 RI-1378

So 160

132

173 117 1

1

M

50

"

Ί · " · | · "

100

150

200

m/i

Figure 16. Mass spectrum of compound XII (synthesized). In Bioactive Volatile Compounds from Plants; Teranishi, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


XIV

XV

XVI

XVII

Figure 18. Pyridine compounds in spearmint oil.



154


tobacco (20) and four 2,4-pyridine compounds, 2-isobutyl-4-methylpyridine, 2isobutenyl-4-methylpyridine,2-isobutanoyl-4-pyridine,and2^ l-propyl)pyridine, in fig absolute (27) have been reported. Table 4 lists the major pyridine compounds identified from Midwest Scotch, Far West Scotch, Far West native spearmint oil and Mitcham peppermint oil (Williamette). Fig. 18 shows characteristic pyridine compounds identified from spearmint oil. As can be seen in Table 4, the main component is 2-acetyl-4isopropenylpyridine in each of the spearmint oils, and the other pyridine compounds which exist in Midwest Scotch are not so different qualitatively when compared with Far West Scotch and native. These three oils have some qualitative differences. In particular, the differences in quality between Scotch and native are quite clear. From the information in this table, it can be observed that the contribution of flavor in the basic components of the individual oil does not seem to determine the organoleptic differences. On the other hand, analysis of the basic component of peppermint oil showed that it compared well with spearmint oil. Although spearmint and peppermint oils have pyridine compounds common to both, there are some differences. Compounds VIII, IX, X and XI are found in spearmint oil but not in peppermint oil. The fact that those compounds are found only in spearmint oil prompted the study of spearmint flavor. Sensory Evaluation of Pyridine Compounds Table 5 shows the odor descriptions evaluated by seven flavorists. The individual compounds were evaluated at the concentration of 5% in alcohol. Of these compounds, 2-Acetyl-4-isopropenylpyridine, which is unique to spearmint and also is the main component in the oil, has a grassy-sweet, minty, somewhat amber-like odor. The terms used in evaluating pyridine compounds have been reported by Maga (13), Vernin (14), Winter et al. (22), Buttery (25), Suyama et al. (24). These terms are green, astringent, bitter earth or burnt note. Acetylpyridine derivatives are considered to have roasted and coffee-like odor. The odor descriptions in Table 5 are green earth, and roasted or brownish, though the characters such as aged, fermented, herbal, cinnamate and somewhat animalic odor are added to the terms already mentioned. Harsh terms such as astringent disappeared in the evaluation of these compounds, and somewhat more benign terms have appeared. Table 6 shows the sensory evaluation of some pyridine compounds associated with spearmint flavor. The standard spearmint flavor is composed of 30 flavor chemicals. To this standard spearmint flavor, the following pyridine compounds were added at ppb levels to evaluate their impact. These compounds were added to improve the spearmint flavor by giving a grassy-sweet note to this spearmint flavor which had a somewhat fishy odor. When 2-acetyl-4-isopenylpyridine was added at the concentration of 40 ppb to the standard spearmint flavor, it imparted a slightly roasted, fermented or aged odor, augmenting a grassy-sweetness, accentuating the sweetness of a terpene top note. On the other hand, 2,4-diisopropenylpyridine and 2-acetyl-4-isopropylpyridine gave characteristic properties to the standard one at the concentration of 2 and 8 ppb, respectively, as listed in Table 6. Further, when 2-acetyl-4-isopropenylpyridine


11. TSUNEYA ET AL.


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Table 4. M^jor Pyridine Compounds in Spearmint and Peppermint OO (ppm)


Compound Pyridine 2-Methylpyridine 2,5-Dimethylpyrazine 2-Acetylpyridine 2-Isopropyl-4-melhylpyridine (II) 4-Isopropyl-2-mcthylpyridinc (ΙΠ) 4-Isopropenyl-2-mcthylpyridine (IV) 3-[(Z)-l-Buten-l-yl]pyridine (V) 3-[(EH-Buten-l-yl]pyridine (VI) 2-EthyM-isopropenylpyridine (VII) Quinolinc 2-Acetyl-4-isopropylpyridine (VIII) 2,4-Diisopropenylpyridine (IX) 2-Acctyl-4-isopropenylpyridine (X) 4-Acetyl-2-isopropenylpyridine (XI) 5-[(Z)-l-Buten-l-yl]-2-propylpyridine(XII) 3-[(Z)-l-Buten-l-yl]-4-propylpyridine (XIII) 3-Phenylpyridine 5-[(£)-l-Buten-l-yl]-2-propylpyridine (XIV) 3-[(£)-l-Buten-l-yl]-4-propylpyridne(XV) 3-Phenyl-4-propylpyridine (XVI) 5-Phenyl-2-propylpyrdine (XVII)

