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BiochemicalSystematicsand Ecology, VoL 24, N~ 3, pp.



Copyright O 1996 Bsevier Science Ltd Printsd in Great Britain.All rights reserved 0305-1978/96 $15.00+0.00

Limonoid and Flavonoid Composition in Varieties of Papeda and Papedocitrus
Bioactive Constituents Research Unit, National Center forAgricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604-3902, U.S.A.

Key W o r d Index--Citrus; limonoids; flavonoids; papeda; Cabuyao; Citrus ladpe~,, C. hystrix;, C.
macrophylla; C. webberii; C. junos; C. wilsonii; C. ichangen$is.
A b s t r a c t - - T h e limonoids of the seeds and the flavonoids of the leaves of a number of species of the Citrus sub-genus Papeda were examined. The seeds contained only the normal citrus limonoids and did not contain ichangensin, the unique limonoid found in C. ichangensis. A flavonoid analysis showed that a number of papeda varieties including C. la~'pes and C. hys~ix may be a papeda hybrid species. C. junos had flavanone neoheperidosides, which are also found in cultivars related to the pummelo. All papedas examined had rutin present, which is characteristic of cultivars related to the citron. Citrus ichangensis had the most complex flavone/flavonol pattern of all the papedas examined. This suggests that Citrus ichangensis may truly be a unique papeda species unlike that of the other papedas. Copyright © 1996 Elsevier Science Ltd

Introduction The papedas represent a number of wild species of Citrus which are found growing in the forests of Eastern Asia. The subgenus Papeda has been classified by Tanaka (1977) into two sections: the section Papeda comprising the typical species and the section Papedocitrus which is intermediate in character between the subgenus Citrus and the section Papeda with flowers much like those in the subgenus Citrus and leaves like those of the section Papeda. The distinguishing characteristics of the papedas are the numerous, often acrid, oil droplets associated with the pulp vesicles and the large winged leaf petioles. The papedas can tolerate lower temperatures than do species of the Citrus subgenus and papeda hybrids may yet prove to be valuable as rootstock material. Because Citrus species hybridize easily, they have been difficult to classify and there have been several proposed classification schemes that are often at odds with one another (Swingle and Reece, 1967; Tanaka, 1969; Barret and Rhodes, 1976; Tanaka, 1977; Scora, 1988; Fang et al., 1991). Limonoids are a group of chemically related triterpene derivatives that have received extensive study in plants of the Rutaceae and Meliaceae families. Among the 36 limonoids isolated from Citrus taxa and their hybrids, limonin is the predominant limonoid and is the primary cause of limonoid bitterness in citrus juices (Hasegawa and Herman, 1992). However, in Citrus ichangensis, a member of the subgenus Papeda, ichangensin is the predominant limonoid and limonin is only accumulated at much lower concentrations (Bennett et aL, 1988; Herman et al., 1989).
*Author to whom correspondence should be addressed. tWork supported by the Citrus ProductsTechnical Committee. tPresent address: Western Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, 800 Buchanan Street, Albany, Califomia 94710, U.S.A.

