Polymethoxyflavones, Synephrine and Volatile Constitution of Peels of Citrus Fruit Grown in Okinawa

Sayuri Inafuku-Teramoto1,2*, Ryuichi Suwa1, Yasunori Fukuzawa1 and Yoshinobu Kawamitsu1

1Faculty of Agriculture, University of the Ryukyus, Okinawa 903-0213, Japan 2The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065, Japan

Shiikuwasha (Citrus depressa Hayata) and other endemic citrus fruits have been cultivated for several hundred years in the Ryukyu Islands. Citrus fruits contain high levels of functional phytochemicals, including specific and appealing aroma compounds. We analyzed the volatile compounds in immature and mature peel of 10 citrus fruits grown on Okinawa by GC-MS analysis. In addition, we used HPLC to quantify synephrine and six polymethoxyflavones (PMFs: sinensetin, hexamethoxyflavone, heptamethoxyflavone, nobiletin, natsudaidain, and tangeretin). All local citrus cultivars showed unique aroma profiles. The volatile compositions of ‘Kabuchii’ (C. keraji hort. ex Tanaka var. kabuchii) had high contents of sesquiterpene hydrocarbons (3.90–5.17%), ‘Keraji’ (C. keraji hort. ex Tanaka) was high in esters (12.15–19.10%), and ‘Ogimikuganii’ (C. depressa Hayata) was high in γ-terpinene (21.17–29.60%) and p-cymene (6.49–9.84%). The highest levels of synephrine were found in immature peel of ‘Tokunibu’ (C. nobilis Lour.) (8.97 mg·gDW−1), ‘Izumibeni’ (C. tangerina hort. ex Tanaka) (7.03 mg·gDW−1), and ‘Ogimikuganii’ (5.17 mg·gDW−1). There were high levels of PMFs in immature peel of ‘Ogimikuganii’ (20.62 mg·gDW−1), ‘Kabuchii’ (20.66 mg·gDW−1), ‘Oto’ (C. oto hort. ex Yu. Tanaka) (12.52 mg·gDW−1), and ‘T-132’ (C. tankan Hayata) (18.95 mg·gDW−1), each of which showed a unique profile. Our results suggested the scope for effective utilization of the waste, including thinned fruits, from shiikuwasha and tankan, that major cultivars in Okinawa.

Key Words: GC-MS, HPLC, Kabuchii (Citrus keraji hort. ex Tanaka var. kabuchii), Keraji (Citrus keraji hort. ex Tanaka), Ogimikuganii (Citrus depressa Hayata).

smaller than that in Europe, but the sales of aromatherapy Introduction goods are increasing every year. Several kind of citrus Citrus fruits are among the most important fruits essential oils are produced on a small scale, including worldwide. Throughout human history, many parts of yuzu (Citrus junos Siebold ex Tanaka), kabosu Citrus plants have been used for diverse purposes: as (C. sphaerocarpa hort. ex Tanaka), hyuganatsu foods, flavorings, spices, medicines, and perfumes. For (C. tamurana hort. ex Tanaka), and shiikuwasha example, mandarin peel is important in Chinese (C. depressa Hayata). medicine, and the peel oils of major citrus species, which Citrus fruits, particularly the peel, also contain high are produced as byproducts from the juice industry, are levels of flavonoids (Nogata et al., 2006; Yamamoto et used as food additives in housekeeping products and as al., 2008), which have diverse functions in human health. aromatherapy oils (Buckle, 2000). Many reports have One of the most important flavonoid groups is the described the volatile components of Japanese citrus polymethoxyflavones (PMFs), which are found at high fruits (Akakabe et al., 2008a, b; Choi and Sawamura, levels in shiikuwasha (Nogata et al., 2006; Teramoto et 2001, 2002; Phi et al., 2006, 2009; Takeuchi et al., 2005) al., 2010; Yamamoto et al., 2008). PMFs have well- and common citrus fruits cultivated worldwide (Belsito known functions and bioactivities, including anti- et al., 2007; Elmaci and Altug, 2005; Fanciullino et al., tumor, anti-inflammation, anti-hyperglycemic, and anti- 2006; Gariele et al., 2009; Shaw et al., 2001; Yadav et rheumatism effects (Choi et al., 2007; Hirata et al., 2009; al., 2004). The Japanese aromatherapy industry is much Hosseinimehr et al., 2009; Juichi, 2005; Kawai et al., 1999; Kunimasa et al., 2009; Wu et al., 2006). Received; March 11, 2010. Accepted; December 1, 2010. Epidemiological research has also been conducted on * Corresponding author (E-mail: [email protected]). synephrine, a citrus peel component that is used as a

214 J. Japan. Soc. Hort. Sci. 80 (2): 214–224. 2011. 215 dietary supplement for metabolic stimulation can be developed from the fruit and juice residues. (Westanmo, 2007). Synephrine is a major component of Here, we aimed to characterize the volatile Chinese medicines formulated for indigestion and components and to quantify PMFs and synephrine in the pharynx troubles (Pellati et al., 2004; Tsujita and Takaku, peel of 10 citrus fruits grown on Okinawa. This 2008). Furthermore, several volatile components have information will be useful for the development of new been shown to have antitumor and lipolytic activities processing methods and products, which will improve (Choi, 2006; Del Toro-Arreola et al., 2005; Mastelic the utilization of these resources. et al., 2008; Patil et al., 2009). Materials and Methods Shiikuwasha originated on Okinawa and many phenotypes occur throughout the Ryukyu Islands. On Plant materials Okinawa, the production of shiikuwasha has increased We selected seven local citrus cultivars and three over recent years, exceeding 3,500 t in 2009. Production major introduced commercial citrus cultivars for analysis now exceeds the amount required for juice production, (Table 1). Various phenotypes of shiikuwasha are grown so new ways of using the resources are needed. Several in Okinawa, and two were analyzed, ‘Ogimikuganii’ and citrus accessions in the Ryukyu Islands, including the ‘Ishikunibu’. The other local citrus cultivars are Okinawa and Amami Islands, remain uninvestigated ‘Kabuchii’ (C. keraji hort ex Tanaka var. kabuchii), (Teramoto et al., 2010; Yamamoto et al., 2006), and little ‘Keraji’ (C. keraji hort. ex Tanaka), ‘Rokugatsumikan’ information about the phytochemicals and volatile (C. rokugatsu hort. ex Yu. Tanaka), ‘Oto’ (C. oto hort. constituents of Okinawa local citrus fruits is available ex Yu. Tanaka), and ‘Tokunibu’ (C. nobilis Lour.). (Nogata et al., 2006; Teramoto and Kawamitsu, 2010; ‘Tokunibu’ was introduced from China or Southeast Asia Yamamoto et al., 2008). Thus, the composition and in the middle of the 15th century (Tanaka, 1948). The functional properties of these local citrus cultivars should introduced commercial citrus cultivars are ‘T-132’ be investigated so that premium, value-added products (C. tankan Hayata), ‘Izumibeni’ (C. tangerine hort. ex

