Technical Paper Antioxidative Activities of Citrus Peel Essential

Food Sci. Technol. Res., 7 (1), 50–56, 2001 Technical paper

Antioxidative Activities of Citrus Peel Essential Oils and Their Components against Linoleic Acid Oxidation

Hee-Sun SONG, Hiroyuki UKEDA and Masayoshi SAWAMURA*

Department of Bioresources Science, Faculty of Agriculture, Kochi University, B-200 Monobe, Nankoku, Kochi 783-8502, Japan

Received June 9, 2000; Accepted November 2, 2000

Antioxidative activities of thirty kinds of citrus essential oils and their fourteen components against linoleic acid oxidation were examined by a thiocyanate method. The tocopherol contents of these oils were determined by high-per- formance liquid chromatography monitored at 290 nm. All the citrus essential oils and the ﬂavor components showed inhibitory effects against linoleic acid oxidation. Highest antioxidative ability was observed in the essential oils of yuzu, lemon, hassaku, sudachi, mochiyu, yuko and Tarocco orange. Antioxidative activities of -pinene, myrcene, -terpinene, -terpinene and decanal were higher or similar to that of d-tocopherol. Tocopherols were contained abun- dantly in the essential oils of limes, Valencia orange, yuzu and lemons. However, there was little correlation between tocopherol contents and antioxidative activities in citrus essential oils. The composition of terpene compounds seems to be a major factor in the antioxidative activities of these oils.

Keywords: antioxidative activity, citrus essential oil, tocopherols, terpenoids, peroxide value, thiocyanate method

Antioxidants have been investigated most intensively as con- however, there have been few studies regarding the antioxidative stituents preventing diseases associated with oxidative damage, activities of citrus essential oils and citrus constituents. and decreasing lipid oxidation during the processing and storage In the present study, the antioxidative activities of citrus essen- of seafood (Boyd et al., 1993). Synthetic antioxidants such as tial oils and their components and the vitamin E content of the butylated hydroxytoluene (BHT), butylated hydroxyanisole oils were determined. (BHA) and propyl gallate (PG) have been used as food additives to inhibit the actions of toxic and carcinogenic substances Materials and Methods (Chang et al., 1977). Natural antioxidants from natural foods Extraction of citrus essential oils and authentic compounds such as herbs (Bracco et al., 1981; Inatani et al., 1982; Pizzocaro Citrus samples used are listed in Table 1, according to Swingle’s et al., 1994), vegetables (Tsushida et al., 1994; Vinson et al., taxonomy (Swingle, 1943). Twenty-six kinds of citrus fruits and 1998), fruits (Nogata et al., 1996; Wang et al., 1996), oilseeds one kind of kumquat were obtained from the Kochi Prefectural (Osawa & Namiki, 1981; Fukuda et al., 1985; Wanasundara et Fruit Tree Experimental Station and the Japanese Ministry of al., 1994; Medina et al., 1999), spices (Shahidi et al., 1994; Agriculture, Forestry and Fisheries’ Okitsu Branch Experimental Chipault et al., 1995; Kramer, 1995; Hettiarachchy et al., 1996), Station in Shizuoka. Bergamot (Italy) was collected in Reggio green tea (Amarowicz & Shahidi, 1996) and cereals (Asamari et Calabria in Italy. Korean yuzu fruits were obtained from the al., 1996; Maillard et al., 1996; Hendelman et al., 1999) have National Citrus Research Institute in Chejudo and from the been studied, and some of them, such as ascorbate and toco- Yuzanara Farm in Chindo. Citrus essential oils were prepared by pherols, are currently used in a variety of food products. a-Toco- the cold pressing method (Njoroge et al., 1996) and were kept at pherol, a form of vitamin E, is widely used as a natural anti- 25˚C until analyzed. Seven kinds of citrus essential oils stored oxidant. It was suggested that vitamin E diet supplementation for 5 years were also used as sample oils. decreased the risk of coronary heart disease and the oxidation of Fourteen kinds of authentic compounds were employed: a- low-density lipoprotein (LDL), platelet adhesiveness and throm- pinene, terpinolene and d-limonene were from Tokyo Kasei bosis (Zhu et al., 1999). g-Tocopherol and sesamol reportedly Kogyo Co., Ltd. (Tokyo); b-pinene, linalol, octanal, decanal and stimulate high antioxidative activity of sesame oil (Fukuda et al., citronellal were from Wako Pure Chemical Industries (Osaka); 1981; 1985). In recent reports much attention has been given to myrcene was from Aldrich Chemical Co. (Milwaukee, WI); a- citrus components, since they present various pharmaceutical ac- terpinene and g-terpinene were from Sigma Chemical Co. (St. tivities of anticarcinogenicity, antimutagenicity, antioxidative ac- Louis, MO) and Extrasynthèse S.A. (Genay, France), respective- tivity and antiaging (Nogata et al., 1996; Miyake et al., 1997; ly; a-, g- and d-tocopherols were from Eisai Co. (Tokyo). Rapisarada et al., 1999; Sawamura et al., 1999). Grapefruit oil Measurement of antioxidative activity Either 1 ml of cit- and lemon extracts have been suggested as the effective natural rus essential oil or 0.01 m mole of authentic compound in 5 ml of antioxidative compounds (Miyake et al., 1997; Tokoro, 1997), 0.1 M linoleic acid-ethanol was diluted to 25 ml with 0.1 M phos- phate buffer (pH 7.0) and incubated at 37˚C. The ﬁnal concentra- *To whom correspondence should be addressed. tion of authentic compound solution was 0.4 M. The reaction E-mail: [email protected] m Antioxidative Activities of Citrus Peel Essential Oils 51