(Received 2 August 1995; accepted 1 November 1995)

. Fong et al. The CH2CI2 extract was spotted onto TLC plates along with standards. The gradient elution schedule consists of an initial 2 rain run of 80% 0.0. 1993)..45 micron syringe filter into an auto sampler vial for injection on to an automated HPLC system. ichangensis. H a s e g a w a and Ifuku. The end p r o d u c t of the ichangensin l i m o n o i d pathway. Park et al. 1994). IL).. C. the limonin biosynthetic p a t h w a y also exists in these cultivars ( H a s e g a w a and Herman. macrophylla were obtained from the Citrus Variety Collection of the Agricultural ExperimentStation. webberii var. Seeds (80 g) were ground into a meal and placed in a Soxhlet extractor. Ohta et al. 4. ichangensis and its hybrids (Bennett et aL. is tasteless and its accumulation at the e x p e n s e of limonin w o u l d eliminate limonin bitterness in citrus juices. The combined extraction mixtures were centrifuged for 1 min in a tabletop micro-centrifuge to pellet the cell debris. (a) cyclohexane:EtOAc (2:3). ichangensis is biosynthesized from nomilin via d e a c e t y l n o m i l i n and deacetylnomilinate. Extraction and analysis of limonoids. Extraction and analysis of flavonoids. hystrix var. 1969. about 250 mg of the dried powder of each sample was extracted three times with 1 ml of a 1:1 DMSO: MeOH mixture at 50°C. Limonoid glucosides were analysed by HPLC with procedures described previously (Herman et al. 1994). 'Montana'. R e s u l t s a n d Discussion Analysis o f the limonoids in papeda taxa All citrus taxa and their hybrids w h i c h have been examined have been s h o w n to accumulate the bitter-tasting l i m o n o i d limonin through the limonin biosynthetic p a t h w a y ( H a s e g a w a and Herman. 1992. Each extraction was allowed to soak for at least 1 h. The relative concentrations were determined from peak areas using the extinction coefficients determined from standards. 1989. 1983. The resulting supernatant of each sample extract was filtered through a 0. Silica gel HLF plates were purchased from Analtech (Newark. H a s e g a w a and Ifuku. Limonoids were detected by UV absorption at 210 nm (Herman et al. 1971. Kefford and Chandler. wilsonii 'lchang lemon'. 1988. 1994). C. For sample extraction. 5 micron particle size) which was used in conjunction with a UV diode-array detector. Citrus is a particularly rich source of f l a v o n o i d s ( H o r o w i t z and Gentili. The meal was washed thoroughly with hexane to remove oils and then extracted with MeOH. The aqueous solution contained limonoid glucosides. C. The CH2CI2fraction was used for analysis of the limonoid aglycones. 'Cabuyao'.01 M H3PO4 and 20% MeOH followed by a linear gradient to 100% MeOH over 55 min at a flow rate of 1 ml per min (Kanes et al. University of California at Riverside. BERHOW ETAL. junos 'Yuzu'.. Collected leaveswere oven-dried at 60°C in brown paper bags for at least 24 h.A. 1989. C. H a s e g a w a and Ifuku. C. 1977). . Nishiura et aL. Kanes et al. (b) CH2CI2:MeOH (49:1) and (c) EtOAc:CH2CI2 (2:3). and C... latipes. DE). 1994. but little w o r k has been d o n e on the f l a v o n o i d c o m p o s i t i o n of the papedas. T h e f l a v o n o i d s are a major class of plant s e c o n d a r y metabolites w h i c h have proved to have c h e m o t a x o n o m i c value. ichangensin. Limonoid concentrations were determined quantitatively by HPLC using a C18 reversed-phasecolumn and isocratic elution with 49% H20. Hashinaga and Hasegawa. and developed with three different solvent systems. Herman et aL. 1990. 41% MeOH and 10% CH3CN at a flow rate of 1 ml/min. A s e c o n d l i m o n o i d biosynthetic p a t h w a y resulting in the formation of the calamin g r o u p of l i m o n o i d s exists in the cultivars related to the genus Fortunella and their hybrids. ODS 3. 1992. B e r h o w et al. In this pathway. Peak area percentages and extinction coefficients are calculated from chromatograms of standards detected at 285 nm. Dried leaveswere ground to a powder with a small coffee bean mill. C. particle size 5 Ilm was purchasedfrom AIItech Associates (Deerfield.6 x 250 mm..4 cm. 1992). A n u m b e r of Citrus surveys identifying and q u a n t i f y i n g s o m e of the major flavonoids based on separations occur in the literature (Albach and Redman. The analytical H PLC column used for limonoid analysis was a C-18 reverse phase. Seeds were dried at 60°C for 3 days prior to analysis. A third l i m o n o i d biosynthetic p a t h w a y exists in the cultivars related to C.238 M. the residue was extracted with a mixture of CH2CI 2 and H=O (1 : 1) at pH 3. 1992. Hashinaga et aL. 1992. Materials and Methods Seeds and/or leaves of Citrus hystrix. 1993).. 1970. 1993a). partisil ODS-3. The HPLC column used in the flavonoid analysis was a Licrosorb C18 reverse phase analytical column (25 x 0. This wavelength was chosen for monitoring becauseall flavonoids examined in this study absorb at this wavelength and extinction coefficients were similar when calculated from several purified flavonoid standards. After the MeOH was evaporatedfrom the extract.

