Conclusions

Citrus juice fermentation induced the formation of sig

nificant amounts of OS. However, their composition was

found to be significantly different from that of OS gener

ated by acid oligomerization. Furthermore, the formation

of ethanol and meso-inositol should distinguish between

fermentation OS and those due to the addition of medium

invert sugar. Nevertheless, care should be taken to assess

the freshness of a juice when quantitating beet medium

invert sugar in citrus juice.

Literature Cited

Cancalon, P. F. 1992a. Oligosaccharides generation in acidic sugar media.

J. Off. Anal. Chem. (in press).

Cancalon, P. F. 1992b. Production of oligosaccharides during sucrose

inversion. 106th AOAC Int. Annu. Meeting.

Cancalon, P. F. and C. R. Bryan. 1991. Quantitation of beet sugar in

citrus juice. 42nd Annu. Citrus Proc. Meeting, Lake Alfred, FL., pp. 40-44.

Echeverria, E. 1990. Developmental transition from emzymatic to acid

hydrolysis of sucrose in acid limes. Plant Physiol. 92, 168-171.

Faville, L. W., E. C. Hill, and E. C. Parish. 1951. Survival of microor

ganisms in concentrated orange juice. Food Technology 5, 33-36.

Faville, L. W. and E. C. Hill. 1951. Incidence and significance of microor

ganisms in citrus juices. Food Technology 5, 423-425.

Hassid, W. Z. and C. E. Ballou. 1957. Oligosaccharides. In: Pigman,

W.(Ed.) The Carbohydrates, Chemistry, Biochemistry, Physiology. Academic Press, New York. pp. 487-533.

McAllister, J. W. 1980. Methods for determining the quality of citrus

juices. In: Nagy, S. and Attaway, ]. A. (eds.) ACS Symposium series 143, Washington, D.C. pp. 291-31*7.

Swallow, K. W., N. H. Low, and D. R. Petrus. 1991. Detection of orange

juice adulteration with beet medium invert sugar using anion-ex-

change liquid chromatography with puse amperometric detection. J. Off. Anal. Chem. 74, 341-345.

White D. R. and P. F. Cancalon. 1992. Detection of beet sugar adultera

tion of orange juice by liquid chromatography amperometric detec

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

Florida Department of Citrus,

Scientific Research Department,

Citrus Research and Education Center

700 Experiment Station Road

Lake Alfred, FL 33850

Additional index words. Flavonoids, limonoids, phenolic

acids, vitamin C, vitamin A.

Abstract. Dietary components present in citrus juices have

been shown to exert protective effects against the induction

and spread of cancer in animals and humans. The components

with the most potent anticarcinogenic activities are mainly

naturally occurring secondary metabolites and include

flavonoids, limonoids, phenolic acids and vitamins. These

phytochemicals react by different mechanisms, namely, by

maintaining cellular oxidation-reduction balance and protect

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

phytochemicals with known anticarcinogenic activities and

various aspects of cancer and anticancer mechanisms are

noted in this review.

Cancer

Initiation

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

genotoxic carcinogens have been identified. Carcinogens

may be of a chemical or viral nature, or may be induced

by radiation (as occurs primarily in skin cancer).

Florida Agricultural Experiment Station Journal Series No. N-00736.

From this initial damage, mutation, duplication or

translocation of normal cellular genes involved in growth

control may portend the development of cancer. By one

mechanism or another, damage to diverse proto-on-

cogenes (normal cellular genes that may become cancer

producing) has been implicated in the genesis of human

tumors. Many oncogenes encode proteins with key roles in

controlling normal growth and development.

Promotion

While a genotoxic carcinogen can initiate or alter the

genetic material of a cell, this event is only the first step in

an elaborate sequence of events leading to neoplastic

growth. Cancers are populations of cells that have acquired

the ability to multiply and spread without normal re

straints. An abnormal cell population needs to achieve a

selective growth advantage in the presence of surrounding

normal cells that are regulated by growth-controlling fac-

Oxidative Damage

Carcinogens

Fibers, phytosterols, terpenes,

sulfides, phenollc8, lignans,

triterpenoids, isoflavones,

cruciferous indolea

Steroid Hormones

Phenolics, solicylates,

flavonoids, polyacetylenes,

sulfides

162

Prostaglandin

(PGS)

Figure 1. The effects of phytochemicals on metabolic pathways as sociated with breast cancer (Pierson, 1992).

