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The Composition of Volatile Aroma Components, Flavanones, and Polymethoxylated Flavones in Shiikuwasha (Citrus depressa Hayata) Peels of Different Cultivation Lines
Yonathan Asikin,† Ikuko Taira,‡ Sayuri Inafuku-Teramoto,‡ Hidekazu Sumi,§ Hideaki Ohta,∥ Kensaku Takara,†,‡ and Koji Wada*,†,‡
† ‡

United Graduate School of Agricultural Science, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan § Ogimi Village Office, 157 Oganeku, Ogimi, Okinawa 905-1392, Japan ∥ Faculty of Nutritional Sciences, Nakamura Gakuen University, 5-7-1 Befu, Johnan-ku, Fukuoka 814-0198, Japan ABSTRACT: Citrus peels are important sources of various pleasant aroma compounds and valuable bioactive substances. To investigate differences in the composition and content of Shiikuwasha (Citrus depressa Hayata) peels from different cultivation lines, the composition of volatile aroma components, flavanones, and polymethoxylated flavones (PMFs) in four Shiikuwasha cultivation lines was examined. The composition of volatile aroma components in cold-pressed extracts of Shiikuwasha peels was analyzed using gas chromatography−flame ionization detection and gas chromatography−mass spectrophotometry. The extracts contained mainly monoterpene hydrocarbons (93.40−97.25%), including limonene (46.52−68.26%) and γ-terpinene (21.48− 30.52%). Differences in the composition of volatile aroma compounds in the Shiikuwasha cultivation lines were revealed using principal component analysis. Additionally, the composition of flavanones and PMFs was determined using high-performance liquid chromatography methods. Neohesperidin (96.58%) was the predominant flavanone in 'Izumi kugani' peel, while the other peels had high hesperidin contents (89.26−98.66%). Moreover, the PMFs of Shiikuwasha peels were composed of nobiletin (56.74−64.77%) and tangeretin (23.17−34.70%). KEYWORDS: Shiikuwasha peel, cultivation line, volatile aroma components, flavanones, polymethoxylated flavones (PMFs)

INTRODUCTION The exocarp of citrus fruits, also called peel or rind, is an important component that contributes many essential properties to the overall quality of the fruits. Citrus peels do not comprise only acceptable and pleasant flavor compounds but also bioactive substances that have beneficial effects on human health.1,2 Hence, there is currently an upsurge of interest in the biochemical properties and functions of citrus peels, as well as their commercial application in foods, nutraceuticals, and pharmaceuticals. These chemical and biochemical characteristics may vary depending on citrus fruit origin, species, cultivation line, and degree of ripening. Shiikuwasha (Citrus depressa Hayata) is a citrus fruit cultivar with about 10 kinds of cultivation lines, including 'Kaachi', 'Izumi kugani', 'Katsuyama kugani', 'Ogimi kugani', 'Ishikunibu', 'Hijyakunibu', 'Kabishi', and 'Fusubuta', which grows naturally on Okinawa Island, Japan. It has small yellowish-green fruits with a very sour taste and strong characteristic aroma.3 The average annual commercial production of Shiikuwasha fruits was around 3000 tons from 2007 to 2009, and about 90% of its products are from 'Kaachi', 'Izumi kugani', 'Katsuyama kugani', and 'Ogimi kugani' lines.4 Shiikuwasha is commonly consumed as fresh fruit or processed into different food products, such as beverages, confectionary items, and food additives. Shiikuwasha juice is an abundant source of ascorbic acid and flavonoids, even when subjected to filtration, sterilization, and bottling processes.5
© 2012 American Chemical Society

A study of the characteristic aroma composition of citrus fruits has been carried out for many years, leading to investigation of the distinctiveness of each type of citrus fruit.6 The volatile aroma components of citrus fruits, which are mainly extracted from the peels, are predominantly monoterpene and sesquiterpene hydrocarbons. Moreover, the subsequently oxygenated compounds derived from these hydrocarbons include alcohols, aldehydes, esters, ketones, ethers, phenols, and oxides of various compositions. These unique aroma compositions have been investigated using several types of extraction systems and analytical techniques.7,8 Additionally, extracts of volatile aroma components of citrus peels, as well as other plant essential oils, have been shown to exhibit various antimicrobial, antiviral, and antioxidant activities.9,10 Flavonoids, along with phenolics, carotenoids, and limonoids, are known as typical and important bioactive substances from citrus peels. Common flavonoid compounds in citrus peels are flavanones, flavones, and PMFs, which possess biological activities.11−13 Of the flavanones, citrus peel-derived hesperidin was observed to have anti-inflammatory and antialcoholic fatty liver effects,14,15 and naringenin enhanced melanin synthesis
Received: Revised: Accepted: Published:
7973

May 1, 2012 July 9, 2012 July 17, 2012 July 17, 2012
dx.doi.org/10.1021/jf301848s | J. Agric. Food Chem. 2012, 60, 7973−7980

