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Lactones Newly Identified in the Volatiles ofPouchong-type Semi-fermented TeaYuriko Nobumotoa, Kikue Kubotaa & Akio Kobayashiaa Laboratory of Food Chemistry, Ochanomizu University, 2–1–1 Ohtsuka, Bunkyo-ku, Tokyo112, JapanPublished online: 12 Jun 2014.

To cite this article: Yuriko Nobumoto, Kikue Kubota & Akio Kobayashi (1993) Lactones Newly Identified in the Volatiles ofPouchong-type Semi-fermented Tea, Bioscience, Biotechnology, and Biochemistry, 57:1, 79-81, DOI: 10.1271/bbb.57.79

To link to this article: http://dx.doi.org/10.1271/bbb.57.79

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Biosci. Biotech. Biochem., 57 (1), 79-81, 1993

Lactones Newly Identified in the Volatiles of Pouchong-type Semi-fermented Tea

Yuriko NOBUMOTO,* Kikue KUBOTA, and Akio KOBAYASHlt

Laboratory of Food Chemistry, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112, Japan Received July 17, 1992

Repeated column chromatography enabled the lactone fraction to be separated from the volatile condensate of pouchong tea. In this fraction, several unidentified lactones were found, and their structures were elucidated mainly by GC-MS and GC-FTIR. 2-Hexen-4-olide, 5-octen-4-olide, 5-octanolide, 2-nonen-4-olide, 7 -decen-4-olide, and 3,7 -decadien-5-olide were finally identified by a comparison with respective standard compounds. These compounds seem to contribute to the characteristic pouchong tea aroma.

Pouchong tea is a kind of semi-fermented tea produced in Taiwan. As the fermentation process is lighter than that for ordinary oolong tea, an infusion of pouchong tea is green in color and has a characteristic floral and elegant aroma. Yamanishi et al. 1

) have identified jasmin lactone, methyl jasmonate, linalool, linalool oxides, benzyl cyanide and indole as the main contributory constituents of the pouchong tea aroma. They also reported that some of them were markedly increased during the manufacturing process.2,3)

The aroma of this above-mentioned mixture of com­pounds can explain some characteristics of the pouchong tea aroma; however, the characteristic sweet and milky aroma of pouchong tea could not be reproduced from a mixture of the same artificial compounds. Therefore, there should be other minor compounds in the essential oil of pouchong tea that would contribute to its aroma.

This paper reports the separation of the oxygenated­compound fraction, which has the characteristic aroma of pouchong tea, and the identification by GC, GC-MS, and GC-FTIR of severallactqnes contributing to the aroma.

Materials and Methods 1) Materials. Pouchong-type semi-fermented tea was prepared from the

spring leaves of Camellia sinensis L. (cultivar; Chin-shin-oo-long) which were plucked at the Bunzan tea estate in Taiwan.

2) Preparation of the aroma concentrates by steam distillation under reduced pressure. Using a standard rotary evaporating system,4) the volatiles were steam-distilled from 100 g of powdered tea with 500 ml of purified water at under 55°C. When almost the whole volume of water had been distilled, the same amount of water was added to repeat the steam-distillation process. The ether extract of the volatile components from the distilled water (ca. 700ml) was dried over anhydrous sodium sulfate and, after evaporating the solvent at under 40°C, the residue was treated as the aroma concentrate (79.3 mg). The oxygenated-compound fraction was eluted with ether through 2.5 g of silica gel after eluting the hydrocarbon fraction with pentane, and again subjected to silica gel column chromatography (2.5 g of silica gel in a 0.7 m Ld. column, with a flow rate of 0.6 ml/min), using a solvent of pentane-ether = 1 : 1. The eluate was fractionated into five portions, whose yields and aroma characteristics are summarized in Table I.

3) Separation and identification of the aroma constituents. a) Gas chromatography (GC). A Shimadzu GC-7A gas chromatograph

equipped with a flame-ionization detector and an FS-WCOT PEG-20M column of 0.25 mm Ld. x 50 m was used with a Shimadzu CR-3A data

processing system. N 2 was used as the carrier gas at a flow rate of 1.2 ml/min with a split ratio of 30: I. The oven temperature was held at 60°C for 4 min and then increased to 180°C at a rate of 2°C/min. The injection temperature was 200°C.

b) Gas chromatography-mass spectrometry (GC-MS). A Hewlett­Packard-5790A gas chromatograph was combined with a JEOL JMS-DX 300 mass spectrometer and a JEOL-DA-5000 data processing system. The GC conditions were the same as those in a), except for using He as the carrier gas and an FS-WCOT PEG-20M column of 0.25 mm i.d. x 60 m. MS was performed in the electron impact (EI) mode at 70eV.

c) Gas chromatography-Fourier transform infrared spectroscopy (GC­FTIR). A Hewlett-Packard 5890A gas chromatograph was combined with a Hewlett-Packard 5965A infrared spectrometer. The GC conditions were as follows: column, ULTRA #1 0.31 mm Ld. x 50m cross-linked methyl silicone; He carrier gas flow rate, 0.9 ml/min; and split ratio, 10: 1. The oven temperature was held at 60°C for 4 min and then increased to 200°C at 2°Cfmin. The injection temperature was 200°C.

