Limonoids and Flavonoids in Juices of Oroblanco and Melogold Grapefruit Hybrids

CHEMISTRY/BIOCHEMISTRY

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 determined 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 similar 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). An aliquot of the juice was mixed reported to increase is the bitter effect of each compound (Guadagni 1:1 with dimethylsulfoxide, centrifuged and filtered through a 0.45 et al., 1974 ). In the case of Oroblanco and Melogold, the presence micron filter to remove particulates before injection. The flavonoids of nomilin even at low concentrations could be expected to enhance were detected by photodiode array detector and quantified by absor- bitterness. bance at 285 nm. Peaks were identified by comparison of retention Pummelo, one of the parents of Oroblanco and Melogold, has a times and spectra with standards. Concentrations were determined severe limonoid bitterness (Ohta and Hasegawa, 1995). Its juice con- by comparison with a standard curve prepared from pure naringin tains very high concentrations of limonin, an average of 18 ppm for for the flavanones and pure rhoifolin for the flavones. 16 cultivars. Bitterness in grapefruit as well as in sour orange is mainly due to bitter flavonoids (Hagen et al., 1966; Castillo et al., 1992). RESULTS & DISCUSSION However, grapefruit, the other parent of these hybrids, also has li- FRUITS WERE HARVESTED AFTER THE OROBLANCO AND MEL- monoid bitterness problem (Scott, 1970; Rouseff, 1982). Thus, it is ogold were fully ripe in February. Like Valencia oranges, most culti- understandable why Oroblanco and Melogold juices have the prob- vars of grapefruit are not harvested until May, June or July in the lem. Riverside area of Southern California. The fruit rinds of grapefruits were nearly completely yellow. The soluble solids and acid concen- Limonoid glucosides trations of the juice were within the range acceptable for mature fruit Limonoid glucoside contents in juices of pummelo and its hy- (data not shown). Our analysis is obviously a single profile base for brids are very low compared to those in sweet oranges. Total limonoid the limonoid glucoside concentrations in these fruit. While relative glucoside concentrations in Oroblanco juice samples ranged from amounts may change during the course of a harvest season or in 97 to 115 ppm with an average of 99 ppm (Table 1). However, these different years, the limonoid glucoside concentrations in grapefruit levels were above those in Melogold juices which contained an av- juices would probably be comparable to those of Oroblanco and erage of 59 ppm. These concentrations were lower than those of grape- Melogold. The glucosidation of limonoid aglycones in citrus fruits fruit averaging about 194 ppm for commercial juices and 191 for 4 grown in California appears to begin in September regardless of har- cultivars (Table 2), Mandarin juices averaging 220 ppm (Ozaki et vest season as found in both navel orange (Hasegawa et al., 1991) al., 1995), and were much lower than those of commercial orange and Valencia orange (Fong et al., 1992). The presence or absence of juices which averaged 320 ppm (Fong et al.,1989 ). a particular compound would also be consistent for the mature fruit Pummelo juices contain a low average of 28 ppm of total limonoid and could be applied as taxonomic markers. glucosides for 16 cultivars (Ohta and Hasegawa, 1995). The low

58—JOURNAL OF FOOD SCIENCE—Volume 63, No. 1, 1998 Table 3—Flavonoidsa in juices of Oroblanco and Melogoldb Samples NRTG NGTG ATG1 NDM NER NRT NRG HES NHS RUT RFN N6M ISR PON Melogold 6C-29-4 14 20 - - - 17 413 - 7 - 28 21 - 8 6C-29-5 15 18 - - - 182 449 - 4 - 17 19 - 7 6C-29-7 17 25 - - - 210 580 - 8 - 25 25 - 10 Average 15 21 - - - 136 480 - 6 - 23 33 - 08 Oroblanco 6A-13-10 10 17 15 - 5 166 489 - 7 - - 28 - 10 6A-15-7 9 14 12 - 4 146 346 - 5 - - 20 - 10 6A-15-6 9 16 12 - 4 176 426 - 6 - - 23 - 10 Average 9 16 13 - 4 163 420 - 6 - - 24 - 10 aNRTG: narirutin 4'glucoside; NGTG: naringin 4'glucoside; ATG1: rhoifolin 4'glucoside; NDM: neodiosmin; NER: neoeriocitrin; NRT: narirutin; NRG: naringin; HES: hesperidin; NHS: neohesperidin; RUT: rutin; RFN: rhoifolin; N6M: naringin-6'-malonate; ISR: didymin; PON: poncerin;. bAverage of triplicate (ppm).