'Katsuyama kugani'. Bedford. particularly flavonoids. extracted from the flavedo. Japan).20 To the best of our knowledge.25 ± 0. with a sharp knife.74 35. as well as their flavanones and PMFs.Journal of Agricultural and Food Chemistry Table 1.53 ± 4. which is defined as flavedo.14 ± ± ± ± 'Katsuyama kugani' 26. All extractions were performed in triplicate.71 1. skin thickness. Flavedo peel samples were freeze-dried and then crushed and ground using a dry blender.84 ± 1. Ltd. 60 m × 0. Japan).org/10. PMFs of citrus peels are known to have a wide range of nutraceutical functions. for Shiikuwasha fruits. Helium was used as the carrier gas. The collected oils were centrifuged at 2900g for 15 min at 4 °C.63 Each morphological trait value is expressed as the mean ± standard deviation (n = 10).51 ± 1. The fruits that come to commercial maturity were collected during the Shiikuwasha harvesting season in December 2009. depressa Hayata) 'Kaachi'. Agric. (Tokyo.23 The GC injector and FID temperatures were both set at 250 °C. neohesperidin. and maintained at 200 °C for 38 min.22 The flavanone and PMF extracts were filtered using a 0. and the resulting supernatant layers were carefully collected.19 1. Japan).61 ± 4.30 8−12 7−9 2.18 Moreover. Plant Materials.78 ± 0.51 34.26 8−12 8−9 1.17.74 29. Chemicals. v/v) at room temperature. and tangeretin standards were from Funakoshi Co. CA) and a flame ionization detector (FID). and narirutin.38 ± 0.93 ± 0. Volatile aroma components were identified by comparing their linear retention indices (RIs) and mass spectra fragmentation patterns with the MS data of corresponding authentic compounds from the National Institute of Standards and Technology (NIST) MS Library. The column and oven programs for GC-MS analysis were as described above. Morphologicala and Chemicalb Traits of Shiikuwasha Fruits fruit traits average fruit weight (g) polar diameter (mm) equatorial diameter (mm) skin thickness (mm) seed number carpel number titratable acidity (%) total soluble content (°Brix) a Article 'Kaachi' ± 3. The aroma compounds were extracted by using a cold-press system for its ability to produce more authentic and unique aroma of citrus essential oils and its simplicity as compared to other extraction methods. Japan.88 ± 0. Briefly.49 1. Agilent J&W.doi. 50 mg of freeze-dried Shiikuwasha flavedo powder was extracted using vortex mixing and sonication for 10 min with 600 μL of a mixture of dimethylsulfoxide and methanol (1:1. hesperidin.60 46.69 'Ogimi kugani' 36.51 5−7 8−10 1.1021/jf301848s | J. 7974 ■ MATERIALS AND METHODS Cold-Press Extraction of Volatile Aroma Compounds. The resulting supernatant layers were dehydrated overnight with anhydrous sodium sulfate at 5 °C and were then filtered. Authentic compounds of n-hexanol and methyl myristate were purchased from Tokyo Chemical Industry. and farming conditions.55 ± 2. and total soluble content (Table 1). Japan) and expressed as °Brix.22 ± 0. The total soluble content of the fruit juices was measured using a hand-held refractometer (model N-1α. Japan).d. Atago Co. All other reagents were supplied by Wako Pure Chemical Industries and were of analytical grade unless otherwise specified.62 7. size. Japan. w/w) or per kilogram of fruit. ionization energy of 70 eV. 60. then increased to 200 °C at a rate of 2 °C/min.24 9−12 8−9 1.23 0. two PMF compounds in particular. from Shiikuwasha peels have been reported to have antiobesity effects. bEach chemical trait value (titratable acidity and total soluble content) is expressed as the mean ± standard deviation (n = 5). A natsudaidain standard was obtained from the National Institute of Fruit Tree Science (Shizuoka. and nobiletin as standards were obtained from Wako Pure Chemical Industries (Osaka. 2012. Shiikuwasha (C. On the other hand. 'Izumi kugani'. Louis.19 and Shiikuwasha juices containing PMFs were also demonstrated to have a hepatoprotective effect against D-galactosamine-induced liver injury..28 26.16 1.11 8.89 ± 1.25 μm.18 1. mass acquisition range of (m/z) 29−450 amu. The mass spectra of volatile aroma compounds were analyzed using an Agilent 7890A GC coupled with an Agilent 5975C mass spectrometer.28 ± 0. The weight percentage of the peak was calibrated by FID responses of the internal standards.38 11. The outermost green surface of the peel. weather. Samples (1 μL) were injected using a split ratio of 1:50. The oven temperature was initially set at 40 °C for 2 min. Santa Clara. Version 2008. neither the characterization of volatile aroma components nor the compositions of flavonoid compounds in Shiikuwasha peels from different cultivation lines have yet been reported. Some of the identifications were confirmed by peak dx. The fruit types were characterized as to average weight. was to distinguish the four different Shiikuwasha cultivation lines from Okinawa.77 ± 0. and tyrosinase activity in cells.66 ± 0. Extraction of Flavanones and PMFs. The mixture was then centrifuged at 15000g for 30 min at 4 °C.17 8. flavanones and PMFs fractions were extracted through vortex and sonication.32 46. 