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

tors through intercellular communication. The intercellu

lar signals that control tissue development and regenera

tion have not been identified, but there are modulating

factors or "promoters" that function by inhibiting this in

tercellular communication. Cell replication depends on en

dogenous and exogenous controlling elements operating

by epigenetic mechanisms which either enhance or retard

the process (Weisburger, 1992).

As a classic example, investigations of breast cancer

have identified oxidative damage, action of steriod hor

mones and the action of certain kinds of prostaglandins as

promoters of neoplastic proliferation (Pierson, 1992; Fig

ure 1). Figure 1 not only demonstrates the sequence of

events in breast cancer, but also shows the influence of

dietary photochemicals (plant chemicals) on modulating

the metabolic pathways associated with this type of cancer.

Invasiveness and Metastasis

The factors responsible for the biochemical and

phenotypic aberrations of a mutated cell are not entirely

understood. An emerging mutated cell may produce a cell population which is benign (non-invasive; respects the

boundaries of other tissue cells) or may acquire the

capabilities for extended proliferation, invasion of adjacent

tissue and metastasis (formation of secondary and tertiary

tumors at different locations in the body).

Evidence (Bishop, 1987) exists for the presence of on-

cogenes which bestow the ability to metastasize on cells

already capable of abnormal proliferation. In exploring

the causes of cancer, a systematic study is needed for in

itiators (genotoxic factors) leading to an abnormal genome

and for promoters (epigenetic factors) involved in the

growth and development of abnormal neoplastic cells and

their further progression to malignancy and metastasis.

Diet and Cancer

A large body of information has accumulated within

the past two decades that strongly suggests individuals who

regularly consume higher amounts of fruits and vegetables

have a lower risk of developing diverse types of cancer

(National Research Council, 1982). Because of these find

ings, the National Academy of Sciences, the American

Cancer Society and the National Cancer Institute have pro

posed many modifications to our diets. One of the most

important is that individuals increase their consumption of

fresh fruits and vegetables, especially citrus fruits and yel

low/green vegetables.

Fruits and vegetables contain naturally occurring com

pounds called "phytochemicals." Extensive studies have re

vealed that about 14 classes (Table 1) of these plant chem

icals possess the ability to modulate specific processes of

oncogenesis and/or carcinogenesis. Chemopreventive phytochemicals can inhibit the formation of a carcinogen

and/or can function by blocking the promotion process

(Figure 1). The biochemical activities ascribed to these

phytochemicals are diverse. These include: (1) direct deto

xification of xenobiotics, (2) protection of cells by scaveng

ing free radical forms of carcinogens, (3) inhibition or

modulation of enzyme systems (microsomal cytochrome P-

450 mono-oxygenase) involved in carcinogenic activation,

(4) stimulation of enzyme systems (glutathione 5-trans-

ferase) involved in detoxification, (5) mudulation of en-

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

Table 1. Fourteen classes of phytochemicals with known anticancer prop

erties and presence in citrus.

Phytochemical group Present in citrus

Carotenoids

Coumarins

Flavonoids

Glucarates

Indoles

Isothiocyanates

Lignans

Monoterpenes

Phenolic acids

Phthalides

Phytates

Polyacetylenes

Sulfides

zyme systems (protein kinase C) involved in abnormal cel

lular proliferation, (6) control of membrane permeability,

and (7) unknown activities, as for example, the modulation

of cellular signals.

Citrus Phytochemicals

Demonstrating Anticarcinogenic Activities

Table 1 lists seven classes of citrus compounds designa

ted by the National Cancer Institute as exhibiting anticar

cinogenic properties (Caragay, 1992). It is beyond the

scope of this paper to discuss the extensive epidemiological

and laboratory studies related to the chemopreventive ac

tivities of all citrus phytochemicals. However, through

select examples of specific phytochemicals, we intend to

demonstrate the beneficial role of citrus in cancer

chemoprevention.

Vitamin A. There has been a growing accumulation of

evidence that indicates an inverse relationship between risk

of cancer and the consumption of foods that contain vita

min A or its precursors (carotenoids in citrus and green

and yellow vegetables; National Research Council, 1982).