Table 1. Samples of the Citrus genus. No. Common name Varieties Speciesa) 1Yuzu (Kochi, Japan) Citrus junos Sieb. ex Tanaka C. ichangenesis 2Yuzu (Cheju, Korea) C. junos Sieb. ex Tanaka C. ichangenesis 3Yuzu (Chindo, Korea) C. junos Sieb. ex Tanaka C. ichangenesis 4 Seedless yuzu C. junos Sieb. ex Tanaka C. ichangenesis 5 Sudachi C. sudachi Hort. ex Shirai C. ichangenesis 6 Sudachi (’94) C. sudachi Hort. ex Shirai C. ichangenesis 7 Mochiyu C. inflata Hort. ex Tanaka C. ichangenesis 8Yuko C. yuko Hort. ex Tanaka C. ichangenesis 9Yuko (’94) C. yuko Hort. ex Tanaka C. ichangenesis 10 Ichang lemon C. Wilsonii Tanaka C. ichangenesis 11 Mexican lime C. aurantifolia Swingle C. aurantifolia 12 Tahiti lime C. latifolia Tanaka C. aurantifolia 13 Bergamot (Italy) C. bergamia Risso var. Fantastico C. aurantifolia 14 Bergamot (Japan) C. bergamia Risso var. Balotin C. aurantifolia 15 Eureka lemon C. limon Burm. f. cv. Eureka C. limon 16 Eureka lemon (’95) C. limon Burm. f. cv. Eureka C. limon 17 Lisbon lemon C. limon Burm. f. cv. Lisbon C. limon 18 Lisbon lemon (’95) C. limon Burm. f. cv. Lisbon C. limon 19 Tosa-buntan C. grandis Osbeck forma Tosa C. grandis 20 Grapefruit C. paradisi Macfadyen C. paradisi 21 Hassaku C. hassaku Hort. ex Y. Tanaka C. paradisi 22 Natsudaidai C. natsudaidai Hayata C. paradisi 23 Daidai C. aurantium Linn. var. Cyathifera Y. Tanaka C. aurantium 24 Daidai (’94) C. aurantium Linn. var. Cyathifera Y. Tanaka C. aurantium 25 Kabusu C. aurantium Linn. forma Kabusu C. aurantium 26 Valencia orange C. sinensis Osbeck forma Valencia C. sinesis 27 Tarocco orange C. sinensis Osbeck var. Sanguinea Tanaka forma Tarocco C. sinesis 28 Tarocco orange (’94) C. sinensis Osbeck var. Sanguinea Tanaka forma Tarocco C. sinesis 29 Iyokan C. iyo Hort. ex Tanaka C. sinesis 30 Hyuganatsu C. tamurana Hort. ex Tanaka C. sinesis 31 Ujukitsu C. ujukitsu Hort. ex Shirai C. sinesis 32 Satsuma mandarin (M) C. unshiu Marcov. forma Miyagawa-wase C. reticulata 33 Satsuma mandarin (I) C. unshiu Marcov. forma Imamura C. reticulata 34 Ponkan C. reticulata Blanco cv. F-2426 C. reticulata 35 Oyu C. ozu Hort. ex Y. Tanaka Unidentified 36 Oyu (’94) C. ozu Hort. ex Y. Tanaka Unidentified 37 Kumquat Fortunella japonica Swingleb) Fortunella japonica a)Classified by Swingle. b)Another genus in the Rutaceae family comprising the Citrus genus.