7973−7980 . The study results provide information regarding the distinctive flavors and the natural sources of bioactive substances.98 ± 1.62 10. The volatile aroma components of Shiikuwasha peels were determined using an Agilent 6890N GC equipped with a bonded-phase fused silica capillary column (DB-Wax.. including antiproliferative and pro-apoptotic effects in cancer cells. was carefully separated from the soft inner layers. film thickness 0.25 ± 2.31 46.59 ± 0. Naringenin. and scan rate of 1.21 The ratio of the neat extract and the two internal standards was 150:1:1 (w/w/w). GC-FID and GC-MS Analysis of Volatile Aroma Components. and the volatile aroma compositions were expressed as the relative concentration (%) or milligrams of volatile compound per 100 g of flavedo on a fresh-weight basis. culture.24 ± 1.45 μm nitrocellulose membrane (Millipore. and the yields were expressed as grams of extract per 100 g of fresh flavedo peel (%. The internal standards were added to the collected extract before injection into the GC. Volatile aroma compounds of Shiikuwasha peels were extracted from the flavedo (500 g) by hand-pressing and collected in a 10 mL of saturated sodium chloride solution on ice. and the linear velocity of the flow rate was 32 cm/s. MA) prior to use. and a methyl myristate standard was used for those emerging after linalool in the eluted order. Food Chem. The MS conditions were as follows: electron impact ion source and interface temperatures of 230 °C. Quantitative analysis was performed using authentic aroma compounds of n-hexanol and methyl myristate as internal standards. respectively. The Shiikuwasha trees were grown under the same climate. or albedo. titratable acidity. according to a previous method. Authentic chemical compounds used as standards for the analysis of volatile aroma components were purchased from SigmaAldrich (St. Tokyo.25 mm i. therefore.16 On the other hand.77 scans/s. Ltd. The collected extracts were stored in sealed vials at −30 °C until analysis. nobiletin and tangeretin. A n-hexanol standard was used for the peaks eluting before linalool.68 'Izumi kugani' 45. and 'Ogimi kugani' fruits were obtained from several farms located in the northern part of Okinawa Island.65 35.89 7. The aim of the present study. based on the composition and content of their volatile aroma components. MO) and Tokyo Chemical Industry (Tokyo.76 41. sinensetin.21 All extractions were performed in triplicate.

11 ± 0. followed by alcohols (0. Food Chem. while the reverse sequence was observed for γ-terpinene and p-cymene.05 ± 0. The highest content of volatile aroma components was found in the peel of 'Izumi kugani' (141. and p-cymene [0. 13 compounds). Shimadzu Corp. ketones. The identified volatile aroma components of Shiikuwasha peels are listed according their order of elution on a polar DB-Wax column.41 mg/100 g of fresh flavedo peel).41−7. 2012.24 with slight modification. might remain in the final extracts. The Shimadzu LC-10A-VP pump was operated in isocratic mode at a flow rate of 1 mL/min with a mobile phase containing a mixture of methanol.20 mg/100 g of fresh flavedo peel)]. v/v/v/v) for flavanone analysis and a mobile phase of 75% methanol containing 10 mM phosphoric acid for PMF analysis.35−1.org/10.00 0.05) in the relative concentration of almost all of the identified compounds were found in the four Shiikuwasha cultivation lines.14 ± 0.24 ± 0. All analyses were carried out in triplicate.00 'Ogimi kugani' 0.30−1. The compositional analyses of volatile aroma compounds. junos). terpinolene.04 'Katsuyama kugani' 0. 125. reticulate). In addition. and contents (Table 3). alcohols. Statistical Analysis. Japan). provide useful information for the citrus flavor and essential oil industries as a basis for estimating yields from Shiikuwasha peel extracts of different cultivation lines during oil extraction. and were observed in the range of 1. 25 of these compounds were also specifically identified by comparing their mass spectra profiles against authentic standards. therefore. Yielda of Cold-Press Extraction from Shiikuwasha Peels extract properties 'Kaachi' 'Izumi kugani' 0.05 ± 0. γ-terpinene [21. the relative concentrations of limonene differed significantly among cultivation lines and follow the rank order: 'Kaachi' > 'Izumi kugani' > 'Katsuyama kugani' > 'Ogimi kugani'. flavanones.. The main volatile aroma components identified were the monoterpene hydrocarbons limonene [46.01 ■ yielda (%. and 63. dx. Redmond. In reference to the literature. The compounds were monitored at 340 nm using a Shimadzu UV−vis detector (model SPD-10VP).d.84. Significant differences (p < 0.12 ± 0. WA).4 in Japanese yuzu (C. and 'Katsuyama kugani' peel extracts were 307.) was used. The percentage yield of each Shiikuwasha cultivation line was measured as the extract per fresh flavedo peel. To establish the differentiation of volatile aroma compounds in the cultivation lines.04 fruit) a b Each value is expressed as the mean ± standard deviation (n = 3).48− 30. pigments. as well as the diversity of the terpene compounds.65%.39 mg/100 g of fresh flavedo peel). the ratio of monoterpene to sesquiterpene hydrocarbons was determined to describe the distinctive aroma combination in each Shiikuwasha peel extract. compositions.84.26% (32.04 0. 