Beta carotene is a strong antioxidant, and high dietary in

takes can prevent cancers arising from oxygen-free radi

cals that damage DNA. Additionally, beta carotene may

prevent cancer because of the way the body converts it into

the potent agent, retinoic acid. Retinoic acid is a weak anti

oxidant but is effective in treating tumors caused by agents

that do not form oxygen radicals, such as cancers of the

blood and bladder. Wang (1992) has shown that both beta

carotene and retinoids have an inhibitory effect on cancer

of the mouth, lung, bladder, and breast.

Citrus does not synthesize vitamin A but produces pre

cursors that can be metabolized into vitamin A by animals

and humans. The most common forms of vitamin A pre

cursors (termed provitamin A) in citrus are a- and 0-carotenes, p-cryptoxanthin and p-apo-8'-carotenal. These

vitamin A precursors are cleaved to form vitamin A al

dehyde in the human intestine by p-carotene 15,15'-

oxygenase. Thereafter, aldehyde reductase reduces this al

dehyde to the all trans vitamin A.

Stewart (1980) tabulated the provitamin A contents of

several types of citrus juices (Table 2). Orange juices (Ham-

lin, Pineapple, Valencia) were found to have the least

amount of provitamin A, whereas Murcott (orange-

tangerine hybrid) contained the highest.

163

Table 2. Provitamin A content of citrus juice.

Juice

Hamlin

Pineapple

Valencia

Robinson

Dancy

Orlando

Murcott

VitaminA(I.U.)z

80

133

83

1142

965

236

3195

RDA percentagey

1.6

2.7

1.7

23.0

19.0

4.7

64.0

'Calculations based on p-carotene equal to 1.667 International Units vita

min A/ug, equals 100%; a-carotene, 52.7%; 0-cryptoxanthin, 57%.

yValues based on 6 oz. juice and calculated to a daily dietary allowance

of 5000 I.U.

In grapefruit juice, there is a wide difference between

the provitamin A contents of white varieties (Duncan,

Marsh, Walters) and the pigmented varieties (Ruby Red,

Flame, Ray Ruby, Star Ruby, Thompson). Ting and De-

szyck (1958) reported red and pink grapefruit juices to

contain about 1667 to 2334 I.U. of vitamin A per 100 g

juice. Recent results by Rouseff et al. (1992) showed (i-

carotene contents of the edible portion (juice vesicles, seg

ment membranes, juice) of pigmented grapefruit to con

tain: Ruby Red (4.2 |xg/g), Ray Ruby (7.0 fxg/g), Flame (8.6

jxg/g) and Star Ruby (9.6 fig/g). These values approximate

700 to 1600 I.U. of vitamin A per 100 g of edible juice and

tissue.

Citrus fruits providing meaningful amounts of provita

min A components include tangerines and their hybrids,

and red and pink grapefruit. Oranges, lemons and limes

provide inconsequential amounts of provitamin A (Gross,

1987).

Vitamin C. L-Ascorbic acid (vitamin C) has been exten

sively studied for its therapeutic effects on cancer under a

diverse set of conditions. One of the principal biochemical

reactions of vitamin C is to destroy toxic free radicals (hy-

droxyl and perhydroxyl) resulting from metabolic prod

ucts of oxygen. Vitamin C has also proved effective in pre

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

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

of carcinogens. What is established is that consumption of

foods rich in vitamin C results in a lower risk for specific

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-

47 mg/100 ml), orange juice from concentrate (35-44 mg/

100 ml), pasteurized orange juice (35-47 mg/100 ml),

grapefruit juice from concentrate (30-37 mg/100 ml) and

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

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

and cancer chemoprevention.

Phenols, Phenolic Acids and Their Conjugates. Plant

phenols, phenolic acids and their conjugates are wide

spread in the human diet. An average daily dietary intake

value may be as high as 1 gram. Early studies by Watten-

berg (1978, 1979) showed that the synthetic phenol, buty-

lated hydroxyanisole, reduced the incidence of neoplasia

induced by several types of carcinogens in laboratory rats

and mice. From these interesting findings, Wattenberg and

co-workers (1980) expanded their studies to show similar

effects with natural plant phenolics - caffeic, ferulic and

p-coumaric acids (all of these acids, including sinapic, are

found widespread in citrus fruits). Unfortunately, Watten

berg and colleagues (1980) offered no specific explana

tions as to the tumor-inhibiting activities of these phenolic

acids.

