Citrus Limonoids - American Chemical Society

Chapter 14

Limonin and Nomilin Inhibitory Effects on Chemical-Induced Tumorigenesis 1

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Luke Κ. T. Lam , Shin Hasegawa , Carl Bergstrom , Sylvia H. Lam , and Patrick Kenney Downloaded by UNIV OF MICHIGAN ANN ARBOR on May 20, 2016 | http://pubs.acs.org Publication Date: July 30, 2000 | doi: 10.1021/bk-2000-0758.ch014

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LKT Laboratories, Inc., 2233 University Avenue West, St. Paul, MN 55114 Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany, CA 94710

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Citrus Limonoids are furan containing triterpenes present in common edible citrus fruits such as lemon, lime, orange and grapefruit. Limonin and nomilin, two of the most abundant limonoids, have been found to inhibit chemical-induced carcinogenesis. Both compounds are inducers of glutathione S -transferase, a major detoxifying enzyme system. The increased enzyme activity was correlated with the ability of these compounds to inhibit carcinogenesis. Nomilin was found to reduce the incidence and number of tumors per mouse of forestomach tumors induced by benzo[a]pyrene (BP). Topical application of the limonoids was found to inhibit both the initiation and the promotion phases of carcinogenesis in the skin of SENCAR mice. Nomilin appeared to be more effective at the initiation stage while limonin was more potent as an inhibitor at the promotion phase of carcinogenesis. Administration of nomilin and limonin to the diet or by gavage inhibited BP-induced and 4-(methylnitrosamino)-l-(3 -pyridyl)-l-butanone-induced lung tumor formations, respectively, in A/J mice. These findings suggest citrus limonoids are potential cancer chemopreventive agents.

Introduction Citrus limonoids are furan containing triterpenes. The most abundant limonoids in Rutaceous plants that include the commonly edible fruits lemon, lime, orange, and grapefruit, are limonin and nomilin (1). These two compounds are present in citrus fruits mostly as their glucosides (2). While the aglycones are intensely bitter and are one of the bitter principles that contribute to the unpleasant taste of the fruits and juices, the glucosides, on the other hand, are not organoleptic. Despite their unpleasant taste which is a major concern in the citrus industry (3), limonin and nomilin are potentially beneficial substances to human health. They have been © 2000 American Chemical Society

Berhow et al.; Citrus Limonoids ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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186 investigated as cancer chemopreventive agents in mice (4-6), hamsters (7-10), and cell culture systems (11). The results so far appear to be encouraging.

Structural Features Of Citrus Limonoids

Downloaded by UNIV OF MICHIGAN ANN ARBOR on May 20, 2016 | http://pubs.acs.org Publication Date: July 30, 2000 | doi: 10.1021/bk-2000-0758.ch014

One of the characteristic features of citrus limonoids is the presence of a furan ring (Fig. 1) (12). The D-ring lactone of a highly oxygenated triterpene is attached to the

Kahweol palmitate

2-Heptylfuran

Figure 1. Furan containing natural products that show cancer chemopreventive potential. 3-position of the furan moiety. The furan function plays an important role that defines the biological activity of citrus limonoids. Mono-substituted furan compounds such as 2-n-heptylfuran are known to inhibit chemically induced carcinogenesis (13). Furan containing natural products such as kahweol and cafestol from coffee beans inhibit carcinogenesis in the hamster cheek pouch (10) and the rat mammary glands (14). The inhibitory activity of these furan compounds is attributed, in part, to their ability to induce the detoxifying enzyme system, glutathione S-


187 transferase (GST) (4-6, 13, 15, 16). Conversion of the furan function to dihydro- and tetrahydro-furan by stepwise hydrogénation eliminates the inducing properties of cafestol acetate (16). The loss of GST inducing property suggests the diminishing activity of cancer chemoprevention as well.