A 0.98 1.25 0.06 0.20 0.05 0.12 0.05 0.10 0.20 0.04 0.09 0.06 0.35 3.34 0.05 0.02 0.14 0.58 0.08 0.26 0.05 0.28

Β 0.12 0.06 0.11 0.14 0.05 0.11 0.02 0.09 0.17 0.03 0.11 0.02 0.26 3.26 t 0.02 0.25 0.34 0.34 0.44 0.03 0.18

C

D

0.46 t 0.58 0.13 0.13 0.14 0.02 0.23 0.59 0.14 0.11 0.04 0.40 3.54 0.03 0.02 0.49 0.57 0.14 0.05 0.07 0.22

0.26 0.02 0.02 0.07 0.34 1.90 t 0.15 0.22 t 0.06

-

-

0.02 0.01 0.41 0.10 t 0.04 0.12

A : Spearmint Midwest Scotch, B: Spearmint Farwest Scotch, C: Spearmint Farwest Native, D: Peppermint Mitcham(Willamette)

was added to toothpaste material together with the standard one, in addition to these evaluations in Table 6, rough properties of the artificial flavor seemed to be smoothed out. It is interesting that these compounds in such low concentrations can contribute towards approximating natural flavor. Other compounds of low threshold values have also been known to cause similar effects. However, these compounds alone should not be considered as representing the exclusive role of the basic component of spearmint oil. Besides this compound, a combination of the components with some other characteristic minor pyridine components had an impact towards approaching a "natural" flavor. In general, a pyridine component has not been evaluated to be as good a contributor as a pyrazine component because pyridine compounds are not as palatable as pyrazine compounds. It should be emphasized that pyridine compounds can enhance natural flavors. They can modify the flavor quality of the synthetic to resemble the natural one. The study on pyridine components in nature will be continued. Genetic Consideration: As can be seen from Table 4, a feature of the basic components of spearmint oil is the existence of the 2,4-disubstituted pyridine derivatives which are rare in nature. These compounds are not found in peppermint oil. A possible mechanism of their characteristic skeleton can be explained as shown in Scheme 4 (Figure 19). The oxidative degradation product



green-bitter, nutty-beany, slightly sweet ether like, browny-acidy, radish(ozone like) slightly nutty, herbal, bitter earthy, slightly seaweed, somewhat citrus earthy green, somewhat sour and citrus amine Eke, ozonous green, violet-perilla herbal, white floral like, minty somewhat rose, fermented beany, wormwood earthy green, green beany, powdery musk like nutty,roastedsoybean, methyl cinnamate like minty, sweet, fermented earthy green tomato leaf, slightly methyl cinnamate like grassy-sweet, minty, somewhat amber like weak herbal green, fermented roast grassy-green leaf, green herbal, somewhat violet

Odor description

2 ppb 8 ppb

2-Acetyl-4-isopropylpyridine (VIII)

Concentration 40 ppb

2,4-Diisopropenylpyridine (DC)

2-Acetyl-4-isopropenylpyridine (X)

Compound

Odor Description slightly roasted and brownish, fermented odor, aged, soft, pull out the sweetness of terpene, augment the bottom note like hay with sweetess, slightly brownish, round out, gorgeous enhancing thickness, sweetness peculiar to spearmint, naturality

Table 6. Sensory Evaluation of Pyridine Compounds to Spearmint Flavor

4-Isopropenylpyridine (I) 4-Isopropenyl-2-methylpyridine (IV) 2-Ethyl-4-isopropenylpyridine (VII) 2,4-Diisopropenylpyridine (DC) 2- Isopropyl-4-methylpyridine (II) 4- Isopropyl-2-methylpyridine (ΙΠ) 3- [(Za£)-l-Buten-l-yl]pyridine (V & VI) 5- [(Za£)-l-Buten-l-yl]-2-propylpyridine (ΧΠ & XIV) 3-[(Za£)-l-Buten-l-yl]-4-propylpyridine (ΧΠΙ & XV) 3-Phenylpyridine 3-Phenyl-4-piOpylpyridine (XVI) 5-Phenyl-2-propylpyridine (XVII) 2-Acetyl-4-isopropenylpyridine (X) 4-Acetyl-2-isopropenylpyridine (XI) 2-Acetyl-4-isopropylpyridine (VIII)

Compound

Table 5. Odor Description of Synthesized Pyridine Compounds


11.

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AL.