The flavonoids found in Oroblanco and Melogold were very sim- conversion of aglycones to their respective glucosides during maturation could be due to the low activity of limonoid glucosyltrans- ilar to those reported in pummelo and grapefruit (Kanes et al., 1993). However, these cultivars did not contain a measurable amount of ferase in the fruit tissue during maturation. The relatively low concentrations of limonoid glucosides in the Oroblanco and Melogold didymin which is present in grapefruit. Apparently, these character- istics were inherited from the parental species. The concentrations juices were apparently inherited from pummelo. For comparison, the compositions of individual limonoid gluco- of total bitter flavonoids, 440 ppm for Oroblanco juice and 495 ppm for Melogold juice would not cause unacceptable bitterness in juic- sides were also determined in juices of grapefruit (Table 2 ). Total limonoid glucoside concentrations in grapefruit juices, average of es. However, the presence of these flavonoids may increase the limonoid bitterness (Guadagni et al., 1974). 194 ppm for commercial juices and 191 ppm for extracted juices at the laboratory, were very similar to reported values (Fong et al.,1989). Limonoid delayed bitterness has been a problem of early-season to mid-season fruit, but not late season fruit. The acceleration of the Limonin glucoside was the predominant limonoid glucoside in all juices comprising 70% of total limonoid glucosides for Oroblan- natural limonoid debittering process through genetic engineering could result in the reduction of bitter limonoid aglycone accumula- co, 52% for Melogold, 60% for commercial grapefruit and 65% for hand extracted grapefruit juices. This trend was very much similar tion in fruit tissues. Limonoid glucosyltransferase, which catalyzes this glucosidation, has been isolated from navel orange albedo tis- to that in commercial sweet orange juices (Fong et al., 1989) and mandarin juices (Ozaki et al., 1995). All the juices also contained sues and its properties characterized (Hasegawa et al., 1997). The insertion of the gene encoding for the glucosyltransferase into com- nomilin glucoside and nomilinic acid glucoside. In addition, a trace amount of obacunone glucoside was detected in grapefruit juices. mercial cultivars through genetic engineering may create transgenic citrus cultivars producing fruit potentially free from limonoid bitter- The limonoid glucosides in fruit tissues are stable (Herman et al., 1991). However, in seeds, they are hydrolyzed during germination ness. Pummelo, Mandarin, and citron are reported to be three of the to liberate glucose molecules by the catalytic action of an enzyme, Citrus limonoid 17ß-D-glucopyranoside ß-glucosidase (Ronneberg et al., original species of . Mandarin orange and its hybrids (sweet oranges) have relatively high levels of limonoid glucosyltransferase 1995). In juice, the glucosides are generally stable, except nomilin glucoside, which converts to nomilinic acid glucoside below pH 3.5 activity. In contrast, pummelo and its hybrids, grapefruit, Oroblanco and Melogold, have low levels of the enzyme. Consequently, such or above 8.0 (Herman et al., 1991). This explains why in commercial grapefruit juices the concentration of nomilinic acid glucoside cultivars would be excellent candidates for such genetic engineering research to improve citrus juice flavor quality. was higher than that of nomilin glucoside, but in freshly prepared juice, the concentration of nomilin glucoside was higher than that of REFERENCES nomilinic acid glucoside (Table 2). Albach, R.F., Juarez, A.T., and Lime, B.J. 1969. Time of naringin production in grapefruit. J. Am. Soc. Hort. Sci. 94: 605-609. Flavonoids Alford, A.R. and Bentley, M.D. 1986. Citrus limonoids as potential antifeedants for the spruce budworm (Lepidoptera: Tortricidae). J. Economic Entomo. 