hanaju and C. 1994). L G . junos '¥uzu.. which have also been shown to have fairly low levels of limonoid glucosides (Fong et aL. DG-17-~-D-gfucopyranoeide. 1991.. or the very high levels of deacetylnomilin. We were able to look at the concentrations of 10 major flavanone glycosides (Table 2) five major identified flavone/ol glycosides and an additional 14 as yet unidentified flavone/ols (Table 3) in the papeda specimens present in the Riverside collection.. T A B L E 1. O G ~ b a c u n o n e UNK---unidentified limonoid aglycone. 1993b). 17-[~-o-glucopyranoside U m o n o i d glucosides in seeds (ppm) UNK 202 ------LG 151 412 260 110 56 1745 256 NAG -412 440 --634 -NG 403 828 480 346 120 2380 1025 DG -------OG ---160 ---- Limonoid aglycones in seeds (ppm) Citrus species C/bus/aO~oes LIM 1728 824 300 1987 810 C/bus webbadi "Montana" 5552 4554 NOM 1512 1098 800 858 550 872 911 OBA 756 261 90 909 120 123 569 -20 476 51 DAN 1512 -60 ICN 227 ----18 -- C#rus hystedx "Cabuyao "° C#rus hystedx Citrus mecrophylla *The 'Cabuyao" cultivars in the R i v e ~ i d e Citrus Variety Collection have been labeled a C.l i m o n i n 1 7 NG~nomilin 17-~-o-glucopyrenoside. the low level of glucosides found in seeds from mature fruit seems to indicate a relationship with the pummelos. N O M . Berhow et al. narirutin and neohesperidin. Berhow et aL.. the hybrids C. Abbreviations: L I M . Hashinaga et aL. Indeed. macroptera variety by Swingle.. L I M O N O I D C O M P O S I T I O N OF SEEDS OF V A R I O U S PAPEDAS A N D P A P E D O C I T R U S IN T H E C I T R U S V A R I E T A L C O L L E C T I O N A T T H E U N I V E R S I T Y O F C A L I F O R N I A A T RIVERSIDE.d e e c e t y l n o m i l i n . 1994). and its corresponding glucoside (Ohta et al. 1992. the presence of the limonin pathway in the papedas C.L I M I N O I D A N D F L A V O N O I D IN CITRUS VARIETIES 239 In order to examine the extent that the genes for the ichangensin pathway are expressed in cultivars related to C. Ohta et aL. webberii 'Montana' is clearly shown by the occurrence of limonin and to a lesser degree the limonin precursors nomilin. hystrix. sudachi and C. The limonoid content of the non-ichangensis papedas resemble more closely that of the 'true' citrus species such as the mandarins. ichangensis. 1989. sphaerocarpa "Mochiyu' and 'Yuko' have also been shown to possess ichangensin and its glucoside derivative. the citrons and the pummelos. latipes and C. Ozaki et aL. obacunone and ichangin in the seeds examined. ichangensin 17-J3-D-glucopyranoside in their fruit tissues and seeds (Hashinaga and Hasegawa. . we have analysed the limonoids in seeds of some of the other papeda taxa. ichangensis. C. that indicates the presence of the initial stages of the ichangensin biosynthetic pathway. but instead accumulate relatively high concentrations of deacetylnomilin. This shows that these other papeda species do not have this pathway and indeed the pathway is only found in cultivars directly related to C. O B A ~ o b a c u n o n e . However. wilsonii 'lchang lemon'--two other C. D A N . macrophylla. hystrix 'Cabayao' and C. 1992. ichangensis hybrids--do not contain ichangensin. the seeds of C. Analysis of the flavonoids in papeda taxa This report is the first thorough examination of a large number of individual flavonoids in the leaves of cultivars in the Papeda subsection. However.l i m o n i n . While there have been some fairly substantial characterizations of the flavonoids in the major commercial species.nomilin. 1990. hesperidin. hyst/ix varieties in the Collection records. at best only one or two of the papeda cultivars have been examined. ichangensis and other citrus taxa. I C N ~ c h a n g i n . none of these seeds contain any ichangensin. As shown inTable 1. C. such as C. NAG~omonilic acid deecetylnornilin 17-[~-o-glucopyranoside. especially naringin. Some of the hybrids between C. but are referred t o as a C. a precursor of ichangensin. Often these examinations have only been in regard to a few of the major flavonoids. J3-D-glucopyranoside.