Other investigations (Newmark and Mergens, 1981)

showed phenolic acids to be highly effective consumers of

nitrite ions, especially at acid pHs. Phenolic acids prevent

nitrosation of susceptible secondary amines and amides to

potent carcinogenic nitrosamines and nitrosamides.

Phenolic acids have also been shown to be effective as anti-

mutagens, especially against aromatic carcinogens, and to

possess moderate to strong activities as inhibitors of neop

lasia development (Newmark, 1992). Plant phenolics can

function as modulators, particularly as inhibitors of the

lipoxygenase pathways of arachidonic acid metabolism and

as cyclo-oxygenase inhibitors (Newmark, 1992; see Figure

1). Phenolic acids (Figure 2) and their bound forms are

found in most citrus fruit parts. Most recent studies on

citrus phenolics were concerned with their roles as precur

sors to a variety of vinyl phenols which contribute desirable

or objectional aroma to citrus products (Rouseff et al.,

1992; Nairn et al., 1992).

The contents of both free and bound forms of hydroxy-

cinnamic acids (HCA) in oranges are listed in Table 3. As

noted, most HCA's are found in bound forms. Highest

concentrations were noted for the peel (flavedo and al

bedo); the endocarp and juice sacs exhibited lower concen-

COOH

OMe

FERULIC ACID CAFFEIC ACID

COOH COOH

p-COUMARIC ACID

H

OMe

SINAPIC ACID

Figure 2. Phenolic acids of the C6-C3 configuration found in all tissues

of citrus fruit.

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

Table 3. Content (mg/kg) of hydroxycinnamic acids (bound and free) in

oranges.7

Table 4. Select list of flavonoids exhibiting anticarcinogenic activity.

Fruit part

Peel

Albedo

Flavedo

Juice sacs

Endocarp

Sinapic

Bound

95.1

46.2

48.9

8.6

10.8

Free

5.4

0.1

5.3

0.1

0.1

Ferulic

Bound

178.4

27.2

151.8

28.0

21.3

Free

3.2

0.5

2.2

0.1

0.1

Coumaric

Bound

76.7

5.1

71.6

5.3

4.4

Free

0.5

0.0

0.5

0.0

0.0

Caffeic

Bound

7.3

3.5

3.8

3.1

1.8

Free

0.2

0.0

0.2

0.0

0.0

Source: Peleg et al. (1991).

zFruits harvested randomly (mid season) from various sections of 4 trees

to provide 1 kg material. Values derived from HPLC analyses (300 nm).

Flavedo values were calculated.

trations. In most cases, the hydroxycinnamic acid contents

were in the following order: ferulic > sinapic > coumaric

> caffeic.

All bound forms of phenolic acids in citrus are not

known. Generally in plants, phenolic acids are found con

jugated to organic acids, sugars, amino compounds, lipids,

terpenoids and other phenolics. In citrus, we have detected

at least five bound forms of ferulic acid, namely, feruloylg-

lucose, feruloylputrescine, feruloylglucaric acid, feruloyl-

galactaric acid and diferuloylglucaric acid (Nairn et al.,

1992). Undoubtedly, other citrus phenolic acids are pres

ent in similar conjugated forms. The efficacy of these

bound phenolic acids as anticarcinogenic agents has not

been explored. Conjugated phenolics should be readily

cleaved by intestinal enzyme systems, but some forms may

be absorbed intact through the intestinal wall and into the

circulatory system. Citrus fruits may eventually play an im

portant role as a dietary source for the phenolics and their

conjugates in cancer chemoprevention.

Flavonoids. Flavonoids are C15 compounds arranged in

a C6-C3-C6 configuration and are widely distributed in

plants. Three types of flavonoids occur in Citrus, namely,

flavanones (including 3-hydroxyflavanones), flavones (in

cluding 3-hydroxyflavones) and anthocyanins (Horowitz

and Gentili, 1977). In citrus, flavanones and flavones may

occur as O-glycosides, C-glycosylflavones and aglycones.