mixture was used for the assay of peroxide value (POV) with the Wakosil 5C18 column (250 mm ¥ 2 mm i.d., Wako Pure Chemi- thiocyanate method, which has been suggested as a proper meth- cal Industries, Osaka) was used. The isocratic mobile phase was od for examination of the antioxidative activities of grapefruit acetonitrile-methanol (85:15 v/v). The ﬂow rate was 0.2 ml/min. oils containing antioxidants of oil or water systems (Tokoro, A 20-ml sample was analyzed by absorbance at 294 nm. 1997). An aliquot of the solution, 0.1 ml, was mixed with 4.7 ml Gas chromatography (GC) and GC/mass spectrometry of 75% ethanol, 0.1 ml of 30% NH4SCN and 0.1 ml of 0.02 M (MS) conditions A Shimadzu GC-14A equipped with a ﬂame

FeCl2 in 3.5% HCl, then allowed to stand for 3 min. The POV ionization detector and a Shimadzu GC/MS QP-5000 were used was estimated by absorbance at 500 nm (Osawa & Namiki, for quantitative determination and identification of flavor compo- 1981; Miyake et al., 1997) and triplication. nents, respectively, of citrus essential oils. These analytical con- Tocopherol assays The tocopherol assays of citrus essen- ditions were the same as those in the previous paper (Song et al., tial oils were carried out by the method described in Osuna- 1999). The data were shown as relative weight percents. García et al. (1998). A mixture of 300 mg of citrus essential oil, Statistical analysis Analytical data triplicated were used 0.25 g of ascorbic acid, 10 ml of Milli-Q water (Millipore, for statistical analysis. Statistical analysis was performed by use Tokyo) and 30 ml of 99.5% ethanol was homogenized for 90 s. of the SAS computer program (SAS System, version 6.12, SAS After 15 min, 6 ml of 50% KOH was added to the reaction mix- Institute Inc.). Contents of tocopherols and peak area percents of ture. The sample flushed with nitrogen was kept overnight at flavor components in citrus essential oils were statistically exam- 30˚C, and for a further 2 h at 50˚C. The cooled sample was fil- ined by analysis of variance (ANOVA), and significant differ- tered and the slurry was rinsed with 30 ml of 50% ethanol and ences of several groups were analyzed by LSD and Tukey’s test extracted three times with 30 ml of hexane. The hexane extract (Gomez & Gomez, 1984). rinsed with Milli-Q water was evaporated under a nitrogen stream. The residue was redissolved in 2 ml of methanol and fil- Results and Discussion tered through a 0.22-mm membrane prior to HPLC. Antioxidative activities of citrus essential oils Relative HPLC operating conditions A Shimadzu LC-10ADVP antioxidative activities of citrus essential oils are shown in Fig. 1. liquid chromatograph fitted with a Shimadzu SPD-10AVP UV- Calculations of their activities were modified. The straight line VIS detector was used to separate and quantify tocopherols. A equations of peroxide values in samples and control were prima- 52 H-S. SONG et al. rily determined by regression analysis (Choi & Han, 1995) for calculation of relative lipid peroxidation rate (RLPR). The peroxide values were measured at intervals during an incubation period of two weeks. RLPRs were calculated with the slopes of the straight line equations (Tsushida et al., 1994). The calculation formula of RLPR is as follows:

As RLPR (%) = ¥100, Ac where, As: the slope of the straight line equation of the sample,

Ac: the slope of the straight line equation of the control. All the citrus essential oils used in the present study had antioxidative activities against linoleic acid peroxidation. It seems

Fig. 2. Correlation between total tocopherol contents and antioxidative activities in citrus essential oils.

