CHEMISTRY/BIOCHEMISTRY Limonoids and Flavonoids in Juices of Oroblanco and Melogold Grapefruit Hybrids WAN-JEAN HSU, MARK BERHOW, GEORGE H. ROBERTSON AND SHIN HASEGAWA ABSTRACT wa et al., 1989; Ozaki et al., 1991 ). Oroblanco and Melogold are hybrids obtained from pummelo Seventeen limonoid glucosides have been isolated and character- and grapefruit. Limonoids and flavonoids in both juices were ized from citrus and its hybrids (Hasegawa and Miyake,1996). In all analyzed. Oroblanco and Melogold juices contained low con- of the glucosides, the glucose moiety is attached via ß-glucosidic centrations of limonoid glucosides, an average of 99 and 59 linkage to the 17-position of the limonoid structure. Limonoid glu- ppm, respectively. However, they contained relatively high cosides are mostly water soluble and tasteless (Hasegawa et al., 1991). concentrations of bitter limonoid aglycones, limonin and The glucosidation is catalyzed by the action of an enzyme, limonoid nomilin, at levels above the limonin bitterness threshold. For glucosyltransferase (UDP-D-glucose: limonoid glucosyltransferase) comparison, limonoid glucosides in juices of grapefruit, an- ( Hasegawa et al., 1997), which is an important in reducing bitter- other pummelo hybrid, were also analyzed. Limonin gluco- ness associated with limonoids. side was the major limonoid glucoside in all juices analyzed. Limonoids have some important biological activities such as an- Nomilin glucoside and nomilinic acid glucosides were also tifeedant activity against insects (Klocke and Kubo, 1982; Alford present. Oroblanco and Melogold juices contained bitter fla- and Bentley, 1986) and anticarcinogenesis in laboratory animals (Lam vonoids normally found in grapefruit and pummelo includ- et al., 1989; Miller et al., 1989). Limonin glucoside has been shown ing naringin, neohesperidin and poncerin in total amount of to inhibit oral carcinogenesis in hamster pouch when it is applied 440 ppm in Oroblanco juice and 495 ppm in Melogold juice. topically (Miller et al., 1992). A mixture of limonoid glucosides iso- They also contained several other nonbitter flavonoids found lated from citrus molasses reduced the number of cancer tumor for- in grapefruit. mations in oral carcinogenesis in hamsters when applied in the diet Key Words: limonoid glucosides, flavonoids, Oro-blanco, (Record et al., 1997). Since limonoid glucosides are present in fruit Melogold tissues and juices in high concentrations (Fong et al., 1989; Ozaki et al., 1995), they may be important in fruit quality and possibly in human nutrition. INTRODUCTION Flavonoid bitterness may also be caused by one or more of the PUMMELO [CITRUS GRANDIS (L.) OSBECK] IS CONSIDERED TO BE four flavanone neohesperidosides that accumulate in citrus species one of three major, original citrus species (Swingle and Reece, 1967). related to the pummelo. Naringin is by far the most dominant fla- Because pummelo can readily hybridize, there are many distinct vonoid bitter principle in grapefruit (Hagen et al., 1966) and neohes- pummelo related cultivars including the economically important peridin is slightly more predominant in sour orange (Castillo et al., grapefruit and sour orange. Oroblanco and Melogold are hybrids of 1992) Neoeriocitrin and poncirin are also bitter and occur in rela- an acidless pummelo (Citrus grandis Osbeck) and a seedy, white tively low amounts in citrus juices (Horowitz and Gentili, 1977). tetraploid grapefruit (Citrus paradisi Macf.) developed at the Uni- Our objective was to analyze both limonoid aglycones and glu- versity of California, Riverside (Soost and Cameron, 1981, 1987). cosides, and flavonoids in juices of two newly developed citrus hy- They mature fully six months ahead of Marsh grapefruit grown in brids, Oroblanco and Melogold. For comparison, individual limonoid California and one month or more earlier than Marsh grapefruit grown glucosides in grapefruit juices were also determined. in Israel. Melogold is much larger in fruit size than Oroblanco or Marsh. Both new cultivars produce fruit that is sweeter and less acidic MATERIALS & METHODS than Marsh and are considered to be non-bitter. Oroblanco (trade- name “ Sweetie”) has been planted in considerable numbers in the Materials past decade in Israel and California and is exported mainly to Japan. Oroblanco, Melogold, and grapefruits were obtained from the Both hybrids are generally consumed when their peel color is still Citrus Variety Collection of the Agricultural Experiment Station, greenish yellow because off-flavors develop in late season (Saunt, University of California at Riverside. Commercial grapefruit juices 1990). were purchased from a local market. Juices were extracted with a The limonoid bitterness is an important economic problem in Sunkist juicer from 12 fruits, which had been randomly harvested commercial citrus juice production. Of the 36 limonoid aglycones from different parts of