Table 1. The phenotypic characters of mature citrus fruits grown on Okinawa.

C. rokugatsu hort. Scientific name C. depressa Hayata C. depressa Hayata C. madurensis Lour. C. oto hort. ex Yu. Tanaka (Local name) (Ogimikuganii) (Ishikunibu) (Shikikitsu) ex Yu. Tanaka (Oto) (Rokugatsumikan) Fruit weight (g) (n = 10) 38.4 ± 2.4 33.8 ± 4.9 24.4 ± 9.1 117.4 ± 19.3 99.7 ± 10.3 Fruit shape index (n = 10) 137 156 111 122 116 Brix (%) (n = 5) 10.0 ± 0.8 11.7 ± 0.5 10.3 ± 0.9 9.6 ± 0.5 9.9 ± 0.4 Acidity (%) (n = 5) 2.5 ± 0.2 1.2 ± 0.2 7.3 ± 1.4 1.45 ± 0.2 1.4 ± 0.2 Average seed per fruit (n = 5) 14.6 8.4 5.4 12.3 12.4 Segment number (n = 5) 8.4 ± 0.8 9.5 ± 0.8 6.8 ± 0.8 8.8 ± 1.1 10.8 ± 1.1 Peel thickness (mm) (n = 5) 1.8 ± 0.2 1.8 ± 0.4 1.8 ± 0.3 6.1 ± 1.0 2.6 ± 0.6 Fruit skin some rough (reentrant) rough smooth rough smooth Peeling easy easy easy easy easy Harvest date (immature) 22nd Sep., 2008 22nd Sep., 2008 11th Oct., 2008 22nd Sep., 2008 23rd Sep., 2008 Harvest date (mature) 28th Nov., 2008 20th Feb., 2009 18th Dec. 2008 18th Mar., 2009 12th Dec. 2008 Type of usage sour and table sour sour table table Fruit skin colorz vivid yellow 2507 dark orange 1307 vivid orange 1605 vivid yellowish orange 1906 vivid yellow 2507 Fresh colorz yellowish orange 1907 dark orange 1307 orange 1606 yellowish orange 2210 orangey yellow 2210

Scientific name C. keraji hort. ex Tanaka C. keraji hort. ex Tanaka C. nobilis Lour. C. tankan Hayata C. tangerina hort. ex (Local name) (Kabuchii) (Keraji) (Tokunibu) (T-132) Tanaka (Izumibeni) Fruit weight (g) (n = 10) 88.5 ± 6.4 79.0 ± 11.8 246.3 ± 24.6 150.3 ± 8.5 85.1 ± 4.4 Fruit shape index (n = 10) 123 113 135 119 138 Brix (%) (n = 5) 9.2 ± 0.3 9.3 ± 0.6 9.1 ± 0.3 11.3 ± 0.7 12.6 ± 1.2 Acidity (%) (n = 5) 1.1 ± 0.2 1.1 ± 0.5 1.0 ± 0.1 1.0 ± 0.1 1.3 ± 0.3 Average seed per fruit (n = 5) 11.0 0.6 4.3 0.4 9.2 Segment number (n = 5) 9.4 ± 1.1 8.7 ± 1.3 11.8 ± 1.3 10.6 ± 0.6 10.4 ± 0.6 Peel thickness (mm) (n = 5) 4.4 ± 0.0 4.0 ± 0.5 5.0 ± 1.0 2.9 ± 0.9 2.1 ± 0.2 Fruit skin rough rough rough slightly rough smooth Peeling easy easy not easy easy easy Harvest date (immature) 23rd Sep., 2008 27th Aug., 2009 22nd Sep., 2008 28th Sep., 2008 28th Sep., 2008 Harvest date (mature) 22nd Oct., 2008 30th Oct., 2008 2nd Dec., 2008 20th Jan., 2008 8th Jan., 2008 Type of usage table table table table table Fruit skin colorz bright yellowish green 3305 dark yellow green 3507 vivid orange 1605 vivid orange 1605 dark orange 1305 Fresh colorz vivid orangey yellow 2204 vivid orangey yellow 2205 vivid orange 2205 orange 1606 vivid orange 1605 z The numbers and colors from the Japanese horticultural plants standard color sheets. 216 S. Inafuku-Teramoto, R. Suwa, Y. Fukuzawa and Y. Kawamitsu

