Conclusions citrus juice. 42nd Annu. Citrus Proc. Meeting, Lake Alfred, FL., pp. 40-44. Citrus juice fermentation induced the formation of sig Echeverria, E. 1990. Developmental transition from emzymatic to acid nificant amounts of OS. However, their composition was hydrolysis of sucrose in acid limes. Plant Physiol. 92, 168-171. found to be significantly different from that of OS gener Faville, L. W., E. C. Hill, and E. C. Parish. 1951. Survival of microor ganisms in concentrated orange juice. Food Technology 5, 33-36. ated by acid oligomerization. Furthermore, the formation Faville, L. W. and E. C. Hill. 1951. Incidence and significance of microor of ethanol and meso-inositol should distinguish between ganisms in citrus juices. Food Technology 5, 423-425. fermentation OS and those due to the addition of medium Hassid, W. Z. and C. E. Ballou. 1957. Oligosaccharides. In: Pigman, invert sugar. Nevertheless, care shouldbe taken to assess W.(Ed.) The Carbohydrates, Chemistry, Biochemistry, Physiology. the freshness of a juice when quantitating beet medium Academic Press, New York. pp. 487-533. McAllister, J. W. 1980. Methods for determining the quality of citrus invert sugar incitrus juice. juices. In: Nagy, S. and Attaway, ]. A. (eds.) ACS Symposium series 143, Washington, D.C. pp. 291-31*7. Literature Cited Swallow, K. W., N. H. Low, and D. R. Petrus. 1991. Detection of orange juice adulteration with beet medium invert sugar using anion-ex- Cancalon, P.F. 1992a. Oligosaccharides generation in acidic sugar media. change liquid chromatography with puse amperometric detection. J. J. Off. Anal. Chem. (in press). Off. Anal. Chem. 74, 341-345. Cancalon, P.F. 1992b. Production of oligosaccharides during sucrose White D. R. and P.F. Cancalon. 1992. Detection of beet sugar adultera inversion. 106th AOAC Int. Annu. Meeting. tion of orange juice by liquid chromatography amperometric detec Cancalon, P. F. and C.R. Bryan. 1991. Quantitation of beet sugar in tion with column switching. J. Off. Anal. Chem. 75, 1-4.

Proc. Fla. State Hort. Soc. 105:162-168. 1992.

ANTICARCINOGENIC ACTIVITY OF PHYTOCHEMICALS IN CITRUS FRUIT AND THEIR JUICE PRODUCTS

Steven Nagy and John A. Attaway From this initial damage, mutation, duplication or Florida Department of Citrus, translocation of normal cellular genes involved in growth Scientific Research Department, control may portend the development of cancer. By one Citrus Research and Education Center mechanism or another, damage to diverse proto-on- 700 Experiment Station Road cogenes (normal cellular genes that may become cancer Lake Alfred, FL 33850 producing) has been implicated in the genesis of human tumors. Many oncogenes encode proteins with key roles in Additional index words. , limonoids, phenolic controlling normal growth and development. acids, vitamin C, vitamin A. Promotion Abstract. Dietary components present in citrus juices have While a genotoxic carcinogen can initiate or alter the been shown to exert protective effects against the induction genetic material of a cell, this event is only the first step in and spread of cancer in animals and humans. The components an elaborate sequence of events leading to neoplastic with the most potent anticarcinogenic activities are mainly growth. Cancers are populations of cells that have acquired naturally occurring secondary metabolites and include the ability to multiply and spread without normal re flavonoids, limonoids, phenolic acids and vitamins. These straints. An abnormal cell population needs to achieve a phytochemicals react by different mechanisms, namely, by selective growth advantage in the presence of surrounding maintaining cellular oxidation-reduction balance and protect normal cells that are regulated by growth-controlling fac- ing cells against free-radical mechanisms, direct detoxifica tion of xenobiotics, control of membrane permeability and by other unknown mechanisms. The chemical structures of citrus Oxidative phytochemicals with known anticarcinogenic activities and Damage various aspects of cancer and anticancer mechanisms are noted in this review. Fibers, phytosterols, terpenes, sulfides, phenollc8, lignans, triterpenoids, isoflavones, Cancer Carcinogens cruciferous indolea

Initiation Steroid Hormones The agents of cancer are many, but most act by damag ing cellular DNA (deoxyribonucleic acid). This step is termed "initiation" and is a genotoxic event. Many Phenolics, solicylates, flavonoids, polyacetylenes, genotoxic carcinogens have been identified. Carcinogens sulfides may be of a chemical or viral nature, or may be induced by radiation (as occurs primarily in skin cancer). Prostaglandin (PGS)

Figure 1. The effects of phytochemicals on metabolic pathways as Florida Agricultural Experiment Station Journal Series No. N-00736. sociated with breast cancer (Pierson, 1992).

