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.