Agric. Biol. Chem., 54 (3), 803-805, 1990 803

Note Tanaka (iyokan) and C. limon Burm. f., cv Lisbon (lemon). The fruit samples grown in the fields of the Kochi Fruit Tree Experimental Station were harvested in mid- to late- Volatile Constituents of Several Vari- December, 1988. Each CPOwas prepared in the same way as that described previously.1] A Shimadzu GC-14A gas eties of Pummelosand a Comparison chromatograph was used, and all other conditions regard- of the Nootkatone Levels in ing the instrumental analysis were the same as those in the Pummelos and Other Citrus Fruits1 previous paper.2) Using a weighedcalibration mixture and two internal standards (/t-heptanol and methyl myristate), we deter- Masayoshi Sawamura,Shigeru Kuwahara,* mined the percentage (by weight to CPO) of 62 com- Ken-ichi Shichiri and Toshikazu Aoki** ponents from the range of CPOs. The componentsof each pummelooil are listed in Table I. Components of less than Department of Agricultural Chemistry, Faculty of 0.01% throughout all the varieties of pummelo have been Agriculture, Kochi University, B-200 Monobe, excluded. Nankoku, Kochi 783, Japan * WakayamaAgricultural Processing Research The composition of Tosa buntan (430g as fresh weight per fruit) CPOwas similar to that from previous data.2) Corporation, Momoyama-cho, Naga-gun , Wakayama 649-61 , Japan The similarity in CPOcomposition between Tosa buntan and suisho buntan (480 g) seems reasonable in that suisho ** Kochi Fruit Tree Experimental Station, buntan is regarded as a hybrid of Tosa buntan (Tosa Asakura, Kochi 780, Japan buntanxC. grandis Osbeck var. Banokan Hort. ex Received August 31, 1989 Tanaka). Sesquiterpenes such as a-copaene, jS-copaene, p- cubebene and (5-cadinene were, however, contained to a lesser extent in suisho buntan than in Tosa buntan. Otachibana (370g) is regarded as a native variety in The pummelo is the largest of the citrus fruits, and is Kagoshima Prefecture and is botanically close to Tosa known as buntan or zabon in Japan. It is commercially buntan. Thus, the CPOcomposition of Otachibana was represented by Tosa buntan with an outstanding fra- also equivalent to that of Tosa buntan, except for the level grance, although there are many other varieties of pum- of linalool. The concentration of linalool in pummelos was melo in south-western Japan. Each variety varies in shape, generally less than one percent, but it was as high as 3%in taste and aroma, but their aroma constituents have re- the case of otachibana. Banpeiyu (1350g) is a very large- mained unknown. The authors have studied the constitu- fruited variety among pummelos,amounting to 2kg. ents of the cold-pressed oil (CPO) of Tosa buntan1> and Myrcenewas an extraordinarily abundant componentin the changes during storage.2) It has been suggested that te banpeiyu oil (about 40%). There have been no reports nootkatone was not only a compoundcontributing to the on citrus oils composed of such a high level of myrcene. characteristic odour of Tosa buntan as well as to that of Hirado buntan (980 g) originated from a chance seedling in grapefruit,3} but also an important compound from the Nagasaki Prefecture, while the origin of uchimurasaki aspect of chemotaxonomy. In the present paper, quan- (800g) is not known in detail. Mato buntan originated in titative determinations are reported on the CPOs of sev- Taiwan and is shaped like sanbokan. Egami buntan is now eral varieties of pummelo, and the results are compared grown in Nagasaki Prefecture, and its flesh is fairly pink with those of other citrus species. extending into the albedo, similar to uchimurasaki. /?- The pummelo samples used were as follows: Citrus Cubebene was comparatively abundant in Hirado buntan, grandis Osbeck forma Tosa-buntan (7osa buntan), C. Mato buntan and uchimurasaki, but aldehydes such as n- grandis Osbeck forma Suisho (suisho buntan), C. ota- octanal, w-nonanal, citronellal and ^-decanal were hardly chibana Hort. ex Y. Tanaka (otachibana), C. grandis found in the three varieties other than banpeiyu. Osbeck forma Hirado (Hirado buntan), C. grandis Osbeck The levels of y-terpinene and terpinolene exceeded 5% forma Banhakuyu (banpeiyu), C. grandis Osbeck forma and 0.28%, respectively, in Tosa buntan, suisho buntan Benikawa (uchimurasaki), C. grandis Osbeck forma Mato and otachibana oils, as well as in the oils of popular species (Mato buntan) and C. grandis Osbeck forma Egami such as Satsuma mandarin, ponkan, lemon, etc. (ranging (Egami buntan). The fruits other than pummelowere as from 5 to 12% for y-terpinene and 0.4 to 1% for terpin- follows: C. unshiu Marcov. {Satsuma mandarin), C. ta- olene), while the levels of the two compoundswere less than murana Hort. ex Takahashi (hyuganatsu), C. natsudaidai 0.22% and 0.02%, respectively, in banpeiyu, Hirado bun- Hayata (natsudaidai), C. hassaku Hort. ex Tanaka (has- tan, uchimurasaki, Mato and Egami buntan. The levels of saku), C. sulcata Hort. ex Takahashi (sanbokan), C. both y-terpinene and terpinolene may be a factor in reticulata Blanco, cv F-2426 (ponkari), C. iyo Hort. ex characterizing the variety amongpummelos. Studies on the Essential Oils of Pummelo. Part III. For Parts I and II, see refs. 1 and 2, respectively. Table I. Composition of Cold-pressed Oil from Pummelos