79: 35-38. Both Oroblanco and Melogold juices contained bitter flavonoids Bennett, R.D., Hasegawa, S., and Herman, Z. 1989. Glucosides of acidic limonoids in Citrus. Phytochemistry 28: 2777-2781. such as naringin, neohesperidin and poncerin (Table 3). Oroblanco Berhow, M.A., Bennett, R.D., Kanes, K., Poling, S.M., and Vandercook, C.E. 1991. A juices contained neoeriocitrin as well. Oroblanco juices contained malonic acid ester derivative of naringin in grapefruit. 30: 4198-4200. Berhow, M.A. and Vandercook, C.E. 1989. Biosynthesis of naringin and prunin in an average of 420 ppm of naringin and Melogold juices 480 ppm. detached grapefruit. Phytochemistry 28: 1627-1630. About 50 to 100-fold lower levels of the other bitter compounds Castillo, J., Benavente, O., and del Rio, J.A. 1992. Naringin and neohesperidin levels during development of leaves, flower buds and fruit of Citrus aurantium. Plant Phys- were present in both juices. They had the major flavonoids found in iol. 99: 67-73. grapefruit cultivars such as Duncan: narirutin, naringin, neohesperi- Chandler, B.V. and Kefford, J.F. 1966. The chemical assay of limonin, the bitter principle of oranges. J. Sci. Food Agric. 17: 193-197. din and poncerin. The levels of naringin were consistent with that Dreyer, D.L. 1965. Citrus bitter principles. III. Isolation of deacetylnomilin and deox- reported in grapefruit juices (Albach et al.,1969; Kanes et al., 1993). ylimonin. J. Org. Chem. 30: 749-751. Fong, C.H., Hasegawa, S., Herman, Z., and Ou, P. 1989. Limonoid glucosides The levels of the flavanone rutinosides were very low. The presence in commercial citrus juices. J. Food Sci. 54: 1505-1506. of the malonylester of naringin in the juice was noted. This com- Fong, C.H., Hasegawa, S., Coggins, C.W., Jr, Atkin, D.R,. and Miyake, M. 1992. Con- tents of limonoids and limonin 17-␤-D-glucopyranoside in fruit tissue of Valencia pound was first reported in young grapefruit leaves (Berhow et al., orange during fruit growth and maturation. J. Agric. Food Chem. 40: 1178-1181. 1991). These juices did not contain measurable levels of the flavanone Guadagni, D.G., Maier, V.P., and Turnbaugh, J.G. 1973. Effects of some citrus constituents on taste thresholds for limonin and naringin bitterness. J. Sci. Food Agric. 24: rutinoside hesperidin which is found in grapefruit, orange and lem- 1277-1288. on. Low levels of narirutin (naringenin 7-O-rutinoside) were present Hagen, R.E., Dunlap, W.J. and Wender, S.H. 1966. Seasonal variation of naringin and certain other flavanone glycosides in juice sacs of Texas Ruby Red similar to those found in other grapefruit cultivars (Kanes et al., 1993). grapefruit. J. Food Sci. 31: 542-547. Note that these cultivars did not contain rutin which has been found Hasegawa, S., Ou, P., Fong, C.H., Herman, Z., Coggins, C.W., Jr., and Atkin, D. R. in cultivars related to citron. 1991. Changes in the limonoate A-ring lactone and limonin 17-ß-D-glucopyrano-