162 Proc. Fla.State Hort. Soc. 105: 1992. tors through intercellular communication. The intercellu Table 1. Fourteen classes of phytochemicals with known anticancer prop lar signals that control tissue development and regenera erties and presence in citrus. tion have not been identified, but there are modulating Phytochemical group Present in citrus factors or "promoters" that function by inhibiting this in tercellular communication. Cell replication depends on en Carotenoids dogenous and exogenous controlling elements operating Coumarins by epigenetic mechanisms which either enhance or retard Flavonoids Glucarates the process (Weisburger, 1992). Indoles As a classic example, investigations of breast cancer Isothiocyanates have identified oxidative damage, action of steriod hor Lignans mones and the action of certain kinds of prostaglandins as Monoterpenes promoters of neoplastic proliferation (Pierson, 1992; Fig Phenolic acids Phthalides ure 1). Figure 1 not only demonstrates the sequence of Phytates events in breast cancer, but also shows the influence of Polyacetylenes dietary photochemicals (plant chemicals) on modulating Sulfides the metabolic pathways associated with this type of cancer.

Invasiveness and Metastasis zyme systems (protein kinase C) involved in abnormal cel The factors responsible for the biochemical and lular proliferation, (6) control of membrane permeability, phenotypic aberrations of a mutated cell are not entirely and (7) unknown activities, as for example, the modulation understood. An emerging mutated cell may produce a cell of cellular signals. population which is benign (non-invasive; respects the boundaries of other tissue cells) or may acquire the Citrus Phytochemicals capabilities for extended proliferation, invasion of adjacent Demonstrating Anticarcinogenic Activities tissue and metastasis (formation of secondary and tertiary tumors at different locations in the body). Table 1 lists seven classes of citrus compounds designa Evidence (Bishop, 1987) exists for the presence of on- ted by the National Cancer Institute as exhibiting anticar cogenes which bestow the ability to metastasize on cells cinogenic properties (Caragay, 1992). It is beyond the already capable of abnormal proliferation. In exploring scope of this paper to discuss the extensive epidemiological the causes of cancer, a systematic study is needed for in and laboratory studies related to the chemopreventive ac itiators (genotoxic factors) leading to an abnormal genome tivities of all citrus phytochemicals. However, through and for promoters (epigenetic factors) involved in the select examples of specific phytochemicals, we intend to growth and development of abnormal neoplastic cells and demonstrate the beneficial role of citrus in cancer their further progression to malignancy and metastasis. chemoprevention. Vitamin A. There has been a growing accumulation of evidence that indicates an inverse relationship between risk Diet and Cancer of cancer and the consumption of foods that contain vita A large body of information has accumulated within min A or its precursors (carotenoids in citrus and green the past two decades that strongly suggests individuals who and yellow vegetables; National Research Council, 1982). regularly consume higher amounts of fruits and vegetables Beta carotene is a strong antioxidant, and high dietary in have a lower risk of developing diverse types of cancer takes can prevent cancers arising from oxygen-free radi (National Research Council, 1982). Because of these find cals that damage DNA. Additionally, beta carotene may ings, the National Academy of Sciences, the American prevent cancer because of the way the body converts it into Cancer Society and the National Cancer Institute have pro the potent agent, retinoic acid. Retinoic acid is a weak anti posed many modifications to our diets. One of the most oxidant but is effective in treating tumors caused by agents important is that individuals increase their consumption of that do not form oxygen radicals, such as cancers of the fresh fruits and vegetables, especially citrus fruits and yel blood and bladder. Wang (1992) has shown that both beta low/green vegetables. carotene and retinoids have an inhibitory effect on cancer Fruits and vegetables contain naturally occurring com of the mouth, lung, bladder, and breast. pounds called "phytochemicals." Extensive studies have re Citrus does not synthesize vitamin A but produces pre vealed that about 14 classes (Table 1) of these plant chem cursors that can be metabolized into vitamin A by animals icals possess the ability to modulate specific processes of and humans. The most common forms of vitamin A pre oncogenesis and/or carcinogenesis. Chemopreventive cursors (termed provitamin A) in citrus are a- and 0- phytochemicals can inhibit the formation of a carcinogen carotenes, p-cryptoxanthin and p-apo-8'-carotenal. These and/or can function by blocking the promotion process vitamin A precursors are cleaved to form vitamin A al (Figure 1). The biochemical activities ascribed to these dehyde in the human intestine by p-carotene 15,15'- phytochemicals are diverse. These include: (1) direct deto oxygenase. Thereafter, aldehyde reductase reduces this al xification of xenobiotics, (2) protection of cells by scaveng dehyde to the all trans vitamin A. ing free radical forms of carcinogens, (3) inhibition or Stewart (1980) tabulated the provitamin A contents of modulation of enzyme systems (microsomal cytochrome P- several types of citrus juices (Table 2). Orange juices (Ham- 450 mono-oxygenase) involved in carcinogenic activation, lin, Pineapple, Valencia) were found to have the least (4) stimulation of enzyme systems (glutathione 5-trans- amount of provitamin A, whereas Murcott (orange- ferase) involved in detoxification, (5) mudulation of en- tangerine hybrid) contained the highest.