Mutagenicity Of Citrus Limonoids The highly oxygenated triterpene moiety of limonoids possesses an epoxide on the Dring and cyclic esters on both the A and D rings. The presence of the epoxide and the cyclic ester functions often raise concerns that they may bind to macromolecules to induce mutation, thereby, causing potential carcinogenic or teratogenic effects. To alleviate such concerns, we have used the bacterial mutagenicity testings, the Ames test and forward mutation method, to determine the mutagenicity of citrus limonoids. The Ames test was performed according to the methods of Maron and Ames (17). Three tester strains, TA98, TA100, and TA1535 were used in the presence of S9. The forward mutation assay was performed according to the method of Thilly and coworkers (18,19) using the S. typhimurium strain TM677 strain. Neither limonin nor nomilin show mutagenic activity at concentrations of 5 μg per plate (Table I).

Glutathione S-Transferase Induction The Phase II enzyme system, GST, is a major detoxifying enzyme that catalyzes the conjugation of the endogenous tripeptide, glutathione, with reactive electrophiles (20, 21). Since many activated carcinogens are electrophiles, the induction of GST is considered an enhancement of carcinogen detoxification by an increase of carcinogen glutathione conjugate formation and their subsequent excretion (21). Many furancontaining natural products including the citrus limonoids have been shown to induce GST to relatively high levels (13, 16, 22). Limonin and nomilin have been found to induce increased GST activity in the liver and small intestinal mucosa of mice given at 5 mg and 10 mg per animal every other day for a total of 3 doses (4,22). The level of GST was determined using the universal substrate chlorodinitrobenzene (CDNB) (21,22). The results showed that limonin, the compound with the orthogonal A ring to the plane of the molecule was inactive in the liver. Nomilin, on the other hand, was quite active even at a low dose of 5 mg per animal (Table II). The relative inducing activity in the small bowel mucosa was higher than that in the liver for both limonin and nomilin. No appreciable elevation of GST activity was detected in the forestomach. The GST enzyme system consists of three main classes of isoenzymes. They are the α, μ, and % class. Using a chromatofocusing fast protein liquid chromatographic (FPLC) technique some of the isoenzymes in the α and m classes can be separated as distinct peaks (5). Limonin given in the diet at 0.25 and 0.50 % altered the FPLC profile in such a way that the relative intensity of the α and μ isoenzymes differed from that of the normal control level. No dose response factor can be observed, however, suggesting that the change is saturated at a very low dose level. Berhow et al.; Citrus Limonoids ACS Symposium Series; American Chemical Society: Washington, DC, 2000.


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a

Table I. Limonoids Mutagenicity Testing using the Ames Test and Forward Mutation Method . b

c

Chemicals

d

d

b

TA98

ΤΑ 1θΦ^

S9 control

18

99

12

94

Limonin

15

97

15

83

Nomilin

13

85

13

89

a. b. c. d. e. f. g.

TA1535 >f TM677 >f>%

The Ames bacterial testing was performed according to the methods of Maron and Ames (9). Forward mutation assay (Thilly and coworkers (10,11)). 5 μg each of limonin or nomilin per plate. The values are number of revertants per plate. Average of 3 experiments. Average of 2 experiments. The mutagenicity is expressed as mutant fractions, i.e. the average number of 8-azaguanine-resistant (mutant) clones from three plates divided by the average number of 8-azaguanine-sensitive clones from three plates.



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Table II. Limonin and Nomilin Induction of Glutathione S-transferase Activity in the Liver and Small Bowel Mucosa of Female ICR/Ha Mice.

b

GST Activity Compounds

a

Dose

Liver

Control

--

1.15±0.19

0.33±0.04

Limonin Limonin

5 mg 10 mg

1.2910.35 1.24±0.23

0.45±0.08 0.45±0.09

N o m i l i n

5

N o m i l i n

1 0

m

g mg

Small Bowel Mucosa

d

2.86±0.60 3.96±0.81

d

1.00±0.03 1.39±0.15

a Limonin and nomilin were given as fine suspension in 0.3 mL cottonseed oil every other day for a total of 3 doses. Control was given cottonseed oil only. b The GST activity (μηιοΐ/min/mg protein) was determined according to the method of Habig et al. using CNDB as the substrate (21). Mean ± S.D. (n = 3). c P< 0.001. d P

Citrus Limonoids - American Chemical Society

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