69.1021/jf301848s | J.57−8.11 in lemon (C. esters. As a whole. such as ethers. 60. These data. representing approximately 99% of the total volatile aroma components in the extracts were identified by their linear RIs and mass spectra fragmentation patterns.. Interestingly. The extracts were comprised of a complex mixture of different component groups. as well as nonvolatile compounds.11%) were higher than those of 'Kaachi' and 'Ogimi kugani' (0. and the oven (Shimadzu CTO10ASVP) was set at 40 °C. water.3−19.25%.Journal of Agricultural and Food Chemistry enrichment on coinjection with authentic aroma standards. 'Ogimi kugani'. lemon).19 ± 0. Regarding chemical composition. The Table 2. The method of HPLC analysis was adapted from Kawaii et al. Kyoto. The mean values of the results of the analyses were subjected to analysis of variance with Fisher's least significant difference (LSD) posthoc test to evaluate the significant differences (p < 0. The injection volume of samples and standards was 5 μL. Article RESULTS AND DISCUSSION Cold-Press Extraction of Shiikuwasha Peels. principal component analysis (PCA) was also implemented.25− 87.52% (15. Agric. The composition of flavanones and PMFs was examined using a HPLC system (SCL10AVP. acetonitrile. respectively (Table 3). All Shiikuwasha peel extracts were found to contain moderate amounts of myrcene.23 The yield of volatile aroma components from Shiikuwasha peels of four different cultivation lines is shown in Table 2.78. eight compounds). followed by 'Katsuyama kugani' (127. wherein the ratios of other cold-pressed citrus peel extracts were 292. 5-μm particle size. there appears to be great variability in the ratio of monoterpene to sesquiterpene hydrocarbons among the Shiikuwasha cultivation lines. The concentrations of flavanones and PMFs were expressed as milligrams of flavanone or PMF compound per 100 g of flavedo on a fresh-weight basis. The ratios for 'Izumi kugani'. Shiikuwasha cold-pressed volatile aroma extract was observed to retain more phenolic content and exhibit superior antioxidant activities as compared to a steamdistillation extract. and 12. and α-thujene. Shimadzu Corp.02 (g extract/kg 0. volatile aroma components of Shiikuwasha peels from different cultivation lines were produced using cold-press extraction.56 mg/100 g of fresh flavedo peel)]. 228. while that from the other cultivation lines varied from 0. The composition of volatile aroma compounds in Shiikuwasha peels was observed to vary depending on the cultivation line. as well as two structural isomers of pinene (α-pinene and β-pinene).24 g extract per kg fruit. The function of concentration against peak area was calibrated by injecting each authentic standard at a range of different concentrations that covered the concentration levels of the extract samples. 15. Discrimination of different Shiikuwasha cultivation lines by the characteristic composition of volatile aroma components can also been seen with other moderate and low level-oxygenated compounds.12 to 0. The highest extract content was found in 7975 'Katsuyama kugani' fruit.05) between Shiikuwasha cultivation lines for each individual compound. with monoterpene hydrocarbons as the predominant component group (93. authentic aroma components. oxides. Linear RIs of the compounds were also calculated using a homologous series of n-alkanes (C7−C30). nine compounds) and sesquiterpene hydrocarbons (0. and other bioactive substances.52−68.6 mm i.05%).62 in Mandarin orange (C. This extraction method is commonly used for the production of essential oils containing volatile aroma compounds from citrus peels. including their RIs.37−39.19 g.25 These chemical compositions. Moreover.36%.00 0. HPLC Analysis of Flavanones and PMFs..02−2. In the present study. 39 compounds. might differ depending on the regulation of the monoterpene and sesquiterpene hydrocarbon biosynthesis pathways in citrus plants. Volatile Aroma Components of Shiikuwasha Peels. yield of the extracts was found to vary depending on the cultivation line.40− 97. which contained 0.11 ± 0. and acetic acid (15:2:2:1. All analyses were carried out in triplicate. 'Kaachi'.68 mg/100 g of fresh flavedo peel)].doi. A Shim-pack VP-ODS column (150 mm × 4. w/w)b 0. sinensis).01 in sweet orange (C. and PMFs were statistically evaluated using Microsoft Office Excel 2007 (Microsoft Corp.50%. The extraction yields of both 'Izumi kugani' and 'Katsuyama kugani' (0.98% (0.17. 7973−7980 . The ratio of extract weight to fresh flavedo peel weight.26 All compounds were quantified using the two internal standards n-hexanol and methyl myristate.1. In the cold-press system.