Fig. 3. Antioxidative activities of authentic compounds relating to flavor components in citrus essential oils. Samples with asterisk (*) were of the concentrations adjusted to the means in citrus essential oils: a-tocopherol, Fig. 1. Antioxidative activities of citrus essential oils. 0.006 mM; g-tocopherol, 0.0002 mM; d-tocopherol, 0.0002 mM. Antioxidative Activities of Citrus Peel Essential Oils 53 possible to group the citrus samples according to three levels of rus classification. These results suggest that citrus essential oils antioxidative activities. The essential oils of yuzu (Japanese and may contain some constituents contributing to their different Korean), Lisbon and Eureka lemons, hassaku, sudachi, mochiyu, antioxidative activities among the species. yuko and Tarocco orange presented high antioxidative activities, Tocopherol contents in citrus essential oils Tocopherols more than 90%. The oils of the second group, showing antioxi- of citrus essential oils were identified and quantified by HPLC. dative activities in the range of 70% to 88% were bergamot (Ital- The preliminary tocopherol assay was performed using standard ian), Hyuganatsu and Tahiti lime, Tosa-buntan, natsudaidai, a-tocopherol. Recovery in the assay was about 90%. The daidai, Valencia orange, Iyokan, Satsuma mandarin (M), ponkan, amounts of tocopherols in citrus essential oils are shown in Table oyu and kumquat. Other oils such as ichang lemon, bergamot 2. Each tocopherol content is given in mg per 100 mg of citrus (Japanese), grapefruit, kabusu, ujukitsu and Satsuma mandarin essential oil. Mexican and Tahiti limes, Valencia orange and yuzu (I) showed comparatively low antioxidative activities in the range essential oils were relatively abundant in tocopherols (about 0.4 of 49% to 60%. Choi et al. (2000) examined free radical-scav- mg to 0.6 mg of total tocopherol content). Mochiyu, ichang lem- enging effects of 37 kinds of citrus essential oils and volatile fla- on, natsudaidai, daidai and kabusu essential oils contained little vor components; radical-scavenging activities of oyu, daidai and a-tocopherol. Lisbon lemon oil was abundant in g-tocopherol, Valencia orange oils were weaker than that of Trolox, however, and daidai, hassaku, Valencia and Tarocco oranges oils in d-toco- yuzu, lemons, limes, ichang lemon, hassaku, sudachi, yuko and pherol. The total tocopherol content of citrus essential oils usu- kabosu oils showed high radical-scavenging effects of over 50%. ally depended on the level of a-tocopherol. These facts of previous reports support the present results that The correlation between total tocopherol contents and antioxi- citrus essential oils have antioxidative activities. From the results dative activities of citrus essential oils was examined by statisti- of Fig. 1 it is possible to suggest that the species C. limon, C. cal analysis, as shown in Fig. 2. There was no significance in grandis, C. paradisi and C. reticulata have high antioxidative their relationship at the low level of correlation coefficient activities. It has been previously reported that the inhibitory (0.0022). Correlation coefficients between each a-, g- and d- effects of citrus extracts on cyclooxygenase and lipoxygenase tocopherol content and antioxidative activities of citrus essential differed in citrus species; extracts of citron and Eureka lemon oils were as low as 0.0058, 0.0530 and 0.0003, respectively. showed selective inhibition effects on cyclooxigenase and those Thus, it is suggested that the antioxidative activities of citrus of yuzu and sudachi did so on lipoxigenase (Nogata et al., 1996). essential oils are little related to their tocopherol contents. They also have shown that the inhibitory compounds on both Antioxidative activities of relating flavor compounds All enzyme metabolites were related to the citrus classification. the authentic compounds tested showed some antioxidative ac- Kawaii et al. (1999) also reported that the contents of 24 kinds of tivities, as shown in Fig. 3. g-Terpinene showed highly antioxida- flavonoids in the edible part of citrus fruits were related to the cit- tive activities, as much as 2.6% of RLPR. The RLPRs of b-