Tanaka), and ‘Shikikitsu’ (C. madurensis Lour.), which volatile citrus components (Akakabe et al., 2008a, b; are grown in Okinawa and across China and Southeast Belsito et al., 2007; Choi and Sawamura, 2001, 2002; Asia. Fanciullino et al., 2006; Gariele et al., 2009; Miyazawa Immature and mature fruits were collected from all et al., 2009; Phi et al., 2006, 2009). cultivars from September 2008 to March 2009. All cultivars grew in Motobu, Nago, and Ogimi, in the PMFs and synephrine analyses using HPLC northern part of Okinawa Island. The fruits were picked A mixture of dimethylsulfoxide and methanol (50 : 50 from the same tree and then peeled with a peeler or by v/v) was used for extraction of PMFs. Distilled water hand. The fresh peel was stored at −20°C until extraction was used for extraction of synephrine. Powdered peel of volatile components. Another peel sample was (50 mg) was vortexed and then sonicated for 10 min in prepared for high performance liquid chromatography 600 μL of each extraction liquid. This first extraction (HPLC) analyses; peels from five fruits were freeze- step was finished by 30 min of centrifugation (15,000 × dried and then ground to a fine powder before being g) at 4°C, and then the supernatant was carefully stored at −20°C until analysis. collected. Each peel sample was extracted three times, and the supernatants were combined for HPLC analysis. Extraction of volatile components from peels These samples were filtered through a 0.45 μm In our previous study (Teramoto and Kawamitsu, nitrocellulose membrane before injection into the HPLC 2010), the volatile constituents of citrus peel did not system (Controller, SCL-10Avp, Shimadzu Co.). The show many differences among distilled extracts, cold- conditions used for PMF and synephrine analyses were pressed extracts and hexane extracts, therefore, we used as follows: ODS reverse phase column (Shim-pack VP- the hexane extract here. Frozen fresh peel (10 g) was ODS, 150 mm × 4.6 mm i.d., Shimadzu Co.) in the CTO- homogenized in an equal volume of distilled water, and 10ASvp oven at 40°C, with a UV detector (SPD-10Avp, then 1 mL hexane was added. The mixture was vortexed, Shimadzu Co.) set at 340 nm for PMFs and 223 nm for sonicated for 10 min, and then centrifuged at 15,000 × g synephrine. The transfer buffer used in PMFs analyses (12,000 rpm) for 30 min at 4°C. The hexane phase, which was 75% methanol - 25% distilled water (v/v) containing was separated as the supernatant, was collected. All 10 mM phosphoric acid; that used for synephrine hexane fractions were dehydrated with sodium sulfate analyses was distilled water containing 5% (v/v) and then filtered (MINISART RC4, 0.2 μm pore size, acetonitrile and 0.5% (v/v) acetic acid. The flow rate for Sartorius Stedim Biotech GmbH, Göettingen, Germany) both analyses was 1.0 mL per min, provided by a LC- before gas chromatography-mass spectrometry (GC-MS) 10Avp pump operated in isocratic mode. Authentic analysis. standards of nobiletin, tangeretin, sinensetin, Hexa- methoxyflavone, and synephrine were purchased from Volatile constitutions analysis using GC-MS Funakoshi Inc., and Wako Pure Chemical Industries, Two types of capillary columns, DB-WAX and DB- Ltd. Natsudaidain and heptamethoxyflavone were 5MS (30 m × 0.32 mm (i.d.), film thickness, 0.25 μm, supplied by the National Institute of Fruit Tree Science J&W Scientific Inc., Folsom, USA) were used for GC- (Shizuoka, Japan). MS analyses (GC-MS 2010 Plus, Shimadzu Co., Kyoto, Results Japan). The initial oven temperature for both columns was 60°C. The temperature was increased by 3°C per Volatile constitution min to a final temperature of 200°C for DB-WAX and Seventy volatile components were identified in the 240°C for DB-5MS. The electron ionization detector of peel (Tables 2 and 3). Each cultivar showed characteristic the mass spectrometer was set to 0.7 kV at 200°C. The constituents, especially in immature peel (Table 2), GC-MS injection temperature was set to 220°C in which contained more minor compounds and a higher constant flow mode. Injections were performed in split percentages of them than mature peel (Table 3). The mode (20 : 1). The analyses were performed in constant contents of monoterpene hydrocarbons increased with flow mode with helium as the carrier gas. Authentic maturity, while those of other minor volatile components standards used for identifying volatile components were decreased. d-Limonene was the most abundant mono- purchased from Wako Pure Chemical Industries, Ltd. terpene hydrocarbon in all peels, and increased at (Osaka, Japan), Nacalai Tesque Inc. (Kyoto, Japan), maturity in all cultivars except ‘Kabuchii’. The next Sigma-Aldrich Co. (St. Louis, USA), Funakoshi Inc. most abundant monoterpene hydrocarbons in mature peel (Tokyo, Japan), and Kanto Chemical Co., Inc. (Tokyo, could categorize the cultivars into three groups: low γ- Japan). Compounds that could not be identified by terpinene and low β-myrcene (type L); high γ-terpinene comparison to authentic standards were identified on the (type T); and high β-myrcene (type M) (Fig. 1). Type L basis of results in the FFNSC and NIST08 libraries included ‘T-132’ and ‘Izumibeni’ which contained (Shimadzu Co.) and the retention index calculated using mainly d-limonene. Type T included all cultivars that the retention time of co-injected n-alkanes. We also originated in Okinawa and ‘Shikikitsu’. Type M included referred to previously published retention indexes of ‘Keraji’ and ‘Tokunibu’. Other monoterpene hydrocar- J. Japan. Soc. Hort. Sci. 80 (2): 214–224. 