08 ± 0.022 1.019 0.86 ± 0.006 ND 0. MS.38 ± 0. RI.01 ± 0.01 ± 0.68 ± 0. RI.03 ± 0.001 c 0.63 ± 0.001 a 0.23 ± 0.035 62.002 0.038 a 60.001 0.159 tr.000 0.004 b 0.00 ± 0.76 ± 0. RI.103 0.09 ± 0.071 b 0.01 ± 0. dx.004 ND 0. RI.009 0.doi.01 ± 0.01 ± 0.002 0.09 ± 0.01 ± 0.16 ± 2.25 ± 3.217 c 1.004 0.001 0.66 ± 0. MS.000 b ND 0.02 ± 0.005 b 0.14 ± 0.01 ± 0.14 ± 0.32 ± 0.008 0.071 0.007 b ND 0. RI.22 ± 0. RI.000 b 0.43 ± 0.304 a 8.23 ± 0.038 a 1. RI.057 c 0.45 ± 0.09 ± 0. RI.066 0.01 ± 0.001 21.42 ± 0.06 ± 0.000 b 0.99 ± 0.274 2.55 ± 0.004 1.15 ± 0.00 ± 0.09 ± 0.011 48.002 c 1.001 0.72 ± 0.001 ND 1.03 ± 0.52 ± 0.65 ± 0.org/10. RI.006 0.88 ± 0.64 ± 0.02 ± 0.008 0.41 ± 0.022 2.03 ± 0.033 a 5. MS MS.06 ± 0.05 ± 0.08 ± 0.61 ± 0.002 c 0.170 1.118 b 0.54 ± 0.005 0.22 ± 0.08 ± 0.000 b 30.02 ± 0.02 ± 0. RI.53 ± 0.143 0.138 b 0.018 0.14 ± 0.01 ± 0.10 ± 0.34 ± 0.07 ± 0.12 ± 0. MS.02 ± 0.174 1.05 ± 0.421 1. MS MS.01 ± 0.004 1.007 0.01 ± 0.36 ± 0.46 ± 0. RI.20 ± 0.001 b 0.01 ± 0.02 ± 0.008 0.06 ± 0.01 ± 0.000 c 0.01 ± 0. RI.403 b 0.006 b 0.060 a 0.076 0.01 ± 0.01 ± 0.04 ± 0. RI.37 ± 1. b 1.000 a 0.05 ± 0.81 ± 0.000 0.052 87.48 ± 0.015 ND 0.001 0. RI.051 c ND 1.21 ± 0.052 0.34 ± 0.15 ± 0.001 b ND 0.09 ± 0.001 d 0.002 0. MS MS MS.01 ± 0.101 a 1.002 b 0.15 ± 0.03 ± 0.000 a 0.814 0.001 0.05 ± 0.61 ± 0.016 b 68.000 b 30. RI.10 ± 0.003 a ND 1.001 MS.000 b 21.20 ± 2.74 ± 0.05 ± 0.005 0.01 ± 0.43 ± 0.03 ± 0.001 a ND 0.06 ± 0.007 0. Food Chem.88 ± 0.02 ± 0.002 a 0.013 c 0.000 b 0.001 d 0. b 2.003 a 0.006 b 0.004 c 0.97 ± 0.01 ± 0.23 ± 0.002 3.01 ± 0.001 b 0.05 ± 0.13 ± 0.20 ± 0.060 ND 1.051 0.054 a 0.008 a 0.000 b 0.098 0.051 a ND 0.25 ± 3.01 ± 0.003 0.02 ± 0.01 ± 0.38 ± 0.002 0.15 ± 0.002 d 0.003 0. RI.000 c 0.10 ± 0.011 0.06 ± 0.599 1. RI.106 0.004 b 0.33 ± 0.8-cineol γ-terpinene p-cymene terpinolene nonanal α-cubebene 4-carene sabinene hydrate α-copaene decanal germacrene d linalool bornyl acetate β-caryophyllene methyl thymol terpinen-4-ol (e)-2-decenal α-caryophyllene terpinyl acetate α-terpineol bicyclosesquiphellandrene α-muurolene l-carvone δ-cadinene perilla aldehyde nerol elemol γ-eudesmol thymol isothymol 1021 1026 1061 1107 1119 1163 1177 1213 1215 1216 1257 1272 1287 1391 1456 1468 1476 1489 1497 1536 1554 1579 1594 1594 1604 1640 1664 1693 1699 1704 1722 1731 1754 1777 1806 2080 2170 2186 2214 RI.007 a 0.98 ± 0. MS.09 ± 0.008 b 0.009 0. RI.04 ± 0.16 ± 0. RI.005 0.815 1.124 31.44 ± 0.026 0. RI.003 b 1.005 0. MS.002 0.009 b 1.007 0.002 b 0.10 ± 0.001 0.001 2.57 ± 0.002 0.002 c 0.175 c 0.68 ± 0. compound PC PC PC PC PC PC PC PC PC PC 7976 PC PC Article 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 α-pinene α-thujene camphene β-pinene sabinene myrcene α-terpinene limonene β-phellandrene 1.07 ± 0.01 ± 0.004 a 0.23 ± 0.001 b 0.003 0.000 c 0.05 ± 0.25 ± 0.002 ND 0.03 ± 0.018 1.001 b 0. RI.18 ± 0.06 ± 0.001 39.36 ± 0.01 ± 0.26 ± 0.009 c ND 0.71 ± 0.00 ± 0.Table 3.51 ± 0.01 ± 0.94 ± 0.98 ± 0.002 0.005 b 0.117 0.001 0. RI.006 a 0.07 ± 0.39 ± 0. RI.140 0.02 ± 0. 