trees during February. Limonoid glucoside isolated from citrus and its hybrids, limonin is the major cause of and limonoid aglycone standards had been isolated from citrus juic- limonoid bitterness (Chandler and Kefford, 1966), though nomilin es and seeds, and characterized by NMR spectrometry (Hasegawa et is also involved (Rouseff, 1982). Natural debittering of limonoids al., 1989; Bennett et al., 1989). through glucosidation of the limonoid aglycones occurs in the fruit tissues and seeds at late stages of fruit growth and maturation (Hase- Analyses of limonoids gawa et al., 1989). Limonoid glucosides are among the major sec- Aglycones were identified by TLC on silica gel plates developed ondary metabolites in mature citrus fruit tissues and seeds (Hasega- in three solvent systems: EtOAc-cyclohexane (3:2), EtOAc-CH2Cl2 (2:3), and CH2Cl2-MeOH (97:3). After drying, the plates were sprayed with Ehrlich’s reagent and exposed to HCl gas (Dreyer,1965). Li- Authors Hsu, Robertson and Hasegawa are with the Western Regional Research monoid aglycones were identified with standards by visual compar- Center, Agricultural Research Service, USDA, 800 Buchanan St., Albany, CA 94710. ison. HPLC was used to quantify aglycones (Ozaki et al., 1991). A Author Berhow is with the National Center for Agricultural Utilization Research, Agricultural Research Service, USDA, Peoria, IL 61604. 10 mL sample of juice was passed through a C-18 Sep-Pak (Waters Associates, Milford, MA). The limonoid aglycones were retained on Volume 63, No. 1, 1998—JOURNAL OF FOOD SCIENCE 57 Limonoids/Flavonoids in Grapefruits Hybrids Juices . Table 1—Limonoid aglycones and glucosides in juices of Oroblanco Table 2—Limonoid glucosides in grapefruit juicesa and Melogolda Limonoid Glucosides (ppm) Glucosides (ppm)b Aglycones (ppm)b Nomilinic Cultivars LG NG NAG Total L N Juices Limonin Nomilin Obacunone acid Total Oroblanco Commercial 6A-13-9 81 24 10 115 8.1 0.8 A 127 21 2 50 200 6A-15-6 56 24 5 85 6.3 0.4 B 102 23 1 49 175 6A-15-7 72 8 17 97 6.9 0.6 C 124 24 3 48 199 D 115 21 2 64 202 Means 70 19 11 99 7.1 0.6 Mean 117 22 2 53 194 Melogold 6C-28-15 31 12 17 60 15.3 1.8 Cultivars 6C-29-4 28 12 17 57 9.5 1.4 Duncan 125 55 3 25 208 6C-29-5 35 15 10 60 7.8 0.9 Hudson Foster135 45 2 18 200 Hall 132 45 5 15 196 Means 31 13 15 59 10.9 1.4 Camulos 103 35 2 20 160 aAverage of two analyses. Numbers under Cultivars represent assigned tree numbers. bL: limonin, N: nomilin, NA: nomilinic acid, G: glucoside Mean 124 45 3 20 191 aAverage of two analyses. the column, rinsed with water, eluted with MeOH and then deter- mined quantitatively by HPLC using a C-18 reversed-phase column (Spherisorb ODS-2 5 um, 250 ∞ 4.6 mm, Alltech Associate, Deer- Delayed (limonoid) bitterness field, IL) and isocratic elution with H2O-MeOH-CH3CN (49:41:10). Freshly prepared juices from both Oroblanco and Melogold fruits Retention times for limonin, nomilin and obacunone were 15, 26, were sweet and nonbitter as noted in a previous publication (Saunt, and 45 min, respectively. 1990). All juice samples, however, became bitter in several hours, Limonoid glucosides were determined by HPLC procedures (Her- suggesting the presence of limonoid delayed bitterness, particularly, man et al., 1990). A 2-mL sample of juice was treated with a C-18 in the Melogold juice. Limonin concentrations in Oroblanco juice Sep-Pak similar to the aglycone analysis, and the resulting sample samples ranged from 6.3 ppm to 8.1 ppm with an average of 7.1 ppm was loaded on the C-18 reverse-phase HPLC column, which had (Table 1). These concentrations were above the limonin bitterness threshold level of 6 ppm (Guadagni et al., 1973). Limonin concen- been equilibrated to 15% CH3CN in 3 mM H3PO4. The column was eluted at 1 mL/min with a linear gradient starting with 15% CH3CN trations in Melogold juice samples were higher than those in Oro- blanco juices, ranging from 7.8 ppm to 15.3 ppm with an average of in 3 mM H3PO4 and ending with 27% CH3CN over 36 min. Li- monoids were detected at 210 nm. Retention times for the gluco- 10.9. These limonin levels clearly confirmed the taste perception. sides of limonin, nomilin, and nomilinic acid and obacunone were Both juices also contained nomilin, another intensely bitter li- 18, 29, 30 and 34 min, respectively. monoid. The threshold of nomilin bitterness in orange juice is simi- lar to that of limonin (Rouseff, 1982). Nomilin has been reported to Analyses of flavonoids be present in grapefruit juices, ranging from 0.1 ppm to 1.6 ppm Juice samples were analyzed for flavonoid content by HPLC us- (Rouseff, 1982). Oroblanco and Melogold juice samples contained ing a C18 reverse-phase column developed with a 20% to 100% an average of 0.6 ppm and 1.4 ppm, respectively. The presence of methanol gradient in 0.01M phosphoric acid over a period of 50 min two bitter compounds, limonin and naringin, in the same juice was (Berhow and Vandercook, 1989).