2011. 217 x y , RI z MS, RI, Co RI, MS, Identification t——MS, RI t——MS, 0.61 0.07 — MS, RI v 0.01 3.40 0.19 0.94 0.03 0.08 MS, RI ‘Keraji’ ‘Kabuchii’ ‘Tokunibu’ ‘Oto’ ‘T-132’ ‘Izumibeni’ mikan’ ‘Rokugatsu 0.01 t 0.01 t — w ‘Ishikunibu’ ‘Shikikitsu’ The volatile constituent of immature Okinawa. The constituent peel grown on citrus volatile ‘Ogimi kuganii’ Table 2. C. depressa C. depressa C. madurensis C. rokugatsu C. keraji C. keraji C. nobilis C. oto C. tankan C. tangerina Subtotal 18.82 3.25 5.85 8.41 14.05 5.31 14.29 21.15 23.48 31.87 Subtotal 2.31 2.65 0.09 1.30 0.84 5.17 0.56 1.98 0.26 0.08 Subtotal 72.75 92.65 73.16 86.44 53.93 83.13 68.86 54.38 73.14 64.82 Local name Scientific name Scientific Freee0.20—————— -Farnesene α Freee———— -Farnesene -Ocimene — 0.10 0.07 2.33 0.56 0.92 0.08 0.43 0.88 0.14 MS, RI β β -Bergamotene 0.10 t — — 0.14 — — 0.07 — — MS, RI t )-Terpinene 4-ol)-Terpinene 1.10 0.10 0.02 0.07 0.15 0.19 0.35 0.47 0.01 0.10RI, Co MS, -Thujene }-Pinene 3.89 5.82 1.39 3.11 1.95 4.99 0.88 1.85 1.40 1.18 MS -Copaene- t 0.01 — 0.03 0.14 0.12 — 0.02 — — MS, RI -Terpineol-Humulene 3.91 0.82 1.63 0.53 0.08 0.97 t 1.70 t 2.94 0.01 3.37 0.12 0.92 0.20 1.42 MS, RI, Co — 0.04 0.02 — RI, MS, Co -Terpinene 1.00 0.89 0.10 0.51 0.33 1.19 0.09 0.25 t 0.07 MS, RI, Co -Caryophyllene 0.36 t — t 0.25 0.05 — 0.07 t — MS, RI, Co -Pinene 3.09 4.20 1.28 1.84 1.06 3.60 0.39 1.00 0.09 0.42 MS, RI, Co -Sesquiphellandrene t t — t — 0.18 0.03 — — — MS, RI -Myrcene 2.73 3.72 6.11 5.42 13.95 4.35 12.55 3.37 5.80 2.36 MS, RI, Co -Phellandrene 0.69 0.49 1.08 0.63 0.82 0.67 0.96 2.90 0.89 0.36 MS, RI -Limonene-Cymene 30.89 40.66 6.49 61.87 9.43 56.36 0.05 26.10 43.19 0.75 47.82 0.14 34.84 1.13 60.66 0.14 54.51 0.75 MS, RI, Co 0.29 0.14 MS, RI -Elemene —— t —————— —MS, RI —— t —————— —MS, -Elemene -Cadinene — 2.59 0.07 0.08 — 0.26 0.31 — — — MS, RI -Sabinene hydrate-Sabinene 1.12 t 0.05 0.03 0.15 0.17 0.31 0.45 — 0.07RI MS, -Cadinene 0.36 0.05 — 0.19 — — 0.03 0.04 0.01 — MS, RI -Terpinene 21.17 23.98 0.26 13.84 7.77 20.03 5.57 7.85 2.58 5.44 MS, RI, Co − -Sabinene hydrate 1.23 0.25 0.01 0.03 0.24 0.38 0.47 0.53 t 0.09 MS, RI Monoterpene hydrocarbons α α c t δ α α β β γ δ α Sesquiterpene hydrocarbons α β β Aliphatic aldehydes α d β γ p Monoterpene alcohols RI (DB-5MS) WAX) RI (DB- 12 10283 1028 1071 931 936 953 Camphene 0.03 t 4 1081 801 Hexanal 0.01 0.02 0.01 t 0.02 0.02 0.03 0.01 0.03 t RI, MS, Co 5 1112 980 67 1124 1165 975 991 Sabinene 0.66 0.73 0.92 0.53 0.41 0.76 0.17 0.54 0.37 t MS, RI 8 1182 1019 9 1207 1038 11 1248 1065 1517 128725 138735 1489 1002 1592 Octanal 1106 1207 Nonanal 1307 Decanal Undecanal 0.57 0.05 0.21 t 0.55 0.04 0.36 — 4.39 1.85 1.69 0.14 0.17 0.01 0.07 0.83 — 0.37 0.59 1.11 0.20 0.07 0.85 0.23 0.34 — 0.02 0.14 0.01 — 0.06 0.05 0.03 0.06 0.32 1.07 0.07 0.06 — MS, RI, Co MS, RI, Co MS, RI, Co — MS, RI 21 1461 1075 20 145532 1338 1577 1410 2834 1547 1589 1102 Linalool 1182 ( 31 1573 1384 11.30 2.05 3.59 7.44 4.94 2.81 9.27 16.21 21.97 29.57 MS, RI, Co 27 1542 1092 24 1479 1370 42 1690 1473 4750 170951 1739 1740 1488 1510 Bicyclogermacrene 1500 (E,E)- 1.21 t — 0.99 0.12 0.96 — 0.19 0.13 — MS, RI 5657 176859 179760 1848 1237 1986 1224 Citronellol 1252 Nerol 1299 Geraniol Perilla alcohol3740 1647 1659 1447 1454 (E)- — — 0.12 0.04 — — 0.03 — 0.05 0.02 0.44 0.04 — 0.04 — 0.27 0.53 2.10 4.97 t — 0.05 0.01 — 0.11 — 0.84 t — — 0.12 0.06 0.04 t 0.28 0.14 0.11 0.37 0.20 MS, RI, Co RI, MS, Co MS, RI, Co — MS, RI 43 1686 1197 53 1756 1518 58 1803 1547 B Germacrene — — 0.02 — 0.06 t — — — t MS, RI 10 12151213 1252 1046 14 1269 1282 1050 1032 (E)- 1065 Terpinolene 2.11 2.63 0.02 1.12 0.83 2.30 0.21 0.60 0.18 0.20 MS, RI RT No. 218 S. Inafuku-Teramoto, R. Suwa, Y. Fukuzawa and Y. Kawamitsu Identification 0.02 — t 0.66 0.06 MS, RI ———— —MS, RI, Co RI, ———— —MS, ————— t MS, RI ————— t MS, ‘Keraji’ ‘Kabuchii’ ‘Tokunibu’ ‘Oto’ ‘T-132’ ‘Izumibeni’ —————— —MS, RI —————— —MS, RI —————— —MS, mikan’ Continued. ‘Rokugatsu Table 2. ‘Ishikunibu’ ‘Shikikitsu’ ————— ‘Ogimi kuganii’ C. depressa C. depressa C. madurensis C. rokugatsu C. keraji C. keraji C. nobilis C. oto C. tankan C. tangerina Subtotal 0.87 0.99 8.16 0.22 2.01 2.23 0.62 0.54 0.17 1.20 Subtotal 0.22 tSubtotal 0.91 1.46 t tSubtotal 1.99 1.43 0.09 0.96 0.01 0.09 0.11 0.90 0.38Subtotal 1.45 0.83 0.03 0.23 0.07 t 0.01 0.07 0.17 0.30 4.49 1.91 0.03 11.57 1.19 t 19.01 19.10 0.07 0.01 0.54 0.98 0.62 0.51 t Subtotal 0.08 0.11 0.93 0.43 7.79 0.24 2.84 0.66 1.91 0.51 Total (%)Total 97.62 99.73 97.03 99.78 97.92 99.54 99.73 98.54 99.58 99.76 Local name retention index). retention