4) Authentic lactone samples. 3,4-Dibromo-hexanoic acid, prepared from (E)-3-hexenoic acid by adding bromine, was converted to 2-hexen-4-olide by debromination and lactonization according to the method of Kuhn et al. 5)

5-0cten-4-olide, 5-octanolide, 2-nonen-4-0Iide, 7-decen-4-olide, and 3,7-decadien-5-0Iide were supplied by Hasegawa Perfumery Company.

The purity and structure of each standard lactone were confirmed by GC, GC-MS, IR, and lH-NMR.

Results and Discussion As shown in Table I, the most abundant fraction was Fr.

1, whose gas chromatogram shows many identifiable peaks of aliphatic aldehydes, alcohols and esters, while Fr. 2 contained the already identified mono- and sesquiterpene alcohols. These constituents of Frs. 1 and 2 corresponded to the respective aroma characters. On the other hand, Fr. 3 had a characteristic pouchong tea aroma with a floral and milky odor, in spite of the medium yield among the

Table I. Fractions of the Oxygenated Compounds from a Pouchong Tea Aroma Concentrate by Silica-gel Column Chromatography

Fraction Volume Yield Aroma character

no. (ml) (mg)

1 20 33.4 Grassy-greenish 2 10 5.2 Citrus-like 3 20 3.8 Most significant pouchong tea aroma 4 20 1.4 Sweet 5 20 1.6 Unpleasant

* Present adress: Tokyo Research Laboratories, Kao .corporation, 2-1-3 Bunka, Sumida-ku, Tokyo 131, Japan. t To whom correspondence should be addressed.

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80 Y. NOBUMOTO, K. KUBOTA, and A. KOBAYASHI

oxygenated fractions. Therefore, the constituents of Fr. 3 were further analyzed by GC, GC-MS and GC-FTIR.

The gas chromatogram of Fr. 3 is shown in Fig. 1, the main constituents being geraniol, benzyl alcohol and phenyl ethyl alcohol. As minor compounds, five lact6nes were identified as 4-hexanolide, 5-decanolide, (Z)-7-decen-5-0lide (jasmin lactone), dihydroactinidiolide, and couma­rin, which have already been found in the pouchong tea aroma. 1) These results suggested that there may have been some unknown lactones in this fraction. Peaks (a) and (c) were identified as 2-hexen-4-olide and octane-5-0Iide, respectively, by a direct comparison with the MS and KI values for authentic samples. As the newly identified lactones, (a) and (c), had relatively simple structures, it was possible to elucidate their structures only from GC-MS data. However, as summarized in Table II, the GC-MS data for the other peaks were not as helpful for identifying the unknown lactones, because the strength of the fragment peaks in MS were variable depending on y- or b-Iactone rings and the presence of a double bond in the cyclic and side chain structures. On the other hand, GC-FTIR data were valuable for identifying these common structures. As GC-FTIR is measured in the gas phase, the absorption bands are different from those obtained from liquid states or solutions, and reference to FT -IR data 7) is necessary to discuss these chemical structures.

o 10 20 30 40 50 60 70 80 min

Fig. 1. Gas Chromatogram of Fraction No.. 3 of the Oxygenated Aroma Compounds from Pouchong Tea.

Table II. Data Used for Identifying Unknown Peaks (b), (d), (e), and (f)

Molecular MS

Compound formula

m/z (%)

The IR data for peak (b) showed an absorption band at 1813 cm -1, which is attributable to a 4-olide without a double bond in the ring. The molecular formula CSH 120 Z was estimated from the molecular ion peak at m/ z 140 in the MS data; therefore, one (Z)-double bond was present in the side chain because of the lack of a 970 cm - 1

absorption band diagnostic of a trans configuration in the IR spectrum. The location of this double bond was finally established by comparing with an authentic sample. Peak (b) was identified as (Z)-5-octen-4-olide, itsMS fragmenta­tion pattern being identical with that of the same compound obtained from tuberose.6)

The IR absorption band at 1806 cm - 1 of peak (d) suggested a conjugated butenolide structure. The molecular formula C9H 140 Z was calculated from the molecular ion peak at m/ z 154. As there is no additional double bond in this formula, the compound was identified as 2-nonen-4-olide, whose IR, KI, and MS data were the same as those of an authentic sample.