66 . 0. 6.1 weight Flavanone concentrations in m g / g d w w e i g h t NRG ERC NER NRT NGN HES 5. (hesperetin-7-neohesperidoside).93 3.. 0. Rouseff et aL. i c h a n g e n s i s C.07 35.99 . NGN---naringin NER---neoeriocitrin (eriodictyol-7HES-(naringenin-7-rutinoside). 1977. The other papeda species have a slightly different genetic makeup but also share a possible common ancestry with the citron and. 5. PON---poncerin (isosakuranetin- 7*neohesperidoside). junos 'Yuzu' all had detectable levels of flavanone neohesperidosides. ichangensis and its hybrid C. Neither the true mandarin or the pummelo cultivars have rutin present in their tissues. 2. .52 1. True mandarins and citrons only accumulate the flavanone rutinosides. .39 1. hystrix varieties in the Collection records. ichangensis. hystrix "Cabuyao. Due to their retention times.39 -0.09 . . -----2.53 0. cultivars related to the pummelo accumulate flavanone neohesperidosides (Nishiura et al. possibly sharing a remote common ancestor with the citron. webberii'Montana' C.240 M . C. hesperidin NRT---narirutin (hesperetin-7-rutinoside). rnacrophylla could not be assessed from either the limonoid or flavonoid data except to exclude C. m g / g fresh DID--<lidymin (isosakurenetin-7-rutinoside). These researchers propose that C.90 6.59 -0. These taxa have a slightly different genetic makeup than the other papedas and possibly share a common ancestor with the pummelo in addition to the citron.66 1. macrophylla .16 12. We feel that C.28 ---UNK --- Citrus species C. hystrix.68 12. junos 'Yuzu" C. most of these flavone/ols appear to be glycosylated. 1983. 1977). 1987. NGM--~aringin-6"-malonate (narigenin-7-neohesperidoside-6"-malonate). Rutin is typically found in the citrus cultivars related to citron (Kanes et al.39 33. 2. C. 1991).. wilsonii.27 -0.06 --NGM --. 1993).92 2.65 NHS --5. ichangensis may be a truly unique wild species of Citrus.89 -. ERC--edocitrin NHS---~eohesperidin (edodictyol-7-rutinoside). The genetic makeup of C. A . 1971. C. --tr .' C. Conclusion One other report in the literature suggests that C.58 -1.16 0. However. 1993).45 2. T A B L E 2.36 1.04 47.40 0.u n k n o w n flavanone peak Rt 33.4 ' . in some specific cases. laO"pes (mature) C. latipe~ (young) C. . The uniqueness of C. ichangensis as a parent.35 9.37 -0.1 min. . wilsonii 'lchang lem.52 *The "Cabuyao" cultivars in the Riverside Citrus Variety Collection have been labeled a C. B E R H O W ETAL. . ichangensis is re-enforced by the fact that it accumulates the unique limonoid ichangensin and a number of additional flavone/ols other than those found in the rest of the papedas.06 6. C.36 1. and are probably not the polymethoxylated flavone aglycones found in the oranges and lemons (Horowitz and Gentili.. All of the papeda species we examined had both hesperidin and rutin. Kanes et aL. neohespe¢idoside). ichangensis may be an evolutionary bridge between the other papeda species and the 'true" citrus species. tr -.01 PON --.02 .. U N K .57 3.58 0. F L A V A N O N E C O M P O S I T I O N IN T H E LEAVES OF V A R I O U S P A P E D A S A N D P A P E D O C I T R U S IN T H E C I T R U S V A R I E T Y C O L L E C T I O N A T THE U N I V E R S I T Y OF C A L I F O R N I A A T RIVERSIDE. Park et al. All the papedas may share a common ancestor with the citron. hystrix C. Abbreviations: N R G ~ n a r i r u t i n . tr -0. The other papedas have lesser amounts of these compounds and nowhere near the levels found in C. (naringenin-7-neohesperidoside).97 --- . Horowitz and Gentili.73 ----4.36 0. macroptera variety by Swingle. tr . DID 4. accumulate detectable levels of nearly all the unidentified flavone/ols listed in Table 3. tr indicates detectable peak at a concentration b e l o w 0.g l u c o s i d e (naringenin-7-rutinoside-4'-glucoside). In general. .96 2. ichangensis may be a separate subgenus of the genus Citrus based on isozyme analysis (Fang et aL.45 tr -0. hystrix "Cabuyao' C. but are referred to as a C. the pummelo.89 0. latipes and C.