Proc. Fla. State Hort. Soc. 105: 1992. 163 Table 2. Provitamin A content of citrus juice. Phenols, Phenolic Acids and Their Conjugates. Plant phenols, phenolic acids and their conjugates are wide Juice VitaminA(I.U.)z RDA percentagey spread in the human diet. An average daily dietary intake value may be as high as 1 gram. Early studies by Watten- Hamlin 80 1.6 Pineapple 133 2.7 berg (1978, 1979) showed that the synthetic phenol, buty- Valencia 83 1.7 lated hydroxyanisole, reduced the incidence of neoplasia Robinson 1142 23.0 induced by several types of carcinogens in laboratory rats Dancy 965 19.0 and mice. From these interesting findings, Wattenberg and Orlando 236 4.7 Murcott 3195 64.0 co-workers (1980) expanded their studies to show similar effects with natural plant phenolics - caffeic, ferulic and 'Calculations based on p-carotene equal to 1.667 International Units vita p-coumaric acids (all of these acids, including sinapic, are min A/ug, equals 100%; a-carotene, 52.7%; 0-cryptoxanthin, 57%. found widespread in citrus fruits). Unfortunately, Watten yValues based on 6 oz. juice and calculated to a daily dietary allowance of 5000 I.U. berg and colleagues (1980) offered no specific explana tions as to the tumor-inhibiting activities of these phenolic acids. In grapefruit juice, there is a wide difference between Other investigations (Newmark and Mergens, 1981) the provitamin A contents of white varieties (Duncan, showed phenolic acids to be highly effective consumers of Marsh, Walters) and the pigmented varieties (Ruby Red, nitrite ions, especially at acid pHs. Phenolic acids prevent Flame, Ray Ruby, Star Ruby, Thompson). Ting and De- nitrosation of susceptible secondary amines and amides to szyck (1958) reported red and pink grapefruit juices to potent carcinogenic nitrosamines and nitrosamides. contain about 1667 to 2334 I.U. of vitamin A per 100 g Phenolic acids have also been shown to be effective as anti- juice. Recent results by Rouseff et al. (1992) showed (i- mutagens, especially against aromatic carcinogens, and to carotene contents of the edible portion (juice vesicles, seg possess moderate to strong activities as inhibitors of neop ment membranes, juice) of pigmented grapefruit to con lasia development (Newmark, 1992). Plant phenolics can tain: Ruby Red (4.2 |xg/g), Ray Ruby (7.0 fxg/g), Flame (8.6 function as modulators, particularly as inhibitors of the jxg/g) and Star Ruby (9.6 fig/g). These values approximate lipoxygenase pathways of arachidonic acid metabolism and 700 to 1600 I.U. of vitamin A per 100 g of edible juice and as cyclo-oxygenase inhibitors (Newmark, 1992; see Figure tissue. 1). Citrus fruits providing meaningful amounts of provita Phenolic acids (Figure 2) and their bound forms are min A components include tangerines and their hybrids, found in most citrus fruit parts. Most recent studies on and red and pink grapefruit. Oranges, lemons and limes citrus phenolics were concerned with their roles as precur provide inconsequential amounts of provitamin A (Gross, sors to a variety of vinyl phenols which contribute desirable 1987). or objectional aroma to citrus products (Rouseff et al., Vitamin C. L-Ascorbic acid (vitamin C) has been exten 1992; Nairn et al., 1992). sively studied for its therapeutic effects on cancer under a The contents of both free and bound forms of hydroxy- diverse set of conditions. One of the principal biochemical cinnamic acids (HCA) in oranges are listed in Table 3. As reactions of vitamin C is to destroy toxic free radicals (hy- noted, most HCA's are found in bound forms. Highest droxyl and perhydroxyl) resulting from metabolic prod concentrations were noted for the peel (flavedo and al ucts of oxygen. Vitamin C has also proved effective in pre bedo); the endocarp and juice sacs exhibited lower concen- venting the reaction of nitrites with amines and amides to form potent carcinogenic nitroso compounds (Mirvish et al., 1975). As an antioxidant, vitamin C may prevent cancer by COOH preventing oxidative damage that could lead to the initia tion and/or promotion phases of cancer. Additionally, it may induce enzyme systems involved in the detoxification OMe of carcinogens. What is established is that consumption of foods rich in vitamin C results in a lower risk for specific CAFFEIC ACID FERULIC ACID cancers, particularly gastric and esophageal cancers (Na tional Research Council, 1982). Citrus fruits and their juices are rich sources of vitamin C (Nagy, 1980). Recently compiled nutrient data on Florida citrus juices by Fellers et al. (1991) showed the following ranges of vitamin C in the following product types: reconstituted frozen concentrated orange juice (38- COOH COOH 47 mg/100 ml), orange juice from concentrate (35-44 mg/ 100 ml), pasteurized orange juice (35-47 mg/100 ml), H grapefruit juice from concentrate (30-37 mg/100 ml) and OMe grapefruit juice (30-38 mg/100 ml). Florida orange juice at a 6 fl-oz serving exceeded 100% of the U.S. RDA for vita p-COUMARIC ACID SINAPIC ACID min C, whereas grapefruit juice provided about 90% of the U.S. RDA. As a major source of vitamin C, citrus fruits and their juices play an important role in human nutrition Figure 2. Phenolic acids of the C6-C3 configuration found in all tissues and cancer chemoprevention. of citrus fruit.