7973−7980 PC PC .004 b 0.014 1.12 ± 0. RI.021 a 0.05 ± 0.07 ± 0.062 a 0.005 0.23 ± 0.01 ± 0.01 ± 0.000 a 0.120 c 1.138 7.06 ± 0.004 0.45 ± 0.016 0.001 0.004 0. RI.001 0.36 ± 0.09 ± 0.06 ± 0. RI.62 ± 0.06 ± 0.003 b ND 0.09 ± 0.13 ± 0.128 1.01 ± 0.06 ± 0.01 ± 0.394 0.703 0.04 ± 0.049 ND 0. MS MS. Agric.003 a 0.54 ± 0. RI.125 a 0.44 ± 0.110 0. 2012.007 a 0.139 a tr.09 ± 0.001 0.04 ± 0.165 6.254 a 0. MS.17 ± 0.002 0. RI.158 0.001 a 2.01 ± 0.004 0.05 ± 0.062 0.009 0.002 a 1.002 c ND 0.02 ± 0.73 ± 0.004 c 0.019 0.035 a 0.022 a 48.001 c 0.03 ± 0.019 ND 0.02 ± 0. 1.001 0. RI.50 ± 0.02 ± 0.01 ± 0.014 d 0. 60.002 0.004 d 0.13 ± 0.012 a 0.180 d 0.007 0. 2.000 0.20 ± 0. RI.02 ± 0.03 ± 0.01 ± 0.024 0.05 ± 0.001 b 0. MS.019 a ND 0.000 15.191 0.001 a 1. RI.11 ± 0. MS.039 bc 0.052 0.005 0.03 ± 0.12 ± 0.007 0.03 ± 0.007 c 1.007 0.03 ± 0.024 0.026 c 0.002 a 0.000 c 0.47 ± 0.289 1.027 0.10 ± 0.10 ± 0.137 de 1.07 ± 0.008 b 1.90 ± 0.003 ND 2.027 a 0.04 ± 0.02 ± 0.01 ± 0.74 ± 0.07 ± 0.005 c 0. MS.011 0.146 2.05 ± 0. MS.004 c 0.57 ± 0.03 ± 0.04 ± 0.35 ± 0.096 0.094 b tr.03 ± 0.04 ± 0.03 ± 0.07 ± 0.02 ± 0.71 ± 0.007 0.006 c 0.03 ± 0.04 ± 0.085 0.038 0.1021/jf301848s | J.08 ± 0.07 ± 0.012 0.016 d 1.002 a 0.074 c 0.01 ± 0. Volatile Aroma Components [Relative Concentration (%) and mg/100 g Fresh Flavedo Weight]a of Shiikuwasha Peels 'Kaachi' RIb % mg/100 g % mg/100 g % mg/100 g % mg/100 g 'Izumi kugani' 'Katsuyama kugani' 'Ogimi kugani' identificationc PC PC PC PC PC PC PC PC PC PC PC Journal of Agricultural and Food Chemistry peak no.052 0.01 ± 0.51 ± 0.12 ± 0. MS.000 a 3.000 ND 0.124 b 0.070 tr.004 a 0. RI.56 ± 1.002 c 0.005 a 46. MS MS.003 a 0.06 ± 0.08 ± 0.08 ± 0.066 0.03 ± 0.005 b 0.14 ± 0.02 ± 0.28 ± 0.001 0.05 ± 0.003 b 0.16 ± 0.040 0.04 ± 0.00 ± 0. RI.01 ± 0.001 0. MS MS MS.08 ± 0.036 d 0.02 ± 0.01 ± 0.002 a ND 0.001 b 0.261 b 1.010 a 0.001 0. RI.07 ± 0. MS.02 ± 0.38 ± 0.82 ± 5.81 ± 0.26 ± 0.12 ± 0.01 ± 0.011 0.06 ± 0.01 ± 0.52 ± 0.80 ± 0.010 d ND 0.16 ± 0.004 ND 1.002 ND 0.006 0.01 ± 0.084 a 1.04 ± 0.65 ± 0.006 0.001 a ND 2.001 c 0.06 ± 0.22 ± 0.018 b 0.20 ± 0. RI.183 0.11 ± 0.008 ND 0.001 b 0.91 ± 0.027 a 0. RI.23 ± 0.007 32.03 ± 0.47 ± 0.003 ND 0.000 b 0. RI.007 0.01 ± 0. MS.001 a 0.001 0.001 d 0.066 b 1.89 ± 0.01 ± 0.001 b 0.004 b 1.001 c 0.080 a 21.32 ± 0.015 a 0. MS.013 a 0.47 ± 0.01 ± 0. MS.44 ± 0.11 ± 0.16 ± 0.10 ± 0.05 ± 0. MS MS MS MS MS.94 ± 0.001 c 0. RI. 1.01 ± 0.79 ± 0. MS MS.03 ± 0.094 0.03 ± 0.04 ± 0.03 ± 0.01 ± 0.025 c 0.100 a 0.001 c 0.01 ± 0. MS MS.02 ± 0.01 ± 0.