Flavanones are the most abundant and occur predomi

nately in the bound form. Only two flavanones, citromitin

and 5-O-desmethylcitrimitin, have been detected in the

nonbound or aglycone form. Many polymethoxylated

flavones occur as aglycones and possess beneficial

therapeutic properties (Robbins, 1980). Since the pioneer

ing studies of Szent-Gyorgyi (1936, 1938) indicated that

citrus flavonoids possessed vitamin-like activity, many sci

entific studies have been conducted relating these biof-

lavonoids to anticancer, antiviral, antiinflammatory and

antiallergic activities, and the ability to inhibit human

platelet aggregation.

Plant flavonoids have been found to inhibit tumor de

velopment in several animal studies and to act by different

mechanisms. Hydroxylated flavonoids have been found to

(1) inhibit the metabolic activation of carcinogens by mod

ulation of cytochrome P-450 enzymes, (2) inactivate ulti

mate carcinogens, (3) inhibit generation of active species

and act as scavengers of active oxygen species, (4) inhibit

arachidonic acid metabolism, (5) inhibit protein kinase C

and other kinase activities involved in cellular prolifera

tion, and (6) reduce the bioavailability of carcinogens

(Huang and Ferraro, 1992).

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

Flavonoids

Nobiletin,

Tangeretin

Quercetin

Nobiletin,

Tangeretin

Tangeretin

Quercetin

Quercetin

Rutin,

Quercetin

Anticancer activity

Protects cultured rat liver epithelial-

like cells against alfatoxin (5-induced

cytotoxicity

Induces aryl hydrocarbon hydroxylase

activity

Inhibits the invasion of malignant

mouse tumor cells into normal tissue

fragments (anti-invasive activity)

Inhibits growth of squamous cell

carinoma

Inhibits growth of human malignant

cells from gastro-intestinal tract

Inhibits mutagenic activity of diol

epoxide tetrahydrobenzo pyrene

Reference

(1)

(2)

(3)

(4)

(5)

(6)

(1) Schwartz and Rate (1979); (2) Wattenberg (1975); (3) Bracke et al.

(1989); (4) Middleton (1989); (5) Yoshida (1990); (6) Huang and Ferraro

(1992).

An extensive coverage of citrus and plant flavonoids

exerting anticancer properties has been recently reviewed

by Attaway (1991). Table 4 lists some important studies on

select flavonoids found in citrus that proved effective in

deactivating carcinogens, modulating metabolic processes,

preventing tumor invasiveness and retarding the metastic

ability of a tumor cell population.

In a study of four flavonoids, nobiletin, tangeretin,

quercetin and tapifolin, Middleton (unpublished results)

noted that nobiletin and tangeretin (Figure 3) were more

effective in retarding growth of a human squamous car

cinoma cell line than the other two flavonoids. The differ

ence in anticancer activity of these flavonoids may be due

to the relatively greater uptake of the more methoxylated

flavonoids (nobiletin and tangeretin) by the cell rather than

tile more hydroxylated flavonoids (quercetin and taxifo-

lin). Figure 4 depicts the structure of an important hydro

xylated flavonoid found in Citrus, namely quercetin, and

its common rutinoside conjugate, rutin.

Well over 60 different types of flavonoids have been

detected in citrus fruits. Because of the complexity of these

phytochemicals, quantitative distribution patterns have not

been undertaken. However, semiquantitative methods (see

Ting and Rouseff (1986) for a listing of methods) have

been developed to obtain an estimate of the flavanone

glycoside contents of orange and grapefruit juices. Hes-

peridin is the principal flavonoid in orange juice, whereas

naringin is the dominant flavonoid in grapefruit juice. The

range of flavanone glycoside contents of Florida juices as

determined by Carter and co-workers (1975) were: Hamlin

juice (69-113 mg/100 ml); Pineapple juice (72-106 mg/100

ml) and Valencia juice (53-88 mg/100 ml). These values

agree with the orange juice flavanone glycoside content

estimated by Beilig et al. (1985) for German RSK values,

namely, 50-100 mg/100 ml juice. An 8-fl oz. serving of

orange juice should provide about 100-200 mg of a diverse

mixture of flavonoids. Citrus fruits are one of the richest

sources of flavonoids bestowing chemopreventive activities

against cancer.

Limonoids. Limonoids are a group of chemically related

triterpene derivatives found in the Rutaceae and Meliaceae

families. The best known compound in this class of

phytochemicals is limonin (Figure 5), and bitterness in cit-

165

OCH3 Table 5. Limonoids in citrus and its hybrids.