The absorption band at 1812cm -1 of peak (e) is the same as that of peak (b), and the molecular formula CloH160Z calculated from the molecular ion at m/ z 168 suggested that this compound was a non-conjugated decen-4-olide. The position of the double bond was established by comparing the spectral data and the retention index with those of an authentic sample.

The absorption band at 1777 cm -1 of compound (f) is attributable to a non-conjugated b-Iactone. From the calculated molecular formula from m/ z 166 and the lack of the absorption of a trans-double bond near 970 cm - 1, this compound had two cis-double bonds in its molecule. The MS fragment peaks appeared strongly at m/z 97 and 69,

O~R H H

b : R = ·C=CC2Hs

H H e : R = ·C2H4C=CC2H5

o~ o~ c

Fig. 2. . Structures of Compounds a, b, c, d, e, and f.

KI

b CSH 1202 140 (M + 9), 111 (100), 56 (33), 55 (30), 29 (28), 85 (25), 1914/1919 2979, 1813, 1464, 1323, 1155, 1029, 910 41 (24), 81 (20), 27 (22), 82 (16)

d C9H 140 2 154 (M + 3), 43 (100), 84 (86), 55 (56), 27 (35), 41 (34), 125 (34), 29 (34), 85 (29), 83 (19)

e Cl0H1602 168 (M + 1), 68 (100), 85 (25), 41 (25), 29 (23), 67 (17), 55 (15), 79 (10),110 (8), 95 (6)

f CloH1402 166 (M + 2), 97 (100), 41 (62), 69 (57), 98 (22), 39 (22), 27 (12), 79 (7), 55 (7), 44 (7)

a Observed Kovats' indices of the sample. b Observed Kovats' indices of the standard. C Vapor-phase spectrum of GC-FTIR.

2007/2014

2110/2114

2130/2139

2941, 1806, 1464, 1321 1031,885, 841 2974, 1812, 1464, 1352 1165, 1054, 898 2950, 1777, 1368, 1210, 1077

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Lactones in Semi-fermented Tea 81

suggesting the presence of the partial structure of 3-penten-5-olide and the (2Z)-pentenyl side chain, respec­tively. The estimated 'structure for (f), (7Z)-3,7-decadien-5-olide, was finally established by comparing with an au­thentic sample.

The unsaturated lactones, (b), (d), (e), and (f), have not previously been identified in any type of tea aroma concentrate; particularly, compound (f) is a dehydrogenated jasmin lactone, which is one of the character impact compounds of the pouchong tea aroma. The introduction of a double bond in the jasmin lactone ring resulted in a change of aroma character.

Through our studies, it has become clear that the combination of GC-MS and GC-FTIR is very effective for identifying volatiles present in small quantities.

A qualitative analysis of these lactones in various teas and their effects on the respective tea aroma are now in progress.

Acknowledgments. We are grateful to Dr. T. Yamanishi, Professor Emeritus of Ochanomizu University, for her encouragement and valuable advice. We also thank Dr. K. Hayashi and Dr. M. Amaike of Hasegawa Perfumery Co. for supplying the lactone samples, and Dr. W. T. F. Chiu, former director of the National Tea Institute in Taiwan, for supplying the pouchong tea samples.

References 1) T. Yamanishi, M. Kosuge, Y. Tokitomo, and R. Maeda, Agric. Bio/.

Chem., 44, 2139-2142 (1980). 2) Y. Tokitomo, M. Ikegami, and T. Yamanishi, Agric. Biol.Chem.,

48, 87-91 (1984). 3) A. Kobayashi, K. Tachiyama, M. Kawakami, T. Yamanishi, I. M.

Juan, and W. T. F. Chiu, Agric. Bioi. Chem.,49, 1655-1660(1985). 4) T. Yamanishi, M. Nose, and Y. Nakatani, Agric. Bioi. Chem., 34,

599-608 (1970). 5) R. Kuhn and D. Jerchel, Ber., 76, 413-419 (1943). 6) B. Maurer and A. Hauser, Helv. Chim. Acta, 65, 462-476 (1982). 7) C. 1. Pouchert (ed.), "Aldrich Library of FT-IR Spectra,," 1st ed.,

Aldrich Chemical Co., Milwaukee, WI (1985).

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