31 and has been known as the main volatile aroma compound in Eucalyptus.80 0.75 1. methyl thymol was solely observed in 'Izumi kugani' peel [1.) and juniper berry (Juniperus drupacea L.21 0.15% (1.22% (1.15 0.).79 Figure 1.01 99. and Chrysanthemum essential oils.15% (1.01 68. and PC.55 0. identificationc Article mg/100 g component groups 134.17 0.39 95.24 0.09 1.doi.30 'Kaachi' % RIb Table 3. identification based on authentic standards analyzed by mass spectrometry.01 127. comprising 25 and 22%.44 'Izumi kugani' % mg/100 g 69. var. e Means in the same row followed by the same letter are not significantly different (p < 0.29 In addition. identification based on the NIST MS library.02 93. esters (bornyl acetate and terpinyl acetate) and aldehydes mg/100 g 66.37 0.69 0. a . 2012.36 141. microcarpa Hassk. and sweet aromas. ND.00 71.78 0. 7977 dx. identification based on RI.32 0. cRI.25 0.75 ND 0.37 ND 0.38 69.27. not detected. MS.41 96.73 0.43 228.84 1.45 2. For instance. This oxide compound was recognized as one of the key aroma components in Pontianak orange (C.21.65 0. while l-carvone produces a minty aroma.97 0. Agric.65 mg/100 g of fresh flavedo peel)].01 99.13 0.29 ND 0.97 1. floral.38 1.57 mg/100 g of fresh flavedo peel)].36 mg/100 g of fresh flavedo peel)] more than 90 times higher as compared to other Shiikuwasha lines. tr.40 0.Journal of Agricultural and Food Chemistry Each value is expressed as the mean ± standard deviation (n = 3). trace amount (<0. This ether compound is somewhat rare in citrus peels but is one of the main volatile aroma components in the essential oils seafennel (Crithmum maritimum L.04 0. contributing citrusy.30 307.09 ND 0. while 'Katsuyama kugani' peel showed a significantly higher level of a ketone compound. Linalool is a key compound in citrus peels.35 0. 60.95 122.27 ND 0.84 0.94 98.57 0.50 63.27 0.06 1.11 1.21 and aldehydes. respectively. Thymus.06 1. a significantly higher relative level of linalool was also found in 'Izumi kugani' peel [1. bRIs relative to n-alkanes on a polar DB-Wax column.94% (1.1021/jf301848s | J.66 ND 0.93 'Ogimi kugani' % 'Katsuyama kugani' mg/100 g % 0.8-cineol [0. 7973−7980 peak no. dRatio of monoterpene hydrocarbons to sesquiterpene hydrocarbons. fresh. l-carvone [1. 'Izumi kugani' peel contained 1.05).35 1. nobilis Lour.30 0. continued monoterpene hydrocarbons sesquiterpene hydrocarbons (Mon/Ses)d alcohols aldehydes esters and ketones ethers oxides total identified compound 97.65 1. PCA plots of volatile aroma components of Shiikuwasha peels from four different cultivation lines: (A) distribution of seven groups of volatile aroma compounds and (B) discrimination of cultivation lines based on the relative concentrations of seven groups of volatile aroma compounds. Food Chem.01%).01 99.74 1.25 0.28 Moreover.org/10.32−34 On the other hand.65 mg/100 g of fresh flavedo peel)] as compared to the other peel extracts.) peel extracts for providing a minty aroma with a relatively high flavor dilution factor30.

Moreover.14 b 'Izumi kugani' ND 3. and perilla aldehyde) were found in relatively small amounts in all Shiikuwasha peel extracts.26 7. followed by 'Katsuyama kugani' and 'Kaachi' (528. PMF Content (mg/100 g Fresh Flavedo Weight)a of Shiikuwasha Peels PMF sinensetin nobiletin natsudaidain tangeretin total a 'Kaachi' 15.31% of the total variance (Figure 1).53 3.35 As shown in Table 4.22−102.73 and 523. hesperidin.87 a a a c 'Katsuyama kugani' 19. the flavanones of Shiikuwasha peels were comprised of narirutin. bMeans in the same row followed by the same letter are not significantly different (p < 0. the compositions of volatile aroma compounds in 'Katsuyama kugani' and 'Ogimi kugani' peels were closely associated (Figure 1B).66% (335. This result is in agreement with Nogata et al. ND. 569 mg per 100 g of fresh flavedo peel). 2012. kinokuni).53 mg/100 g of fresh flavedo peel. This low differentiation ability of PCs is in agreement with the compositions of volatile aroma compounds in 'Katsuyama kugani' and 'Ogimi kugani' peel extracts presented in Table 3. These differences might impact the entire aroma profile of peel extracts and..74 a 1. and neohespiridin. and oxide groups were closely related to each other and were more positively associated to the PCs (Figure 1A).08−474. 355 mg).50 a 39.33 ± 6. Flavanone and PMF Compositions of Shiikuwasha Peels.18 ± 0. 