164 Proc. Fla. State Hort. Soc. 105: 1992. Table 3. Content (mg/kg) of hydroxycinnamic acids (bound and free) in Table 4. Select list of flavonoids exhibiting anticarcinogenic activity. oranges.7 Flavonoids Anticancer activity Reference Sinapic Ferulic Coumaric Caffeic Fruit part Bound Free Bound Free Bound Free Bound Free , Protects cultured rat liver epithelial- (1) Tangeretin like cells against alfatoxin (5-induced Peel 95.1 5.4 178.4 3.2 76.7 0.5 7.3 0.2 cytotoxicity 3.5 0.0 Albedo 46.2 0.1 27.2 0.5 5.1 0.0 Nobiletin, Induces aryl hydrocarbon hydroxylase (2) 3.8 0.2 Flavedo 48.9 5.3 151.8 2.2 71.6 0.5 Tangeretin activity Juice sacs 8.6 0.1 28.0 0.1 5.3 0.0 3.1 0.0 Tangeretin Inhibits the invasion of malignant (3) Endocarp 10.8 0.1 21.3 0.1 4.4 0.0 1.8 0.0 mouse tumor cells into normal tissue fragments (anti-invasive activity) Source: Peleg et al. (1991). zFruits harvested randomly (mid season) from various sections of 4 trees Quercetin Inhibits growth of squamous cell (4) to provide 1 kg material. Values derived from HPLC analyses (300 nm). carinoma Flavedo values were calculated. Quercetin Inhibits growth of human malignant (5) cells from gastro-intestinal tract (6) trations. In most cases, the hydroxycinnamic acid contents Rutin, Inhibits mutagenic activity of diol Quercetin epoxide tetrahydrobenzo pyrene were in the following order: ferulic > sinapic > coumaric > caffeic. (1) Schwartz and Rate (1979); (2) Wattenberg (1975); (3) Bracke et al. All bound forms of phenolic acids in citrus are not (1989); (4) Middleton (1989); (5) Yoshida (1990); (6) Huang and Ferraro known. Generally in plants, phenolic acids are found con (1992). jugated to organic acids, sugars, amino compounds, lipids, terpenoids and other phenolics. In citrus, we have detected An extensive coverage of citrus and plant flavonoids at least five bound forms of ferulic acid, namely, feruloylg- exerting anticancer properties has been recently reviewed lucose, feruloylputrescine, feruloylglucaric acid, feruloyl- by Attaway (1991). Table 4 lists some important studies on galactaric acid and diferuloylglucaric acid (Nairn et al., select flavonoids found in citrus that proved effective in 1992). Undoubtedly, other citrus phenolic acids are pres deactivating carcinogens, modulating metabolic processes, ent in similar conjugated forms. The efficacy of these preventing tumor invasiveness and retarding the metastic bound phenolic acids as anticarcinogenic agents has not ability of a tumor cell population. been explored. Conjugated phenolics should be readily In a study of four flavonoids, nobiletin, tangeretin, cleaved by intestinal enzyme systems, but some forms may quercetin and tapifolin, Middleton (unpublished results) be absorbed intact through the intestinal wall and into the noted that nobiletin and tangeretin (Figure 3) were more circulatory system. Citrus fruits may eventually play an im effective in retarding growth of a human squamous car portant role as a dietary source for the phenolics and their cinoma cell line than the other two flavonoids. The differ conjugates in cancer chemoprevention. ence in anticancer activity of these flavonoids may be due Flavonoids. Flavonoids are C15 compounds arranged in to the relatively greater uptake of the more methoxylated a C6-C3-C6 configuration and are widely distributed in flavonoids (nobiletin and tangeretin) by the cell rather than plants. Three types of flavonoids occur in Citrus, namely, tile more hydroxylated flavonoids (quercetin and taxifo- flavanones (including 3-hydroxyflavanones), (in lin). Figure 4 depicts the structure of an important hydro cluding 3-hydroxyflavones) and anthocyanins (Horowitz xylated found in Citrus, namely quercetin, and and Gentili, 1977). In citrus, flavanones and flavones may its common rutinoside conjugate, rutin. occur as O-glycosides, C-glycosylflavones and aglycones. Well over 60 different types of flavonoids have been Flavanones are the most abundant and occur predomi detected in citrus fruits. Because of the complexity of these nately in the bound form. Only two flavanones, citromitin phytochemicals, quantitative distribution patterns have not and 5-O-desmethylcitrimitin, have been detected in the been undertaken. However, semiquantitative methods (see nonbound or aglycone form. Many polymethoxylated Ting and Rouseff (1986) for a listing of methods) have flavones occur as aglycones and possess beneficial been developed to obtain an estimate of the flavanone therapeutic properties (Robbins, 1980). Since the pioneer glycoside contents of orange and grapefruit juices. Hes- ing studies of Szent-Gyorgyi (1936, 1938) indicated that peridin is the principal flavonoid in orange juice, whereas citrus flavonoids possessed vitamin-like activity, many sci naringin is the dominant flavonoid in grapefruit juice. The entific studies have been conducted relating these biof- range of flavanone glycoside contents of Florida juices as lavonoids to anticancer, antiviral, antiinflammatory and determined by Carter and co-workers (1975) were: Hamlin antiallergic activities, and the ability to inhibit human juice (69-113 mg/100 ml); Pineapple juice (72-106 mg/100 platelet aggregation. ml) and Valencia juice (53-88 mg/100 ml). These values Plant flavonoids have been found to inhibit tumor de agree with the orange juice flavanone glycoside content velopment in several animal studies and to act by different estimated by Beilig et al. (1985) for German RSK values, mechanisms. Hydroxylated flavonoids have been found to namely, 50-100 mg/100 ml juice. An 8-fl oz. serving of (1) inhibit the metabolic activation of carcinogens by mod orange juice should provide about 100-200 mg of a diverse ulation of cytochrome P-450 enzymes, (2) inactivate ulti mixture of flavonoids. Citrus fruits are one of the richest mate carcinogens, (3) inhibit generation of active species sources of flavonoids bestowing chemopreventive activities and act as scavengers of active oxygen species, (4) inhibit against cancer. arachidonic acid metabolism, (5) inhibit protein kinase C Limonoids. Limonoids are a group of chemically related and other kinase activities involved in cellular prolifera triterpene derivatives found in the Rutaceae and Meliaceae tion, and (6) reduce the bioavailability of carcinogens families. The best known compound in this class of (Huang and Ferraro, 1992). phytochemicals is limonin (Figure 5), and bitterness in cit-