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side content of navel oranges during fruit growth and maturation. J. Agric. Food nin 17-ß-D-glucopyranoside. Nutrition and Cancer 17: 1-7. Chem. 39: 262-265. Ohta, H. and Hasegawa, S. 1995. Limonoids in Pummelos [Citrus grandis (L.) Os- Hasegawa, S. and Miyake, M. 1996. Biochemistry and biological functions of citrus beck]. J. Food Sci. 60: 1284-1285. limonoids. Food Rev. Int. 12: 413-435. Ozaki, Y., Ayano, S., Inaba, N., Miyake, M., Berhow, M.A,. and Hasegawa, S. 1995. Hasegawa, S., Suhayda, C.G., Hsu, W., and Robertson, G.H. 1997. Purification of li- Limonoid glucosides in fruit, juice and processing by-products of Satsuma manda- monoid glucosyltransferase from navel orange albedo tissues. Phytochemistry 46: rin (Citrus unshiu Marcov) J. Food Sci. 60: 186-189 33-37. Ozaki, Y., Fong, C.H., Herman, Z., Maeda, H., Miyake, M., Ifuku, Y., and Hasegawa, Hasegawa, S., Bennett, R.D., Herman, Z., Fong, C.H. and Ou, P. 1989. Limonoid glu- S. 1991. Limonoid glucosides in citrus seeds. Agric. Biol. Chem. 55: 137-141. cosides in citrus. Phytochemistry 28: 1717-1720. Record, M.T., Miller, E.G., Binnie, W.H., and Hasegawa, S. 1997. Systemic effects of Herman, Z., Fong, C.H., Ou, P., and Hasegawa, S. 1990. Limonoid glucosides in or- limonoid glucosides on oral carcinogenesis. Abstract. IARR, Orlando, FL ange juices by HPLC. J. Agric. Food Chem. 38: 1860-1861. Ronneberg, T. A., Hasegawa, S., Suhayda, C., and Ozaki, Y. 1995. Limonoid glucoside Herman, Z., Fong, C.H., and Hasegawa, S. 1991. Biosynthesis of limonoid glucosides b-glucosidase activity in lemon seeds. Phytochemistry 39: 1305-1307. in navel orange. Phytochemistry 30: 1487-1488. Rouseff, R.L. 1982. Nomilin, a new bitter component in grapefruit juice. J. Agric. Horowitz, R.M. and Gentili, B. 1977. Flavonoid constituents of Citrus. In Citrus Sci- Food Chem. 30: 504-507. ence and Technology, Vol. 1, S. Nagy, P.E. Shaw, and M.K. (Ed.), p. 397-426. Avi Saunt, J. 1990. Citrus Varieties of the World. Sinclair International Limited, Norwich, Publishing Co., Inc., Westport, CT. England. Kanes, K., Tisserat, B., Berhow, M., and Vandercook, C. 1993. Phenolic composi- Scott, W.C. 1970. Limonin in Florida citrus fruits. Proc. Fla. State Hort. Soc. 82: 270- tion of various tissues of Rutaceae species. Phytochemistry 32: 967-974. 277. Klocke, J. A. and Kubo, I. 1982. Citrus limonoid by-products as insect control agents. Soost, R.K. and Cameron, J.W. 1981. Grapefruit hybrid. U.S. Patent 4645. Entomol. Exp. Appl. 32: 299-301. Soost, R.K. and Cameron, J.W. 1987. Grapefruit hybrid named Melogold. U.S. Patent Lam, L.K.T., Li, Y., and Hasegawa, S. 1989. Effects of citrus limonoids on glutathione 6001. S-transferase activity in mice. J. Agric. Food Chem. 37: 878-880. Swingle, W.R. and Reece, P.C. 1967. The botany of citrus and its wild relatives. In The Miller, E.G., Fanous, R., Rivera-Hidalgo, F., Binnie, W.H., Hasegawa, S., and Lam, Citrus Industry, Vol. 1, W. Reuther, H.J. Webber, and L.D. Batchlor (Ed.), p. 190- L.T K. 1989. The effect of citrus limonoids on hamster buccal pouch carcinogenesis. 430. University of California, Berkeley. Carcinogenesis 10: 1535-1537. Ms received 8/1/97; revised 9/24/97; accepted 9/28/97. Miller, E.G., Gonzales-Sanders, A.P., Couvillon, A.M., Wright, J.M., Hasegawa, S., and Lam, L.K.T. 1992. Inhibition of hamster buccal pouch carcinogenesis by limo- We thank Drs. M.L. Roose and T.L. Kahn, Univ. of California at Riverside for advice and help.

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