Table 2. Tocopherol contentsa–d) in Citrus essential oils. Citrus essential oil a-Tocopherol g-Tocopherol d-Tocopherol Total amount Yuzu 0.384ab 0.001bc — 0.385abcd Seedless yuzu 0.149bc 0.001c 0.150cde Sudachi 0.078c 0.001bc — 0.079e Mochiyu — tr — tr Yuko 0.096c tr — 0.096e Ichang lemon — 0.001c 0.025cde 0.026e Mexican lime 0.624a 0.010bc 0.003f 0.637a Tahiti lime 0.578a —0.002f 0.580ab Bergamot (Fantastico)0.007c 0.002bc — 0.009e Bergamot (Balotin)0.036c 0.001c — 0.037e Eureka lemon 0.098c 0.008bc — 0.106de Lisbon lemon 0.150bc 0.111a — 0.261bcde Tosa-buntan 0.230bc 0.003bc — 0.233cde Grapefruit 0.195bc 0.002bc — 0.197cde Hassaku 0.011c 0.003bc 0.047ab 0.061e Natsudaidai — 0.010bc 0.006ef 0.016e Daidai — 0.005bc 0.057a 0.062e Kabusu — 0.011b 0.015def 0.026e Valencia orange 0.391ab —0.033bcd 0.424abc Tarocco orange 0.032c 0.002bc 0.037abc 0.071e Iyokan 0.006c 0.003bc — 0.009e Hyuganatsu 0.020c 0.003bc — 0.023e Ujukitsu 0.007c 0.002bc — 0.009e Satsuma mandarin (Miyagawa-wase)0.043c 0.001c 0.003f 0.047e Satsuma mandarin (Imamura)0.172bc 0.001c 0.002f 0.175cde Ponkan 0.171bc 0.005bc — 0.176cde Oyu 0.016c — — 0.016e Kumquat 0.007c 0.002bc — 0.009e Stdd) 0.1650.0005 0.1640.0005 0.2260.0005 0.5220.0005 a) Values are given in mg of tocopherol per 100 mg of citrus essential oil. b) tr is less than 0.001 mg. c)Means within columns having the same superscript letters are not significantly different (p<0.05). d)Standard deviation. 54 H-S. SONG et al. pinene, myrcene, a-terpinene and decanal were less than 10%. ing high antioxidative activities have the isopentenyl group (A in The RLPR of terpinolene was 18.6%. It was reported that terpi- Fig. 4) in their chemical structures. The conjugated double-bond nolene had an inhibitory effect on the oxidation of clove oil and and hydrophobicity of authentic flavor components including nutmeg oil (Dorman et al., 2000). a-Pinene and citronellal were octanal and decanal seem to be important to antioxidation. How- not as antioxidative, 26.1% and 42% of RLPR, respectively. In ever, the antioxidative mechanism among these authentic com- the previous report the radical-scavenging effects of g-terpinene, pounds is not clear in the present study. a-terpinene, terpinolene, geraniol, citronellal, citral and nootka- Standard samples of both a- or g-tocopherol of 0.4 mM (0.01 m tone were much higher than that of Trolox (Choi et al., 2000). It mole in 25 ml of the test solution) were not as antioxidative, is suggested that flavor components have high antioxidative ac- about 30% of RLPR; however, that of d-tocopherol showed anti- tivities even at a low level of concentration because the concen- oxidative activity of as low as 8% of RLPR. Several extracts trations of authentic flavor components used in this study (0.4 from sesame oil and sesamol showed lower antioxidative effects mM) were much lower than those in natural citrus essential oils. than g-tocopherol as given in the previous report (Fukuda et al., a-Pinene, b-pinene, g-terpinene and decanal, for instance, com- 1985). On the other hand, tested solutions comprising an individ- monly occur in yuzu essential oils at higher levels (0.14 M; 1.94 ual isomer of a-, g- and d-tocopherols, which were preliminarily mg, 0.06 M; 0.79 mg, 0.69 M; 9.39 mg, 0.57 M; 0.09 mg/100 mg adjusted to the mean concentrations in citrus essential oils of of yuzu oil, respectively), and those are not the major component 0.006 mM, 0.0002 mM and 0.0002 mM, respectively, showed com- like limonene (5 M; 72.25 mg/100 mg of yuzu oil) (Song et al., paratively low antioxidative activities, although above 31% of 1999). RLPR. It has been reported that all constituents extracted from From Fig. 3 and Fig. 4 we can suggest one of the relations grapefruit oil as antioxidants showed higher antioxidative effects between the antioxidative activities of flavor components and than a-tocopherol, even as high as BHT (Tokoro, 1997). These their chemical structures. The authentic flavor compounds show- results suggest that the tocopherols enhance the antioxidative