Proc. Fla. State Hort. Soc. 105: 1992. 165 OCH3 Table 5. Limonoids in citrus and its hybrids.

Neutral limonoids

1. Limonin 2. Nomilin — OCH 3 3. Obacunone 4. Deacetylnomilin 5. Ichangin 6. Deoxylimonin 7. Deoxylimonol 8. 7 a-Limonol 9. Limonyl acetate 10. 7a-Obacunol CH3O 11.7 a-Obacunyl acetate 12. Ichangensin 13. Citrusin 14. Calamin 15. Retrocalamin 16. Cyclocalamin OCH3 0 17. Methyl deacetylnomilinate 18. Isocyclocalamin 19. 6-keto-7p-Deacetylnomilol 20. 6-keto-7p-Nomilol 21. Methyl isoobacunoate diosphenol 22. l-(10-19) Abeo-obacun-9(l l)-en-7a-yl acetate Tangeretin 23. 1 -(10-19) Abeo-7a-acetoxy-1 Op-hyddroxyisoobacunoic acid 3,10-lactone Acidic limonoids 1. Deacetylnomilinate 2. Nomilinate 3. Isoobacunoate 4. Epiisoobacunoate 5. trans-19-Hydroxyobacunoate 6. Isolimonate OCH OCH 3 7. Limonoate A-ring lactone 8. Deoxylimonate 9. 17-Dehydrolimonoate A-ring lactone 10. Calaminate 11. Retrocalaminate 12. Isoobacunoate 13. Obacunoate diosphenol — OCH 3 14. 19-Hydroxydeacetylnomilinate 15. Cyclocalaminate

Source: Hasegawa et al. (1992). CH3O Recently, limonoids have been found to exert anticar- cinogenic activity in laboratory animals (Lam et al., 1989; OCH 3 0 Lam and Hasegawa, 1989; Miller et al., 1992). Eight limonoids were tested by Lam and coworkers (1989), nomi lin, limonin, deacetylnomilin, limonol, obacunone, Nobiletin deoxylimonin, isoobacunoic acid and ichangin; all were found to stimulate the detoxifying enzyme, glutathione S- Figure 3. Two important methoxylated flavonoids possessing anti- transferase (GST). GST enzymes are one of the major en cancer activity. zyme systems responsible for the detoxification of xenobio- tics; additionally, they catalyze the adduct formation of rus juice is primarily attributed to this compound (Maier glutathione with electrophiles, including reactive car et al., 1977). Thirty-eight limonoid aglycones - 23 neutral cinogenic species, to water-soluble substances that are ex and 15 acidic - have been isolated from Citrus and its hyb creted from the body (Chasseaud, 1979). Substances that rids (Table 5). Additionally, citrus tissues and juices con can elicit increased activity of GST may be potential anti- tain very high concentrations of limonoid glucosides, of which 17 have been isolated (Hasegawa et al., 1989; Ben nett et al., 1989). All limonoid glucosides isolated to date contain one D-glucose molecule attached via a P-glucosidic linkage to the C-17 position of the aglycone (see Figure 5; limonin 17-P-D-glucopyranoside).