Scientific name Scientific =

-Elemol 0.32 t 0.15 — 0.02 — — — 0.01 — MS, RI -Sinensal -Sinensal —-Eudesmol 0.03 — — — — — 0.10 1.59 — — 0.05 — 0.49 — — 0.05 MS, RI — — — MS, RI -Limonene oxide — — 0.04 — t 0.01 — — t — MS, RI, Co -Eudesmol — — 0.17 t — 0.04 — t — t MS, RI -Neroridol —-Limonene oxide — 0.01 0.01 — 0.02 0.16 — 0.01 0.15 — — — t — — MS, RI — 0.02 t tRI, Co MS, Aldehydes β Sesquiterpene alcohols t α γ β Ketones, Phenols and Phenol methyl ether Esters and Oxides c t α Aliphatic alcohols RI (DB-5MS) 0.01%.

WAX) RI (DB- 44 1696 1410 Dodecanal 0.03 0.02 0.05 t 0.13 0.05 — — — — MS, RI 2236 1470 1627 1154 1263 Citronellal Dec-(2)-enal3039 155755 1659 1763 1073 1-Octanol 117461 1274 1-Nonanol62 2033 1-Decanol 2040 1567 t — D 1559 4-ol Germacrene 26 —33 t 149445 158146 1698 1147 1707 1229 Camphor 0.57 1268 Thymol methyl ether t 0.22 — 1242 Piperitone — Carvone 0.591618 t 1371 — — 1435 t — 1371 Heptyl acetate 1136 0.97 1.26 0.28 — 0.21 0.07 t 0.02 — 0.11 0.95 t — 0.55 0.01 — — 0.09 — 0.07 t t 0.21 t 0.07 0.03 t 0.02 1.25 t 0.06 0.05 0.27 — 0.01 0.11 t 0.38 0.01 t 0.03 0.02 — 0.17 — 0.14 — MS, RI, Co 0.01 0.03 0.02 — 0.02 t 0.01 0.30 — — — t MS, RI, Co 11.45 0.01 0.01 MS, RI 0.01 18.62 MS, RI, Co t — — 0.05 — 0.07 0.01 — 0.01 MS, RI MS, RI MS, RI, Co — t MS, RI 4148 166554 171569 1757 1238 2293 1266 Neral 1272 Geranial 1728 Perillaldehyde6465 2068 2077 1564 0.07 1541 — 0.01 — 0.08 —6667 2180 0.29 2203 — 1290 0.07 1296 Thymol Carvacrol — 0.3423 0.0929 147038 1551 0.60 2.3549 3.58 1654 120152 1716 1251 Octyl acetate63 1751 1360 Linaryl acetate — 0.10 2042 1359 Citronellyl acetate — 1379 acetate Neryl 1400 acetate Geranyl 0.41 Methyl-N-methyl anthranilate 2.42 — 0.28 0.49 0.38 t 0.02 0.01 t — t — — — 0.76 0.43 t 0.20 t 0.03 — — — — 0.29 0.73 0.14 — t 0.07 MS, RI, Co 0.04 0.03 0.66 MS, RI — 0.02 — MS, RI, Co 2.95 — 0.50 — t — 1.84 t 1.19 t 0.01 0.04 0.12 0.26 — 12.99 — — — 5.80 0.11 — t t 0.26 0.01 — t 0.01 0.21 — 0.01 0.90 0.05 0.32 — 0.24 MS, RI, Co 0.02 t — 0.28 0.22 — 0.03 0.26 MS, RI — t — — MS, RI, Co — RI, MS, Co MS, RI RI, MS, Co RI, MS, Co 68 2207 1695 19 1437 1138 70 2392 1749 RT Detected No. Identification by Kovats index (RI index by Kovats Identification co-injected. compound Standard Not detected. Identification by GC-MS search. library by Identification z y x w v J. Japan. Soc. Hort. Sci. 80 (2): 214–224. 2011. 219 x y , RI z MS, RI, Co RI, MS, Identification tMS, RI, Co RI, tMS, 0.01 MS, RI v ttttt—MS, RI, Co RI, ttttt—MS, ‘Keraji’ ‘Kabuchii’ ‘Tokunibu’ ‘Oto’ ‘T-132’ ‘Izumibeni’ t t 0.02 t 0.01 t t MS, RI mikan’ ‘Rokugatsu w ——————— t ‘Ishikunibu’ ‘Shikikitsu’ The volatile constituent of mature citrus peel grown on Okinawa. mature of peel grown citrus constituent The volatile ‘Ogimi kuganii’ Table 3. C. depressa C. depressa C. madurensis C. rokugatsu C. keraji C. keraji C. nobilis C. oto C. tankan C. tangerina Subtotal 0.26 11.14 1.90 9.35 8.58 6.59 0.76 2.91 1.24 2.72 Subtotal 98.36 84.89 97.05 87.82 69.23 83.48 95.62 92.57 98.05 96.53 Subtotal 0.22 1.17 0.32 0.06 0.19 3.90 1.13 1.22 0.10 0.10 ‘Local name’ Scientific name Scientific -Farnesene — 1.07 — — t — — 0.76 t — MS, RI α -Farnesene 0.03 t t — — 1.69 0.34 t — 0.01 MS, RI -Ocimene — 0.07 0.04 0.93 0.14 1.11 0.09 0.83 0.16 0.16 MS, RI β β -Bergamotene 0.02 t — — — 0.15 — 0.08 t — MS, RI t )-Terpinene 4-ol)-Terpinene 0.03 0.65 0.03 0.03 0.01 0.28 0.02 0.05 t tRI, Co MS, -Thujene }-Pinene 4.19 4.93 1.53 1.51 1.04 6.26 1.79 4.62 0.50 0.41 MS -Copaene- 0.02 t — — 0.06 0.16 0.02 0.03 t 0.01 MS, RI -Terpineol-Humulene 0.15 2.90 0.49 0.27 — 0.38 — 1.06 t 0.17 — 0.29 t — 0.11 0.03 MS, RI, Co t 0.04 0.01 t RI, MS, Co -Terpinene 0.58 0.69 0.10 0.22 0.09 0.72 0.25 0.82 — -Pinene 2.81 3.21 1.46 