Fig. 4. Chemical structures of authentic ﬂavor components. Antioxidative Activities of Citrus Peel Essential Oils 55

Table 3. Vo latile flavor componentsa–d) of Citrus essential oils. Sudachi Yuko Eureka lemon Lisbon lemon Daidai Tarocco orange Oyu Stdd) Fresh ‘94 Fresh ‘94 Fresh ‘95 Fresh ‘95 Fresh ‘94 Fresh ‘94 Fresh ‘94 ef g b a d d e c ji ji ji i h h a-Pinene 1.52 1.29 3.13 3.66 2.27 2.31 2.19 2.43 0.49 0.49 0.46 0.55 0.78 0.78 1.04 0.005 ef ef d d b c a b fg fg j j g f g b-Pinene 0.51 0.32 1.49 1.76 12.71 12.03 12.47 13.30 0.40 0.08 tr tr 0.01 0.11 5.590.005 Myrcene 2.07b 0.34f 1.55bcd 2.74a 1.70bc 1.73bc 1.71bc 1.68bcd 1.27cde 0.82ef 1.22cde 1.32cde 0.75ef 1.04de 0.670.005 c c a c b b b b a-Terpinene 0.02 tr 0.57 tr 0.17 0.16 0.16 0.15 00 00 000.160.005 Limonene 62.80e 46.72i 59.94gh 57.42h 59.43g 61.24f 59.59g 57.03h 84.54b 84.83ab 85.68a 85.64b 81.12d 83.40c 13.630.005 g j b a e f c d h h h h h h g-Terpinene 4.99 tr 18.57 19.79 7.89 7.79 8.83 8.55 0.23 0.18 0 0 0.15 0.22 6.680.005 Terpinolene 0.23g 0.02h 0.80b 0.86a 0.33e 0.32f 0.37c 0.36d tri tri 0.01i 0.01i 0.01i 0.01i 0.290.005 Octanal 0.12b 0.37b 0.08b 0.04b 0.08b 0.05b 0.19b 0.02b 0.76a b 0.35b 1.44a 0.21b trb trb 0.570.005 Citronellal 0.21a 0.06b 00 0.06b 0.06b 0.05b 0.03b 0.08b 0.05b 0.11b 0.02b 0.02b 0.01b 0.090.005 Decanal 0.09c 0.09c 0.04e 0.02f 0.07d 0.07d 0.04e 0.02g 0.17a 0.10b 0.09c 0.08c trh 0.01g 0.040.005 Linalol 0.62b 0.79a 0.26d 0.20ef 0.24d 0.22d 0.26d 0.27c 0.18h 0.24e 0.26d 0.20g 0.11i 0.11i 0.130.005 a)Values are given in relative weight percent (in mg of flavor component per 100 mg of citrus essential oil). b)tr is less than 0.005%. c)Values within rows having the same superscript letter are not significantly different (p<0.05). d)Standard deviation.