7973−7980 .46 'Katsuyama kugani' 49. however.62 a Each value is expressed as the mean ± standard deviation (n = 3).63 cb b a b 'Izumi kugani' 26. Separation of 'Izumi kugani' and 'Kaachi' was clearly observed. while the alcohol. decanal.25 b 474.50 ± ± ± ± 0.35 b 335.05).37 In the analysis of PMFs. The observed amounts were at the same level as other Japanese citrus fruits (in 100 mg of fresh flavedo peel): 139 mg of nobiletin and 65 mg of tangeretin in Shunkokan (C.13 8.15 b 20. aurantium.98 ± 1. the first two principle components (PCs) were identified. ether. with the aim of using the composition and content of these components as an approach for distinguishing different cultivation lines. nobiletin.27 b a c a Each value is expressed as the mean ± standard deviation (n = 3).70 to 294.70 a 723. for instance.03 b 339.22 mg/100 g of fresh flavedo peel)]. The total PMF contents of Shiikuwasha peels examined in this study ranged from 226.doi.24 95. The distribution of these flavonoid compounds in citrus fruits is affected by genetic variation and differential expression of genes in flavonoid biosynthesis. Flavanone Content (mg/100 g Fresh Flavedo Weight)a of Shiikuwasha Peels flavanone narirutin naringenin hesperidin neohesperidin total 'Kaachi' 39.98 9. shunkokan) and 118 mg of nobiletin and 108 mg of tangeretin in Kishu (C.16 523. yatsushiro. 60.69 b 0.08 ± 15. In reference to the reported literature. Japanese Iyo (C. 2-decenal. The PMFs of Shiikuwasha peels were mainly composed of nobiletin [56.58% (698. The highest flavanone content was found in 'Izumi kugani' (723.89 0.46 mg/100 g of fresh flavedo peel).29 163. application of advanced extraction systems. 363 mg). except for 'Izumi kugani' peel.24 128.95 ± ± ± ± 2.20 0. and Tahitian lime (C.30 6. supercritical fluid extraction. the other Shiikuwasha peels were observed to have high hesperidin contents [89.63−169.68 a ND 528.36 Furthermore. These compounds are the most common flavanones in citrus peels. has been developed and is reported to allow optimum yield of these valuable PMFs from Shiikuwasha dx.09 0. Significant differences in the level of each PMF component were also observed in the different Shiikuwasha cultivation lines.07 60. The result shows that volatile aroma components of Shiikuwasha peels from different cultivation lines can be distinguished in a valid manner. four compounds (sinensetin. The relations among groups of volatile aroma compounds. Flavanones are known as the predominant flavonoid compounds in various citrus peels. naringenin.22 259.20 4.53 mg/100 g of fresh flavedo peel)] and tangeretin [23.89 b a b a 'Ogimi kugani' 19. 462 mg).30 77. while the amount of hesperidin in the other Shiikuwasha peels was at the same level as other citrus varieties (in 100 mg of fresh flavedo peel). not detected.52 226.22 294.05).58 ± 0.63 5. Table 5. such as Japanese Yatsushiro (C.65 3.69 ± 90. iyo. latifolia. while neohesperidin was completely absent from the 'Katsuyama kugani' peel.70% (60. respectively.82 ± 50. the aroma characteristics of Shiikuwasha fruits from different cultivation lines.59 mg per 100 g of fresh flavedo peel.80 c 698. The composition and content of flavanones differed between 7978 the four Shiikuwasha cultivation lines. Agric. (nonanal. narirutin was not detected in either 'Izumi kugani' or 'Ogimi kugani' peel. bMeans in the same row followed by the same letter are not significantly different (p < 0. which accounted for 98.88 467. which contained a significant amount of neohesperidin [96. By application of PCA to the relative concentrations of seven volatile aroma compound groups in Shiikuwasha peels from four different cultivation lines (Table 3).17 169.67 102.94 4. Food Chem.29 168.69 mg/100 g of fresh flavedo peel)].70 ± ± ± ± 0.53 ± ± ± ± 4.73 Article 'Ogimi kugani' ND 4.17−34. and tangeretin) were detected in the Shiikuwasha peels (Table 5).16 a 4.org/10.37 5.35 ± 0. consequently.08 5. can be examined from the corresponding loading plots of PC1 and PC2.41 bb 2.82 mg/100 g of fresh flavedo peel)].18 11.1021/jf301848s | J. as well as relations between the group compositions and the cultivation lines.Journal of Agricultural and Food Chemistry Table 4.77% (128.11. Interestingly.31 5. the monoterpene hydrocarbons group was found to be negatively correlated to the PCs.36 who reported that the amounts of nobiletin and tangeretin in an undefined cultivation line of Shiikuwasha were approximately 122 and 71 mg per 100 g of fresh flavedo peel.33 281.79 ± 3.36 the amount of neohesperidin in 'Izumi kugani' peel was as high as in sour orange (C.74− 64. Of the volatile aroma compounds.26−98.59 ± ± ± ± 1. natsudaidain. The flavanone and PMF contents of four Shiikuwasha cultivation lines were examined.20 0. respectively).41 5.

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