0.86 0.42 4.26 0.77 2.70 0.02 0.02 MS, RI, Co -Caryophyllene 0.09 t t — 0.03 0.18 t 0.06 0.01 0.01 MS, RI, Co -Phellandrene 0.12 0.43 0.21 0.22 0.38 1.63 0.81 0.52 0.01 0.01 MS, RI -Myrcene 1.64 2.70 2.19 2.68 23.17 6.37 27.52 4.11 2.63 2.53 MS, RI, Co -Sesquiphellandrene — 0.04 — — — 0.09 t — t t MS, RI -Limonene 47.74 38.86 74.75 67.82 37.17 40.78 53.48 54.80 94.47 93.24 MS, RI, Co -Cymene 9.84 9.63 2.56 0.22 0.03 2.17 0.51 1.82 0.02 0.02 MS, RI -Elemene —0.06———————t MS, RI MS, —0.06———————t -Elemene -Cadinene 0.05 t t — 0.07 0.28 0.77 — 0.06 — MS, RI -Sabinene hydrate-Sabinene — 0.68 — — — 0.20 — 0.02 — tRI MS, -Cadinene — — 0.32 — 0.03 0.41 t 0.12 0.02 — MS, RI -Terpinene 29.60 22.02 13.59 12.70 6.38 17.01 9.48 20.10 0.13 0.13 MS, RI, Co − -Sabinene hydrate 0.07 1.17 t — 0.03 0.26 t 0.02 — t MS, RI Monoterpene hydrocarbons β α α c t δ α α β Sesquiterpene hydrocarbons d β β α α γ δ β γ p Monoterpene alcohols α Aliphatic aldehydes RI (DB-5MS) WAX) RI (DB- 5 1112 980 12 10283 1028 1071 931 936 953 Camphene 0.03 0.01 t 4 1081 801 Hexanal 0.01 0.01 t — 9 1207 1038 67 1124 1165 975 991 Sabinene 0.43 0.53 0.16 0.19 0.14 0.94 0.29 0.56 0.10 t MS, RI 8 1182 1019 21 1461 1075 20 145532 1338 1577 1410 2834 1547 1589 1102 1182 Linalool ( 31 1573 1384 t 5.571517 1287 1.3825 138735 1489 1002 1592 1106 Octanal 8.72 1207 Nonanal 1307 Decanal Undecanal 2.97 4.70 0.25 2.51 t t — — 1.22 0.67 0.09 0.49 t 2.64 0.49 0.05 0.06 MS, RI, Co — 0.23 — 0.04 — 0.46 0.28 0.11 0.91 — 0.84 0.23 0.04 0.04 0.16 t 0.02 — 0.04 t t 0.01 — 0.01 — t — 0.01 RI, MS, Co MS, RI, Co MS, RI, Co 0.01 MS, RI 27 1542 1092 24 1479 1370 10 1215 1046 5657 176859 179760 1848 1237 1986 1224 Citronellol 1252 Nerol 1299 Geraniol Perilla alcohol3740 1647 1659 1447 1454 (E)- — 0.01 t t 0.14 0.02 0.01 — — — — — 0.33 0.71 2.84 1.64 0.07 0.01 0.01 — 0.05 0.27 — t 0.02 0.01 0.01 t 0.01 0.02 MS, RI, Co 0.01 MS, RI, Co MS, RI, Co 43 1686 1197 4247 169050 170951 1739 1473 53 1740 1488 1756 1510 Bicyclogermacrene 1500 1518 (E,E)- — — — 0.06 t 0.79 — 0.13 t 0.06 MS, RI 11 1248 1065 1213 125214 1269 1282 1050 1032 (E)- 1065 Terpinolene 1.38 1.81 0.46 0.47 0.27 2.21 0.63 1.68 0.01 0.01 MS, RI 58 1803 1547 B Germacrene 0.01 t — — t 0.04 — t — 0.01 MS, RI RT RT No. 220 S. Inafuku-Teramoto, R. Suwa, Y. Fukuzawa and Y. Kawamitsu Identification 0.02 MS, RI 0.01 t RI, MS, Co 0.050.02 0.06 0.02 MS, RI MS, RI 0.02 t0.160.03 — — t t 0.01 t0.11 — — — — MS, RI — 0.02 0.06 MS, RI MS, RI — — MS, RI ‘Keraji’ ‘Kabuchii’ ‘Tokunibu’ ‘Oto’ ‘T-132’ ‘Izumibeni’ tttt——tMS, RI, Co RI, tttt——tMS, mikan’ Continued. ‘Rokugatsu ——————— —MS, RI ——————— —MS, ——————— ———— t — Table 3. —————— t — tMS, RI, Co RI, —————— t — tMS, ‘Ishikunibu’ ‘Shikikitsu’ ————— ———— t ———— t ————— t — t ——————— t ‘Ogimi kuganii’ C. depressa C. depressa C. madurensis C. rokugatsu C. keraji C. keraji C. nobilis C. oto C. tankan C. tangerina Subtotal 0.04 0.30 0.14 1.53 12.15 0.35 1.38 t 0.09 0.02 Subtotal 0.01 1.29 0.60 0.27 0.85 2.17 0.20 0.06 0.04 0.04 Subtotal 0.01 0.02 t 0.03Subtotal t 0.29 0.25 0.18 — 0.09 0.54 t t 0.06 1.08 0.01 0.01 2.21 t 0.02 Subtotal 0.22 0.05 t 0.19 0.23 1.01 t 0.06 0.01 0.03 Subtotal 0.01 0.26 t 0.11 8.10 0.16 t 0.10 0.12 0.14 Total (%) 99.42 99.37 99.98 99.90 99.33 98.92 99.19 99.13 99.71 99.61 ‘Local name’ retention index). retention

Scientific name Scientific =

Eeo 0.04tttt -Elemol -Sinensal -Eudesmol — 0.05 — — — t — — — — MS, RI -Sinensal -Limonene oxide — 0.05 — — — t — — t t RI, MS, Co -Eudesmol — — t — — 0.01 — t t — MS, RI -Neroridol -Limonene oxide t 0.02 t — — t t — t tRI, MS, Co Aldehydes Sesquiterpene alcohols α γ β Ketones, Phenols and Phenol methyl ether Esters and Oxides β Aliphatic alcohols t c t α RI (DB-5MS) 0.01%.