Limonoate A-ring lactone Oh Limonin OH 0

Quercetin

0-3-D-glucose

'O-Rutinose OH 0

Rutin (Rutinose=6—0—CX—L—rhamnosyl—D—glucose) Limonin 17- jB-D-glucopyranos'^de

Figure 4. An important hydroxylated flavonoid and its rutinoside de Figure 5. Three forms of a limonoid, as depicted by limonin, found rivative with important therapeutic properties. in Citrus.

166 Proc. Fla. State Hort. Soc. 105: 1992. carcinogens in the inhibition of chemically induced cancer finally, to an ultimate cascading effect of the tumorous formation. cells. The furan moiety attached to the D-ring of limonoids In the prevention of cancer, the importance of diet is was most likely responsible for induction of GST activity now well established through many epidemiological (Hasegawa et al., 1992). Previous studies by Lam et al. studies. Of the many foods that have been tested by the (1982) with kahweol and cafestol suggested that the furan National Cancer Institute, citrus fruits and juices have moieties of those molecules were responsible for inducing shown very positive results as a chemopreventive food GST activity in various tissues of mice. Nomilin was shown (Pierson, 1992). The phytochemicals present in Citrus pos by Lam et al. (1989) to be the most potent inducer of GST sessing anticancer properties are currently being evaluated activity in the liver and in the small intestinal mucosa. In by several private, university and governmental vivo tumor protection experiments confirmed that nomilin laboratories. Citrus has always been considered a nutritious is an inhibitor of benzo (a) pyrene-induced neoplasia in food. One day it may be considered as a modern age the forestomach of mice (Lam and Hasegawa, 1989). Limo- medicinal food. nin, which has a structure different from nomilin by the Literature Cited presence of A and A' rings, was not as effective in inducing Attaway, J. A. 1991. Medical benefits of juice flavonoids. pp. 207-219. GST as nomilin. Lam and coworkers (1989) suggested that International Congress of Fruit Juice. Sao Paulo, Brazil. the A- and A'-ring moieties of limonoids also appear to Beilig, H. J., W. Faethe, J. Koch, S. Walbrauch, and K. Wucherpfennig. play a role in the induction of GST activity (see Figure 5). 1985. Standard values and ranges of specific reference numbers for Additional research on limonoids by Miller et al. (1992) orange juice and grapefruit juice. Confructa 29:191-207. showed that limonin 17-P-D-glucopyranoside inhibited the Bennett, R. D., S. Hasegawa, and Z. Herman. 1989. Glucosides of acidic limonoids in Citrus. Phytochemistry 28:2777-2781. development of oral tumors in hamsters. Interestingly, this Bishop, J. M. 1987. The molecular genetics of cancer. Science 235:305- glucoside had no effect on GSH activity of oral epithelial 311. cells. This was the first reported study noting an anticancer Bracke, M. E., et. al. 1989. The flavonoid tangeretin inhibits invasion of activity for a conjugated limonoid. The water solubility of MO4 mouse cells into embryonic chick heart in vitro. Clin. Exp. Metas tasis 7:283. these glucosidic conjugates may be of considerable impor Caragay, A. B. 1992. Cancer-preventive foods and ingredients. Food tance when considering the method of intake by humans. Technol. 65-68. Questions remain as to whether these glucosides are ab Carter, R. D., B. S. Buslig, and J. A. Cornell. 1975. Statistical inferences sorbed intact through the intestine or whether they are on processed orange juice quality from the 1972-73 and 1973-74 juice definition program. Proc. Fla. State Hort. Soc. 88:358-370. cleaved by intestinal flora prior to absorption. Chasseaud, L. F. 1979. The role of glutathione and glutathione S-trans- Commercial citrus juices contain low levels (about 2-8 ferases in the metabolism of chemical carcinogens and other elec- ppm) of free, nonconjugated limonoids but high levels of trophilic agents. Adv. Cancer Res. 29:175-274. limonoid glucosides. Table 6 shows the contents of the Fellers, P. J., S. Nikdel, and H. S. Lee. 1991. Five-year data base for the major limonoid glucoside (limonin 17-P-D- content of eleven essential vilamins and minerals for nutrition labeling in the major processed Florida citrus juice products. Proc. Fla. State glucopyranoside) and the total limonoid glucosides in Hort. Soc. 104:84-94. orange, grapefruit and lemon juices (Fong et al., 1989). Fong, C. H., S. Hasegawa, Z. Herman, and P. Ou. 1989. Limonoid The large quantity of limonoid glucosides in Citrus high glucosides in commerical citrus juices. J. Food Sci. 54:1505-1506. lights these juices as important sources for chemopreven- Gross, J. 1987. Pigments in Fruits. Academic Press, Orlando, FL. Hasegawa, S., R. D. Bennett, Z. Herman, C. H. Fong, and P. Ou. 1989. tive limonoids. Limonoid glucosides in Citrus. Phytochemistry 28:1717-1720. Hasegawa, S., M. Miyake, and Y. Ozaki. 1992. Biochemistry of limonoids, Conclusion anticarcinogens, in Citrus. National Meeting of the American Chemical Society, New York, Abst. 182. Cancer is the result of the interplay of many variables. Horowitz, R. M. and B. Gentili. 1977. Flavonoid Constituents of Citrus, The sequence of events leading to tumor formation and pp. 397-426. In: S. Nagy, P. E. Shaw, and M. K. Veldhuis (eds.). Citrus Science and Technology, Vol. 1. AVI Publishing Co., Westport, CT. the resulting cascading effects of the metastases have not Huang, M. T. and T. Ferraro. 