18 C.21 m -- C.0 0. .95 -- -- C. . F L A V O N E / F L A V O N A L IRF~isorhoifolin COMPOSITION IN T H E L E A V E S O F V A R I O U S P A P E D A S A N D P A P E D O C I T R U S I N T H E RIVERSIDE.56 1. . z T A B L E 3. .35 3"48 -3.26 -- -- O.6 tr 0.73 32.06 0. w / / s o n / / " l c h a n g lem..39 I I I O'~ IRF --1.65 -- RFN 26. I I I in m g / g d r y w e i g h t NDM 21.46 C.0 tr 0. . 27. . 31.02 1.75 tr . .45 . /at/peS (mature) 1. 28.1 m g / g f i ' u h w e i g h t Identified flavonelob concentrations U n k n o w n f l e v o n e / o l c o n c e n t r a t i o n s in m g / g d r y w e i g h t DSM 20. .4 25.n ~ d ~ d e ) " DSk~--di~in (di~tin-7-~incek:le). . . . species 22. ~ 'Cabuyao' 11. 1. 0. k:hJngwl~ 0.0 2. . RFN---cI~Iin ( a p i g i n i n . .51 .~ 1.7 .58 tr -tr .0 25.17 0.06 2"68 0"71 0.72 1. t r indicates d e t e c t a b l e I x m k at a c o n c e n t r a t i o n b e l o w 0. ~ C.46 0. hy~/r/x --0"25 1.81 -tr .65 . . .5 tr 0. ..0 4. .1 -tr --0.21 0.2 0. CITRUS V A R I E T Y C O L L E C T I O N A T T H E U N I V E R S I T Y O F C A L I F O R N I A A T (al~ginin-7-~n~kle).11 -- -- C.7 .37 -- .33 -- -- 0'35 0"62 --0.34 -- -- C.24 -0.11 I 1 "07 I I -0.91 --0. .' 0.06 1.74 0"76 -1. . ] u n o s "Yuzu" 1. w e b b e d / " M o n t a n a " 1.67 1. -- 1.n e o d i o s m i n ( d i o # m l e t i n .r- o z _o c~ Z./agpes (young) 2.98 -.34 1.45 .5 0. macrophy//a 4. 24. Oe51 C / ~ u .2 tr --- c. Ablxevletions: RUT~rutin (quwcetin-3-rutinoside).23 3. 27.67 -. tr .n e o h e s p e d d o s i d e ) . .6 tr -0.1 2 RUT 23. ~ X :o N D M .61 -- -- -I --0'46 0"33 -tr -0"54 ---1T I 0"~ .46 -- -- C.18 1.

Pasadena. G. Shaw. Ohta. Chem. Univ.. M. 58. and neohesperidin in Citrus. (1991 ) Ichangensin glucoside in Citrus junos. and Batchelor. S. Bennett. Zhang. Fruit and Vegetable Chemistry Laboratory.. 3905-3907. M. (1967) The botany of Citrus and its wild relatives. Berkeley. S. D. R. (1994) Limonoids in seeds of three Citrus hybrids related to Citrus ichangensis. T. Guangzhou. eds). Food Chem. and Chandler. Hasegawa. H. W. Hashinaga. and Hasegawa. and Jackson. Herman. Ci~us Syrup. Int.. Ser. Barret. Israel. Xiao. Y. L. (1993b) Limonoids in Pummelo. Beijing. P. and Redman. (1990) Limonoids in seeds of Yuzu. Tisserat. J. (1993a) Limonoids and their glucosides in Valencia orange seeds during fruit growth and development. (1977) Flavonoid constituents of Citrus.. Fong. 139-145. and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable. D. Miyake. S. H. Phytochemisb7 32. Coggins. J. E. and Reece.. the USDA neither guarantees nor warrants the standard of the product. and Veldhuis. Agric. 35. 237-246. 10.. Japan. S. 112-115. 21. pp.. Fong. W.. C. (1991 ) Studies on the taxonomy and evolution of Citrus species by isozyme analysis. Avery. and Hesegawa. S. C. (1994) Nippon Shokuhin Kogyo Gakkaishi41. ARS. Osaka Pref. Herman. and Esaki. 1-6.. 98-105. H. Tanaka. 