WAX) RI (DB- 44 1696 1410 Dodecanal — 0.03 t — t 0.15 t t 0.02 0.02 MS, RI 6465 2068 2077 1564 1541 68 2207 1695 2236 1470 1627 1154 1263 Citronellal Dec-(2)-enal3039 155755 1659 1763 1073 1174 1-Octanol61 1274 1-Nonanol62 2033 1-Decanol 2040 — 1567 — D 1559 4-ol Germacrene 26 0.18 0.08 1494 1147 — Camphor 0.01 — — 0.1816 0.0218 1371 1435 t t 1.20 1371 t 1136 Heptyl acetate 0.07 t t — — t 0.03 0.19 t t 0.02 t 0.23 — 0.16 — 0.81 t t t 0.09 — — — t t 0.06 MS, RI, Co t 0.03 — 0.01 — 0.02 0.01 — RI, MS, Co 0.01 MS, RI 0.02 MS, RI, Co — — — t MS, RI 4148 166554 171569 1757 1238 2293 1266 Neral 1272 Geranial 1728 Perillaldehyde 0.01 — t3345 1581 t46 1698 —66 1707 1229 t67 2180 1268 Thymol methyl ether 2203 1242 Piperitone t 1290 Carvone — 1296 Thymol — Carvacrol 23 0.03 0.0829 1470 —38 155149 1654 1201 —52 — 4.44 1716 1251 Octyl acetate63 1751 1360 Linaryl acetate 2.46 2042 1359 Citronellyl acetate 0.06 1379 acetate Neryl t t 1400 acetate Geranyl 0.01 0.03 Methyl-N-methyl anthranilate 0.25 — 0.03 0.03 t — t 0.18 0.02 0.02 — — 0.01 0.08 0.02 — — — — 0.02 — — 0.01 0.03 t — — 0.03 t 0.01 0.54 0.09 t 0.11 0.05 MS, RI, Co 0.01 — t t t 0.14 MS, RI t — 0.01 — MS, RI, Co — 0.95 1.36 0.01 0.17 0.06 — 0.09 10.34 t t 1.72 — — 0.05 0.16 2.09 0.12 — — 0.28 — t t 1.10 MS, RI t t t — t t MS, RI 0.02 — 0.05 0.01 MS, RI, Co 0.01 0.01 t t MS, RI, Co MS, RI RI, MS, Co RI, MS, Co 19 1437 1138 70 2392 1749 RT RT Detected No. Identification by Kovats index (RI index by Kovats Identification co-injected. compound Standard Not detected. Identification by GC-MS search. library by Identification z y x w v J. Japan. Soc. Hort. Sci. 80 (2): 214–224. 2011. 221 bons (α-, β-pinene and p-cymene) were found at high 5.17% total sesquiterpene hydrocarbons, and immature levels in shiikuwasha (‘Ogimikuganii’ and ‘Ishikunibu’), ‘Keraji’ contained 19.10% total esters. Aliphatic alcohols ‘Kabuchii’, and ‘Oto’. All immature samples had high and aliphatic aldehydes are keynote aromatic compounds levels of linalool, the major monoterpene alcohol. of citrus fruits, but both groups decreased at maturity, Local cultivars had higher levels of the other minor although they remained high in ‘Ishikunibu’, ‘Kabuchii’, aromatic compounds than common oranges or grape- and ‘Keraji’. fruits. For example, immature ‘Kabuchii’ contained

Fig. 1. The correlation of γ-terpinene with β-myrcene content in mature citrus cultivars grown on Okinawa.

Table 4. Polymethoxyflavones and synephrine content in citrus peel grown on Okinawa. (mg·gDW−1)

Scientific name (Local name) C. depressa C. depressa C. madurensis C. rokugatsu C. oto Compound name (Ogimikuganii) (Ishikunibu) (Shikikitsu) (Rokugatsumikan) (Oto) IMz My IM M IM M IM M IM M Sinensetin 1.61 1.14 0.74 0.46 0.28 0.18 0.63 0.26 0.50 0.24 Hexamethoxyflavone —x —————0.04—0.240.13 Nobiletin 12.77 8.46 5.77 3.68 1.03 0.77 3.31 1.34 1.66 0.77 Heptamethoxyflavone — — — — — — 1.78 0.79 5.46 2.20 Natsudaidain 0.06 0.05 — — 0.04 0.08 2.16 0.82 3.35 1.83 Tangeretin 6.18 3.34 2.46 1.67 0.48 0.36 0.56 0.22 1.31 0.53 PMFs total 20.62 12.99 8.97 5.81 1.83 1.39 8.48 3.43 12.52 5.70 Synephrine 5.17 3.16 3.54 2.21 1.71 1.35 4.46 2.13 2.71 1.74

Scientific name (Japanese name)

ompound name C. keraji C. keraji C. nobilis C. tankan C. tangerina (Kabuchii) (Keraji) (Tokunibu) (T-132) (Izumibeni) IM M IM M IM M IM M IM M Sinensetin 0.84 0.72 0.95 0.55 0.42 0.15 1.63 0.75 1.03 0.62 Hexamethoxyflavone — — — — — — — — — — Nobiletin 6.66 6.83 3.43 1.89 2.04 0.28 10.65 5.80 7.42 4.83 Heptamethoxyflavone 4.04 3.98 6.66 3.53 10.10 3.54 3.04 2.05 — — Natsudaidain 0.51 0.51 1.45 0.99 — — 0.35 0.17 0.53 0.10 Tangeretin 8.61 7.88 3.66 1.80 1.33 0.39 3.28 1.47 2.94 0.23 PMFs total 20.66 19.92 16.15 8.76 13.89 4.36 18.95 10.24 11.92 5.78 Synephrine 2.67 2.19 2.87 1.06 8.97 2.19 5.10 2.01 7.03 4.07 z IM: immature y M: mature x Not detected 222 S. Inafuku-Teramoto, R. Suwa, Y. Fukuzawa and Y. Kawamitsu