CH3O

OCH3 0

Tangeretin

OCH

— OCH 3

OCH 3

— OCH 3

Neutral limonoids

1. Limonin

3. Obacunone

5. Ichangin

7. Deoxylimonol

9. Limonyl acetate

11.7 a-Obacunyl acetate

13. Citrusin

15. Retrocalamin

17. Methyl deacetylnomilinate

19. 6-keto-7p-Deacetylnomilol

21. Methyl isoobacunoate diosphenol

22. l-(10-19) Abeo-obacun-9(l l)-en-7a-yl acetate

2. Nomilin

4. Deacetylnomilin

6. Deoxylimonin

8. 7 a-Limonol

10. 7a-Obacunol

12. Ichangensin

14. Calamin

16. Cyclocalamin

18. Isocyclocalamin

20. 6-keto-7p-Nomilol

23. 1 -(10-19) Abeo-7a-acetoxy-1 Op-hyddroxyisoobacunoic acid 3,10-lactone

Acidic limonoids

1. Deacetylnomilinate

3. Isoobacunoate

5. trans-19-Hydroxyobacunoate

7. Limonoate A-ring lactone

9. 17-Dehydrolimonoate A-ring lactone

11. Retrocalaminate

13. Obacunoate

14. 19-Hydroxydeacetylnomilinate

2. Nomilinate

4. Epiisoobacunoate

6. Isolimonate

8. Deoxylimonate

10. Calaminate

12. Isoobacunoate

diosphenol

15. Cyclocalaminate

CH3O

OCH 3 0

Nobiletin

Figure 3. Two important methoxylated flavonoids possessing anti-

cancer activity.

rus juice is primarily attributed to this compound (Maier

et al., 1977). Thirty-eight limonoid aglycones - 23 neutral

and 15 acidic - have been isolated from Citrus and its hyb

rids (Table 5). Additionally, citrus tissues and juices con

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).

Source: Hasegawa et al. (1992).

Recently, limonoids have been found to exert anticar-

cinogenic activity in laboratory animals (Lam et al., 1989;

Lam and Hasegawa, 1989; Miller et al., 1992). Eight

limonoids were tested by Lam and coworkers (1989), nomi

lin, limonin, deacetylnomilin, limonol, obacunone,

deoxylimonin, isoobacunoic acid and ichangin; all were

found to stimulate the detoxifying enzyme, glutathione S-

transferase (GST). GST enzymes are one of the major en

zyme systems responsible for the detoxification of xenobio-

tics; additionally, they catalyze the adduct formation of

glutathione with electrophiles, including reactive car

cinogenic species, to water-soluble substances that are ex

creted from the body (Chasseaud, 1979). Substances that

can elicit increased activity of GST may be potential anti-

Oh Limonin

OH 0

Quercetin

'O-Rutinose

OH 0

Rutin

(Rutinose=6—0—CX—L—rhamnosyl—D—glucose)

Figure 4. An important hydroxylated flavonoid and its rutinoside de

rivative with important therapeutic properties.

166

Limonoate A-ring lactone

0-3-D-glucose

Limonin 17- jB-D-glucopyranos'^de

Figure 5. Three forms of a limonoid, as depicted by limonin, found

in Citrus.

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

carcinogens in the inhibition of chemically induced cancer

formation.

The furan moiety attached to the D-ring of limonoids

was most likely responsible for induction of GST activity

(Hasegawa et al., 1992). Previous studies by Lam et al.

(1982) with kahweol and cafestol suggested that the furan

moieties of those molecules were responsible for inducing

GST activity in various tissues of mice. Nomilin was shown

by Lam et al. (1989) to be the most potent inducer of GST

activity in the liver and in the small intestinal mucosa. In

vivo tumor protection experiments confirmed that nomilin

is an inhibitor of benzo (a) pyrene-induced neoplasia in

the forestomach of mice (Lam and Hasegawa, 1989). Limo-

nin, which has a structure different from nomilin by the

presence of A and A' rings, was not as effective in inducing

GST as nomilin. Lam and coworkers (1989) suggested that

the A- and A'-ring moieties of limonoids also appear to

play a role in the induction of GST activity (see Figure 5).