380-382. Food Chem. J. S. R. B. CT. 923-925. California. China. J. F. D. 2659-2661. Swingle0 W. S. Citrus Research Conference Abstracts of Papers. C. Hashinaga. Maeda. M. Y. M. 37. Kanes. R. Ozaki. Z.. Suhayda. T. S. In Secondary Metabolite Biosynthesis and Metabolism (Petroski.242 M. Westport. Names are necessary to report factually on available data. 1. Kefford.. G. H. F. and Mendel. Food Chem. taxonomy and evolution of modern cultivated citrus. (1988) Ichangensin: a new citrus limonoid. (1969) Composition and inheritance of flavanones in citrus fruit. Phytochemis~/ 36. 277-289. pp. P. Balaban Publishers. 967-974.. Soc. R. Bot. Scora. Hort. eds). Citrus sudachi. K. F. Fong. and Hasegawa. O. (1969) Misunderstandings with regards to Citrus classification and nomenclature. D. and Hasegawa. 397-426. and Hasegawa. AVI Publishing Company.. (1992) Analysis of limonids in citrus seeds. and Atkin. and Hasegawa.. F. In Proceedings of the Sixth International Citrus Congress (Goren. (1983) Identification of bioflavonoids from Citrus. C. F. 14. Fong. P. Ozaki. Herman. H. Nishiura. (1987) Quantitative survey of narirutin. Bennett. Plenum Press. Germany. H. Studia Ci~ilogica 14. Springer-Verlag. R. Bull. H.. J. L. (1993) Phenolic composition of various tissues of Rutaceae species. Y. M. and Hasegawa. C. . F. H. Berlin. pp. Inc. BERHOWETAL. 1027-1030. W. S. M. (1992) Limonoids in seeds of Citrus hanaju. Herman. Omura. M. Phytochemis~y 31. C. 190-430. Sci. California. 105-136. 227229.. S. M. Biol. F. J. K. B Agric. Syst. C. Food Technol. (1970) The Chemical Constituents of Citrus Fruits.. Hasegawa.. and C~us sphaerocarpa.. (1977) Fundamental discussion of Citrus classification. S. eds). 1990 (Bangyan. Park.. References Albach. C. Berhow. H. 305-317. V. (1971 ) Flavonoids in Citrus and related genera III. Tel Aviv. B. New York. M. L. CA. S. A. Kamiya. Phytochemistry 30. pp.. S. and Hasegawa. H. Z.. R. (1989) Limonoids in Citrus ichangensis. Z. 1543-1 545.. Bennett. P. C.. C. Webber. 1 (Reuther. New Series Vol. Berhow. Y. and Rhodes.. eds). 34. In The Citrus Industry Vol. and Herman. and Wan. M.. Martin. K. J. Acknowledgements---Research conducted at the USDA. and Youtsey. Agric.. Byers. S. eds). November. L. naringin. Fang.. Proc. Phytochemistry 27. M. Horowitz.. S. 361375. R.. Nippon Shokuhin Kogyo Gakkaishi 37. 850-851. New York. and Nelson. H. S. Fong. H. and Ou. R. Hasegawa.. Pasadena. University of California. In Modern Methods of Plant Analysis. Berhow. C. and Qian. Z. Y.. R. A. China. 35. B. Z. M. 1691-1706..A. B. H. Agric. D. and Ifuku. (1976) A numerical taxonomic study of affinity relationships in cultivated Citrus and its close relatives. and Gentili. and Vandercook.. Miyake.. and McCormick. R. hesperidin. Y. J. eds). Z. H. pp. 372. Ifuku. Ohta. Herman.. S. Ozaki. Rouseff. International Academic Publishers. (1989) Limonoids in seeds of Sudachi. D. (1988) Biochemistry.. Agric. (1992) Biosynthesis of limonoids in Citrus. S. S. Berhow. Academic Press.. W. Seed Analysis (Linskens. Flavonoid pattern and Citrus taxonomy. closed by the USDA in 1994.. pp. Hasegawa. Tanaka. however. p. Phytochemistry 8. R. In Citrus Science and Technology Vo/. D. I (Nagy. 127-143.