1992. Phenolic compounds in food and been completely elucidated. What has been charted are cancer prevention, pp. 8-34. In: M. T. Huang, C. T. Ho and C. Y. various steps beginning with an insult to genetic cellular Lee (eds.). Phenolic Compounds in Food and Their Effects on Health. material (genotoxic event; known also as "initiation") and II. American Chemical Society, Washington, D.C. then to production of abnormal DNA (nongenotoxic Lam, L. K. T. and S. Hasegawa. 1989. Inhibition of benzo (a) pyrene-in duced forestomach neoplasia by citrus limonoids in mice. Nutr. event; known also as "promotion"). The transformation of Cancer. 12:43-47. the altered cell may lead to the proliferation of cells with Lam, L. K. T., Y. Li, and S. Hasegawa. 1989. Effects of citrus limonoids invasive (malignant) or noninvasive (benign) qualities. on glutathione S-transferase activity in mice. J. Agric. Food Chem. From a confined region of tissue, cancer spreads to other 37:878-880. tissues (developing secondary tumors or metastases), and Lam, L. K. T., V. L. Sparnins, and L. W. Wattenberg. 1982. Isolation and identification of kahweol palmitate and cafestal palmitate as active constituents of green coffee bean that enhance glutathione S-trans ferase activity. Cancer Res. 42:1193-1198. Table 6. Concentrations of limonin 17-p-D-glucopyranoside (LG) and Maier, V. P., R. D. Bennett, and S. Hasegawa. 1977. Limonin and other total limonoid glucosides in commercial citrus juices. limonoids. pp. 355-396. In: S. Nagy, P. E. Shaw, and M. K. Veldhuis (eds.). 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Proc. Fla. State Hort. Soc. 105: 1992. 167 Mirvish, S. S., A. Cardesa, L. Wallcave, and P. Shibik. 1975. Induction of Rusznyak, S. and A. Szent-Gyorgyi. 1936. Vitamin P: flavonols as vita mouse lung adenomas by amines or ureas plus nitrite and by N-nitroso mins. Nature. 138:27-37. compounds. Effect of ascorbate, gallic acid, thiocyanate and caffeine. Schwartz, A. G. and W. R. Rate. 1979. Inhibition of aflatoxin J$l-induced J. Nad. Cancer Inst. 55:633-636. cytotoxicity and binding to DNA in cultured rat liver cells by naturally Nagy, S. 1980. Vitamin C contents of citrus fruit and their products. J. occurring flavones. J. Environ. Path. Toxicol. 2:1021-1028. Agric. Food Chem. 28:8-18. Stewart, I. 1980. Color as related to citrus quality, pp. 127-149. In: S. Nairn, ML, U. Zehavi, S. Nagy, and R. L. Rouseff. 1992. Hydroxycinnamic Nagy and J. A. Attaway (eds.). Citrus Nutrition and Quality. American acids as off-flavor precursors in citrus fruits and their products, pp. Chemical Society, Washington, D.C. 180-191. C. T. Ho, C. Y. Lee, and M. T. Huang (eds.). Phenolic Com Szent-Gyorgyi, A. 1938. Method for determination of citrin. Hoppe- pounds in Food and Their Effects on Health I. American Chemical Seyler's Z. Physiol. Chem. 255:126-131. Society, Washington, D.C. Ting, S. V. and E. J. Deszyck. 1958. The internal color and carotenoid National Research Council. 1982. Diet, Nutrition, and Cancer. National pigments of Florida red and pink grapefruit. Proc. Am. Soc. Hortic. Academy Press, Washington, D.C. Sci. 71:271-277. Newmark, H. L. 1992. Plant phenolic compounds as inhibitors of Ting, S. V. and R. L. Rouseff. 1986. Citrus Fruits and Their Products: mutagenesis and carcinogenesis. pp. 48-52. In: M. T. Huang, C. T. Analysis and Technology. Marcel Dekker, New York. Ho and C. Y. Lee (eds.). Phenolic Compounds in Food and Their Wang, X. D. 1992. Anti-cancer effects of beta carotene linked to Effects on Health II. American Chemical Society, Washington, D.C. metabolism. Food Chem. News. October 19, pp. 48. Newmark, H. L. and W. J. Mergens. 1981. Inhibition of Tumor Induction Wattenberg, L. W. 1975. Effects of dietary constituents on the metabolism and Development, pp. 127-168. In: M. S. Zedeckand M. Lipkin (eds.). of chemical carcinogen. Cancer Res. 35:3326-3330. Plenum Press, New York. Wattenberg, L. W. 1978. Inhibitors of chemical carcinogenesis. Adv. Peleg, H., M. Nairn, R. L. Rouseff, and U. Zehavi. 1991. Distribution of Cancer Res. 26:197-226. bound and free phenolic acids in oranges and grapefruit. J. Sci. Food Wattenberg, L. W. 1979. Inhibitors of chemical carcinogens, pp. 241-263. Agric. 57:417-426. In: P. Emmelot and E. Kriek (eds.). Environmental Carcinogenesis. Pierson, H. 1992. Diet as a factor in cancer and cancer prevention. Cancer Elsevier, Amsterdam. Med., in press. Wattenberg, L. W., J. B. Coccia, and L. K. T. Lam. 1980. Inhibitory Robbins, R. C. 1980. Medical and nutritional aspects of citrus biof- effects of phenolic compounds on benzo (a) pyrene-induced neoplasia. lavonoids. pp. 43-59. In: S. Nagy and J. A. Attaway (eds.). Citrus Cancer Res. 40:2820-2823. Nutrition and Quality. American Chemical Society, Washington, D.C. Weisburger, J. H. 1992. Mutagenic, carcinogenic, and chemopreventive Rouseff, R. L., S. Nagy, M. Nairn, and U. Zehavi. 1992. Off-flavor devel effects of phenols and catechols. pp. 35-47. In: M. T. Huang, C. T. opment in citrus juice products, pp. 211-227. In: G. Charalambous Ho, and C. Y. Lee (eds.). Phenolic Compounds in Food and Their (ed.). Off-Flavors in Foods and Beverages. Elsevier, New York. Effects on Health. II. American Chemical Society, Washington, D.C. Rouseff, R. L., G. D. Sadler, T. J. Putnam, andj. E. Davis. 1992. Deter Yoshida, M. 1990. The effect of quercetin on cell cycle progression and mination of (3-carotene and other hydrocarbon carotenoids in red growth of human gastric cancer cells. Fed. European Biochem. Soc. grapefruit cultivars. J. Agric. Food Chem. 40:47-51. 260:10-13.