P eak _ , Varieties of pummelos Compound no. Tosa buntan Suisho buntan Otachibana Banpeiyu Hirado buntan Uchimurasaki Mato buntanfl Egami buntan0

a-Pinene 1.44±0.024 1.03±0.010 1.97±0.068 0.26±0.003 0,55±0.026 0.43 ±0.002 0.43±0.001 0.39±0.001 0.46 ± 0,003 0.72±0.043 0.15±0.003 0.50±0.038 0.31 ±0.001 0.60±0.002 j8-Pinene 0.52±0.037 0.17±0.001 0.79±0.004 ( + )-Sabinene 0.12±0 3.18±0.047 0.12±0.013 0.09±0.001 0.14±0 0.29±0.001 0.20±0.001 0.22±0.001 Myrcene 2.23±0.043 87.24±0.626 2.40±0.60 39.30+0.651 2.46±0.093 3.18+0.021 3.26±0.008 3.15+0.010 Limonene 78.24±1.119 5.81 ±0.050 72.21 ±2.207 55.41 ±0.249 86.31±1.088 92.33 ±0.460 90.45±0.174 94.13±0.373 0.19±0.001 y-Terpinene 6.04±0.207 0.36±0.001 9.31 ±0.342 0.22±0.002 0.ll ±0.003 .0.ll ±0.001 0.11±0.001 0.16±0.001 /?-Cymene 0.05 ±0.002 0.36±0.005 0.12±0.003 0.01±0 0.01 ±0.001 0.04±0.001 0.06 ±0.002 tr Terpinolene 0.28±0.014 0.46±0.018 0.02+0.001 tr tr tr «-Octanal 0.28 ±0.005 0.18±0.001 0.02±0.001 «-Nonanal 0.07±0.004 0.05±0.001 0.02±0.001 tr 0.12+0.012 0.04±0.001 0.07±0.002 (Z)-Limonene oxide 0.17±0.021 tr (£)-Limonene oxide 0.30±0.030 0.01 ±0.001 0.10±0.002 0.17±0.017 Citronellal 0,19±0.012 0.1 1 +0.007 0.02±0.004 tr tr > «-Decanal 0.08±0.010 0.17+0.018 0.04+0.002 0.06±0.001 0.01 +0.001 0.11±0.001 tr > a-Copaene 0.07±0.001 0.32±0.032 0.04±0.002 tr tr - 0.08±0.001 tr j8-Copaene 0.60±0.001 0.16±0.016 0.01 ±0.001 - tr - 0.07±0.001 0.22±0.001 > Linalool 0.01 ±0.001 3.26±0.1-50 0.39±0.015 0.24±0.013 0.21 ±0.001 0.17±0.001 0.17±0.003 ^-Elemene 0.03±0.001 å - - - 0.12±0.001 - /?-Caryophyllene 0.06±0.001 0.09±0.016 0.12±0.029 0.01 ±0 0.05±0.001 0.06±0.001 0.05±0.001 Terpinen-4-ol 0.02±0.002 tr 0.02±0.005 0.13±0.001 tr tr 0.02±0.001 0.03 ±0.001 0.04 ± 0.006 a-Humulene 0.07±0.008 -. -- - t r - 0,19±0.018 /7-Decyl acetate 030 + 0.048 0.01 ±0 0.01 ±0.001 - 0.13±0.001 0.06±0.001 0.12+0.001 Neral 0.03+0 0.22+0.018 0.13±0.015 tr tr 0.11±0.070 tr a-Terpineol 0.39+0.054 å 0.15+0.032' 0.06±0.005 0.08±0.009 0.07±0.001 ".0.05±0.001 0.08±0.001 ^-Cubebene 0.09±0.016 0.22±0.020 1.52±0.189 1.16±0.006 0.96±0.008 0.08±0.001 Valencene - - -à" 0.03 ±0.001 0.02±0.001 tr Geranial 0.13±0.013 0.09±0.017 0.33±0.012 0.19±0.020 0.23±0.003 0.17±0.002 0.14±0.001 0.03±0.001 0.06±0.001 0.23 ±0.043 0.08 ±0.005 0.05 ±0.005 tr 0.05 ±0.002 0.17±0.001 (5-Cadinene 0.03±0.001 0.09±0.001 0.02±0.001 Citronellol 0.09±0.001 0.05±0.001 tr Nerol 0.10±0.003 0.02±0.001 0.04±0.003 tr tr Geraniol 0.01 ±0.001 0.04+0.001 0.02±0,001 0.01 ±0.001 tr 2-Dodecenal 0.04±0.001 - : - - 0.04±0.001 -- tr Nootkatone 0.12+0.009tr 0.04+0.007 0.06±0.005 0.06±0.007 0. 14±0.001 0.95 ±0.007 0:30±0.002