PMFs and synephrine acetate. It could find use as a new essential oil for Immature fruits contained higher levels of PMFs than aromatherapy, with some similarities to bergamot mature fruits (Table 4). The total PMF content was (C. bergamia Risso) (Belsito et al., 2007; Verzera et al., highest in immature and mature ‘Kabuchii’, almost as 2003), lemon balm (Melissa officinalis L.) (Koliopoulos high in immature ‘Ogimikuganii’, and third highest in et al., 2010), and lemon verbena (Aloysia triphylla immature ‘T-132’. Nobiletin was presented at 12.77 (L’ Her.) Britt.) (Argyropoulou et al., 2007). Indeed, mg·gDW−1 in immature ‘Ogimikuganii’, 10.65 mg·gDW−1 ‘Keraji’ is easy to grow in the Ryukyu Islands and could in immature ‘T-132’, and 6.66 mg·gDW−1 in immature therefore be a valuable resource. ‘Kabuchii’. Tangeretin was present at 8.61 mg·gDW−1 in ‘Shikkitsu’ is often used as a cheaper shiikuwasha immature ‘Kabuchii’, and at 6.18 mg·gDW−1 in immature juice extender in Okinawa, but our results showed that ‘Ogimikuganii’. Hexamethoxyflavone was detected only the aromatic compositions of these two fruits clearly in ‘Oto’ and ‘Rokugatsumikan’. Sinensetin was present differ. The results of GC-MS or GC analysis suggested at low levels in all samples. Heptamethoxyflavone was that volatile components could be used to determine present at 10.10 mg·gDW−1 in immature ‘Tokunibu’ and whether ‘Shikikitsu’ has been mixed into shiikuwasha at 6.66 mg·gDW−1 in immature ‘Keraji’. A slight amount juice. of natsudaidain was found in immature peel of ‘Oto’ Shiikuwasha has been overproduced in recent years, (3.35 mg·gDW−1) and immature ‘Rokugatsumikan’ and a large amount of residue from juice factories (over (2.16 mg·gDW−1). ‘Shikikitsu’ had the lowest levels of 1,000 t in 2009) is not used efficiently. The pleasant total PMFs. aroma of ‘Ogimikuganii’ suggests that its essential oil Synephrine was highest in immature ‘Tokunibu’ from juice residue could find uses in various products. (8.97 mg·gDW−1), followed by immature ‘Izumibeni’ Yamamoto et al. (2005) reported that the peel of (7.03 mg·gDW−1), ‘Ogimikuganii’ (5.17 mg·gDW−1), and immature citrus fruits contains much greater quantities ‘T-132’ (5.10 mg·gDW−1). The content of PMFs and of PMFs than that of mature citrus fruits; we found this synephrine decreased with maturity in all cultivars same tendency in all cultivars (Table 4). Each cultivar (Table 4). showed a unique PMF profile (Nogata et al., 2006; Teramoto et al., 2010), and recent epidemiological and Discussion medical studies (Ohtani et al., 2007) indicated that most We previously reported some citrus accessions in local cultivars on Okinawa may have beneficial effects Okinawa (Teramoto and Kawamitsu, 2010; Teramoto et on human health. al., 2010). We characterized some of them further here Large quantities of unharvested or thinned fruit of (Table 1). shiikuwasha and tankan are currently abandoned. Citrus species have characteristic aromatic profiles. Shiikuwasha is known as a rich source of nobiletin; in Japanese sour citrus, citron, and lime have low levels of addition, our results suggest that other cultivars except d-limonene and high levels of esters or aldehydes for ‘Shikikitsu’ are also good sources of PMFs and (Akakabe et al., 2008a; Belsito et al., 2007; Choi and synephrie, especially thinned immature fruits (Table 4). Sawamura, 2001, 2002; Choi et al., 2002; Gariele et al., As well as the cost savings that would result from 2009; Phi et al., 2006, 2009; Sawamura, 2008; Sawamura efficient utilization of waste streams, these local citrus et al., 2004; Takeuchi et al., 2005; Yadav et al., 2004). could become valuable resources for the development Both immature and mature ‘Ogimikuganii’ showed the of products with various uses in dietary supplements, same high levels of γ-terpinene as lime and lemon. cosmetics, and foods. In particular, their monoterpene ‘Ishikunibu’, ‘Kabuchii’, and ‘Oto’ also showed high hydrocarbons and monoterpene alcohols have antibac- levels of γ-terpinene in both stages. The high percentages terial and antiviral activities and are used to support liver of α-, β-pinene seemed to contribute the typical fresh function and accelerate metabolism (Buckle, 2000; Choi, woody note in the shiikuwasha (‘Ogimikuganii’ and 2006; Del Toro-Arreola et al., 2005; Sawamura, 2008). ‘Ishikunibu’), ‘Kabuchii’, and ‘Oto’ aromas (Tables 2 Thus, our results will contribute to the development of and 3) throughout maturation. On the other hand, mature new foods or functional products, and will thereby ‘T-132’ and ‘Izumibeni’ showed the same citrus aroma benefit the Okinawa citrus industry. profiles typical of common sweet orange (Fig. 1). This Acknowledgements difference highlights their foreign origin. ‘Kabuchii’ contained a high amount of sesquiterpene We thank Dr. K. Ogawa (National Institute of Fruit hydrocarbons, which may contribute to its strong aroma. Tree Science), and the Instrumental Research Center of The essential oil might therefore prove useful for University of the Ryukyus for GC-MS analyses. We also aromatherapy. thank Mr. H. Kinjyo, Mr. H. Sumi and Mr. H. Ikemiyagi The most distinctive citrus in this study was ‘Keraji’, for supplying materials. which originates from Kikai Island in the Amami Islands (Yamamoto et al., 2005, 2008, 2010), because it contained high levels of geranial, geraniol, and geranyl J. Japan. Soc. Hort. Sci. 80 (2): 214–224. 2011. 223

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