Proc. Fla. State Hort. Soc. 105:168-173. 1992.

EFFECT OF GIBBERELLIC ACID AND POSTHARVEST STORAGE ON QUALITY OF FLORIDA NAVEL ORANGES

Mohamed A. Ismail and Diana L. Wilhite1 of October were more effective in delaying peel color develop Florida Department of Citrus ment than applications made in November or December. Scientific Research Department Citrus Research and Education Center Navel orange is one of the world's premier citrus vari Lake Alfred, FL 33850 eties. It is popular for its distinct flavor, seedlessness and ease of peeling. In Florida, navel orange is treasured by Gift Fruit Shippers as one of the most popular fruit in gift Additional index words, senescence, color, firmness, 2,4-D, packages shipped to all parts of the U.S. and Canada. Or peel. dinarily, the season for Florida navels starts in mid November and ends in late December or mid January. Abstract. Gibberellic acid (GA) and 2,4-dichlorophenoxyacetic The growth regulators gibberellic acid (GA) and 2,4- acid (2,4-D) were applied to 10 to 25-year old navel orange dichlorophenoxyacetic acid (2,4-D) are widely used on cit trees in June, October, November or December to evaluate rus in many parts of the world to retard peel senescence their effect on peel color and fruit quality in later than normal and reduce fruit drop, respectively. Bevington (1973) re harvests. GA significantly delayed peel color development for ported that Washington navel oranges treated with GA two months beyond normal harvest time. It did not, however, were less susceptible to rind injury, water spots, puffing affect total soluble solids, % citric acid or internal segment and creasing, and decay caused by green mold. El-Otmani, drying. GA was marginally effective in maintaining peel M'Barek and Coggins (1990) tested various citrus cultivars firmness as measured by resistance to puncture. Storage at and demonstrated GA and 2,4-D could be used to reduce 38° or 40°F also extended fruit shelf life by an additional fruit drop and delay rind softening. While preharvest ap month. Application of the growth regulators during the month plication of GA can reduce aging, creasing and rind soften ing, it does not influence the juice quality as demonstrated by Coggins and Henning (1988), Kokkalos (1981), Beving 'The authors wish to thank Ellen C. Wheeler and Eric C. Voigt for their assistance in conducting field and laboratory work. ton (1973) and Gilfillan et al. (1981). When GA is applied

168 Proc. Fla. State Hort. Soc. 105: 1992.