ValuesThe CPO(w/wwas%)preparedare the aftermeans±3 standardmonths oferrorstorage.of triplicates. Volatile Constituents of Several Varieties of Pummelos 805

Nootkatone has been known to be a character-impact proportional to the change in the level of valencene.2) It is, compoundof grapefruit, which is a hybrid of pummelo therefore, assumed that there might be another biosyn- and sweet orange.4) We also reported previously that thetic pathway for nootkatone in pummelosbesides that nootkatone was predominant in the characteristic flavor of via valencene. Consequently, it is proposed that the level Tosa buntan.li2) It is, then, assumed that nootkatone may of nootkatone in a citrus CPO should be an indication be a key compound which distinguishes the species of defining the identity of pummelosand their relatives. pummelos as well. In the present experiment, each kind of pummelowas always found to contain nootkatone at a considerable level. The CPOs from several kinds of citrus R eferenc es fruits other than pummelos were quantitatively deter- 1) M. Sawamura and T. Kuriyama, /. Agric. Food mined as well. Nootkatone was also detected in several Chem., 36, 567 (1988). kinds of hybrid buntan such as Natsudaidai, hassaku and 2) M. Sawamura, T. Tsuji and S. Kuwahara, Agric. sanbokan, their contents being 0.11%, 0.40% and 0.03%, Biol. Chem., 53, 243 (1989). respectively. On the other hand, it was not or hardly 3) C. W. Wilson, III and P. E. Shaw, J. Agric. Food detected in the CPOs from Satsuma mandarin, hyuga- Chem., 28, 919 (1980). natsu, ponkan, iyokan and lemon, as well as in those from 4) H. E. Nursten, in "Progress in Flavour Research," C. sinensis Osbeck {Fukuhara orange),5} C. junos Tanaka ed. by D. G. Land and H. E. Nursten, Applied (yuzu),6) Eureka lemon, mandarin and lime7) and overripe Science, London, 1979, pp. 337-355. orange,8) where all of these species are knownto have no 5) K. Uchida, A. Kobayashi and T. Yamanishi, Nippon relation to the pummeloat all. There has been a report Nogeikagaku Kaishi, 58, 691 (1984). that nootkatone was formed in vivo from valencene in cell 6) N. Shinoda, M. Shiga and K. Nishimura, Agric. Biol. suspension cultures of a kind of lemon (C. limon cv. Chem., 34, 234 (1970). Ponderosa).10' Most citrus fruits contain a minute level of 7) A. Baaliouamer, B. Y. Meklati, D. Fraisse and C. valencene: natsudaidai, 0.06%; hassaku, 0.02%; sanbokan, Scharff, /. Sci. Food Agric, 36, 1145 (1985). 0.05%; ponkan, trace; iyokan, 0.03%; lemon, 0.05%. Thus, 8) P. E. Shaw, J. Agric. Food Chem., 27, 246 (1979). it is possible that nootkatone is synthesized from va- 9) M. G. Moshonas and P. E. Shaw, J. Agric. Food lencene. Nevertheless, valencene was absent or its con- Chem., 27, 1337 (1979). centration was too low to accurately measure in the 10) F. Drawert, R. G. Berger and R. Godelmann, Plant pummelo oils. In addition, the significant increase of Cell Reports, 3, 37 (1984). nootkatone during the storage of Tosa buntan was not