activities of citrus essential oils only slightly, the flavor compo- ponkan, orange, grapefruit and buntan peel extracts (Miyake et nents being primarily responsible for antioxidation. al., 1997). Major flavor components of several citrus essential Effects of stored oil Antioxidative activities of stored cit- oils As shown in Table 3, the major flavor components of sev- rus oils were considerable (Fig. 1 and 2, and Table 3), and merit eral citrus essential oils were identified. Significant differences further discussion. The antioxidative activities of Lisbon and among their peak area percents were statistically analyzed. The Eureka lemons essential oils showed little difference between the relationship between antioxidative activities of citrus essential stored and the fresh oils. The RLPRs of stored daidai, Tarocco oils and the compositions of their flavor components through orange and oyu oils were 72%, 16.6% and 28.7%, respectively, antioxidative activities of authentic flavor compounds can be twice as high as those of fresh samples. The a-pinene contents of seen in Figs. 1 and 3 and Table 3. The essential oils of yuko, Lis- stored Lisbon lemon and yuko oils (2.43% and 3.66%, respec- bon and Eureka lemons and sudachi were abundant in a-pinene tively) were significantly higher than in their fresh oils (2.19% and myrcene (1.5% to 3.1%, and 1.6% to 2.1%, respectively). and 3.13%, respectively). Both fresh and stored Lisbon lemon Lemon oils were significantly abundant in b-pinene accounting oils were abundant in g-terpinene as high as about 8.8% and for about 12.5% of weight percent. Daidai, Tarocco orange and 8.6%, respectively, of which RLPR was the lowest (2.6%). oyu oils contained little a-terpinene, and their antioxidative ac- Myrcene and terpinolene were significantly more abundant in the tivities were no higher than those of sudachi and lemons oils. stored yuko oil than in the fresh oil. b-Pinene in fresh Eureka and The content of limonene, which is a major flavor component in Lisbon lemon essential oils were more abundant than those in the citrus oils, ranged from 59% to 63% in Lisbon and Eureka lem- stored ones (12.71% and 12.47%, respectively). The contents of ons, yuko and sudachi; 84.5%, 85.7% and 81.1% in Tarocco limonene and a-pinene were not significantly different between orange, daidai and oyu, respectively. Yuko oil was considerably stored and fresh essential oils including daidai, Tarocco orange rich in g-terpinene and terpinolene (18.57% and 0.80%, respec- and oyu. These results suggest that the composition of terpene tively). The terpinolene contents in Lisbon and Eureka lemons compounds seems to be a major factor in the antioxidative activi- and sudachi oils were significantly higher than those in daidai, ties of citrus essential oils. Tarocco orange and oyu oils. Mochiyu and hassaku showing Studies on the stability of essential oils, especially citrus high antioxidative activities, contained much more monoterpene essential oils, have been focused on their flavor changes resulting hydrocarbons in their essential oils (Sawamura et al., 1999). from deterioration of terpene compounds (Usai et al., 1992; Weight percents of octanal in daidai and Tarocco orange were Njoroge et al., 1996; Haleva-Toledo et al., 1999). However, from significantly as high as 0.76% and 1.44%, respectively. The per- the results of the present study terpene compounds or citrus cent of linalol was markedly high in sudachi (0.62%) but was essential oils can be shown as antioxidative compounds, al- significantly low in oyu (0.11%). Unexpectedly, sudachi essential though those compounds have been reported to be unstable as oil was abundant in citronellal, as much as 0.21%, of which flavoring materials (Usai et al., 1992; Njoroge et al., 1996) RLPR was the highest (42%). High antioxidative activities of the In conclusion, it was demonstrated that the antioxidative activ- citrus essential oils lemons, yuko and sudachi seem to result ities of citrus essential oils, as measured by the thiocyanate meth- from the abundance of terpene hydrocarbons g-terpinene, b- od, resulted from their flavor compositions. It is suggested that pinene and myrcene, because these authentic components the role of tocopherols in citrus essential oils is not significantly showed high antioxidative activities. related to their intrinsic antioxidative activities. It is expected that Other compounds included in citrus fruits such as phenolic citrus essential oils and their related flavor components may con- compounds, flavonoids, cumarins and carotenoids, which have tribute to the prevention of oxidation in foods and inhibit lipid some inhibitory effects on cancer, may contribute to the antioxi- oxidation which results in coronary heart disease in the body. dative activities of citrus oils (Yano, 1998; Kawaii et al., 1999; Rapisarda et al., 1999). An antioxidant, eriocitrin, was abundant Acknowledgement This work was partly supported by a grant from the in lemon and lime peel extracts, however, it was scarce in yuzu, Mitsukan Foundation for Research. 56 H-S. SONG et al.

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