157

[Ο]

OHC , 0

Ο

Ο

[A]

ACarvone


NHj

Dehydrogenation

Aromatization

Ο

Ο

X

[Β]

VIII

Figure 19. Scheme 4. Possible formation mechanism of 2-acetyl4-isopropenylpyridine.

of 1-carvone, which is a main component of spearmint oil, reacts with ammonia to give dihydropyridine intermediate Β through A. Aromatization of Β affords 2acetyl-4-isopropylpyridine VIII. On the other hand, dehydrogenation of Β produces 2-acetyl-4-isopropenylpyridine X. If pyridine compounds were proved to be formed genetically from terpene compounds in this way, it could be said that it is quite rare in nature. The formation of the genetic mechanism of pyridine compounds common in spearmint and peppermint oil is another topic of interest. Study on this matter will be continued. Literature Cited: 1. Canova, L. The composition of Scotch spearmint oil. In 5th International Congress of Essential Oils, Abstract paper, QT/b-22, Brazil, An. Acad. Bras. Cienc., 1971, pp 273-277. 2. Tsuneya, T.; Yoshioka, Α.; Shibai, T.; Shiga, M . Koryo, 1973, 104, 23-26. 3. Takahashi, K.; Muraki, S.; Yoshida, T. Agric. Biol. Chem., 1981, 45, 129132. 4.

Ichimura, N . ; Matsura, Y.; Kato, Y . , 25th Terpene, Essential Oils and Aroma Congress, 1981, 18-20.

5. Sakurai, K.; Takahashi, K.; Yoshida, T. Agric. Biol. Chem., 1983, 47, 1249-1256. 6. Surburg, H.; Kopsel, M . Flavor and Fragrance Journal, 1989, 4, 143-147.



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7. Shimizu, S.; Shibata, H . ; Karasawa, D., Kozaki, T. J. Ess. Oil Res., 1990, 2, 81-86. 8. Sakurai, K . ; Takahashi, K.; Yoshida, T. Agric. Biol. Chem., 1983, 47, 2307-2317. 9. Lawrence, Β. M . Monoterpene interrelationship in the mentha genus: a biogenetic discussion. In Essential Oils, Mookherjeem B. D.; Mussinan, D. J., Eds., Allured, Wheaton, Ill., 1981, pp 1-81. 10. Maarse, H . ; Visscher, C. Α.; Willemsens, L . C. Volatile Compounds in Foods--Qualitative and Quantitative, TNO-CIVO Food Analysis Institute, Zeist, The Netherlands, 1989, pp 110-136. 11. Comins, D. L . ; Abdullah, A. H. J. Org. Chem., 1982, 47, 4315-4319. 12. Minisci, F.; Bernardi, R.; Bertini, F.; Galli, R; Perchinummo, M., Tetrahedron, 1971, 27, 3575-3579. 13. Giordano, C; Minisci, F.; Vismara, E.; Levi, S. J. Org. Chem., 1986, 51, 536-537. 14. Comins, D. L . ; Smith, R. K.; Stroud, E. D. Heterocylces, 1984, 22, 339344. 15. Maga, J. A. J Agric. Food Chem., 1981, 29, 895-898. 16. Vernin, G. Perfumery and Flavorist, 1982, 7, 23-35. 17. Toyoda, T; Muraki, S.; Yoshida, T. Agric. Biol. Chem., 1978, 42, 19011905. 18. Sakurai, K.; Toyoda, T.; Muraki, S.; Yoshida, T. Agric. Biol. Chem,, 1979, 43, 195-197. 19. Vitzhum, Ο. B.; Werkhoff, P.; Hubert, P. J. Agric. Food Chem., 1975, 23, 999-1003. 20. Neurathe,G. B. Beitr. Tabakforsch., 1969, 5, 515-518. 21. Kaiser, R. New natural products of structural and olfactory interest identified in fig leaf absolute (Ficus carica L.). In Prog. Essent. Oil. Res., Walter de Gruyter & Co., Berlin, 1986, pp 227-239. 22. Winter, M.; Gautschi, F.; Flament, I; Stoll, M . ; Goldman, I. M . 1975, U. S. Patent 3,900,582. 1972, U. S. Patent 3,702,253, Flament, I.; Stoll, M.; 1976, U. S. Patent 3,931,246; 1976, U. S. Patent 3,931,245. 23. Buttery, R. G.; Ling, L. C.; Teranishi, R.; Mon, T. R. J. Agric. Food Chem., 1977, 25, 1227-1229. 24. Suyama, K.; Adachi, S. J. Agric. Food Chem., 1980, 28, 546-549. RECEIVED September 28, 1992


Trace Components in Spearmint Oil and Their Sensory Evaluation

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