Additional research on limonoids by Miller et al. (1992)

showed that limonin 17-P-D-glucopyranoside inhibited the

development of oral tumors in hamsters. Interestingly, this

glucoside had no effect on GSH activity of oral epithelial

cells. This was the first reported study noting an anticancer

activity for a conjugated limonoid. The water solubility of

these glucosidic conjugates may be of considerable impor

tance when considering the method of intake by humans.

Questions remain as to whether these glucosides are ab

sorbed intact through the intestine or whether they are

cleaved by intestinal flora prior to absorption.

Commercial citrus juices contain low levels (about 2-8

ppm) of free, nonconjugated limonoids but high levels of

limonoid glucosides. Table 6 shows the contents of the

major limonoid glucoside (limonin 17-P-D-

glucopyranoside) and the total limonoid glucosides in

orange, grapefruit and lemon juices (Fong et al., 1989).

The large quantity of limonoid glucosides in Citrus high

lights these juices as important sources for chemopreven-

tive limonoids.

Conclusion

Cancer is the result of the interplay of many variables.

The sequence of events leading to tumor formation and

the resulting cascading effects of the metastases have not

been completely elucidated. What has been charted are

various steps beginning with an insult to genetic cellular

material (genotoxic event; known also as "initiation") and

then to production of abnormal DNA (nongenotoxic

event; known also as "promotion"). The transformation of

the altered cell may lead to the proliferation of cells with

invasive (malignant) or noninvasive (benign) qualities.

From a confined region of tissue, cancer spreads to other

tissues (developing secondary tumors or metastases), and

Table 6. Concentrations of limonin 17-p-D-glucopyranoside (LG) and

total limonoid glucosides in commercial citrus juices.

Juice LG(ppm)

Total

glucosides (ppm)

Orange

Grapefruit

Lemon

180 ±25

120 ±21

54 ±2

320 ± 48

190 ± 36

82 ±9

Source: Fong et al. (1989).

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

finally, to an ultimate cascading effect of the tumorous

cells.

In the prevention of cancer, the importance of diet is

now well established through many epidemiological

studies. Of the many foods that have been tested by the

National Cancer Institute, citrus fruits and juices have

shown very positive results as a chemopreventive food

(Pierson, 1992). The phytochemicals present in Citrus pos

sessing anticancer properties are currently being evaluated

by several private, university and governmental

laboratories. Citrus has always been considered a nutritious

food. One day it may be considered as a modern age

medicinal food.

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EFFECT OF GIBBERELLIC ACID AND POSTHARVEST STORAGE ON

QUALITY OF FLORIDA NAVEL ORANGES

Mohamed A. Ismail and Diana L. Wilhite1

Florida Department of Citrus

Scientific Research Department

Citrus Research and Education Center

Lake Alfred, FL 33850

Additional index words, senescence, color, firmness, 2,4-D,

peel.

Abstract. Gibberellic acid (GA) and 2,4-dichlorophenoxyacetic

acid (2,4-D) were applied to 10 to 25-year old navel orange

trees in June, October, November or December to evaluate

their effect on peel color and fruit quality in later than normal

harvests. GA significantly delayed peel color development for

two months beyond normal harvest time. It did not, however,

affect total soluble solids, % citric acid or internal segment

drying. GA was marginally effective in maintaining peel

firmness as measured by resistance to puncture. Storage at

38° or 40°F also extended fruit shelf life by an additional

month. Application of the growth regulators during the month

'The authors wish to thank Ellen C. Wheeler and Eric C. Voigt for

their assistance in conducting field and laboratory work.

168

of October were more effective in delaying peel color develop

ment than applications made in November or December.

Navel orange is one of the world's premier citrus vari

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

packages shipped to all parts of the U.S. and Canada. Or

dinarily, the season for Florida navels starts in mid

November and ends in late December or mid January.

The growth regulators gibberellic acid (GA) and 2,4-

dichlorophenoxyacetic acid (2,4-D) are widely used on cit

rus in many parts of the world to retard peel senescence

and reduce fruit drop, respectively. Bevington (1973) re

ported that Washington navel oranges treated with GA

were less susceptible to rind injury, water spots, puffing

and creasing, and decay caused by green mold. El-Otmani,

M'Barek and Coggins (1990) tested various citrus cultivars

and demonstrated GA and 2,4-D could be used to reduce

fruit drop and delay rind softening. While preharvest ap

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

ton (1973) and Gilfillan et al. (1981). When GA is applied

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