Food Factors in Health Promotion and Disease Prevention - American

Downloaded by UNIV LAVAL on July 7, 2014 | http://pubs.acs.org Publication Date: June 19, 2003 | doi: 10.1021/bk-2003-0851.ch019

Chapter 19

Inhibitory Effects of Oral Administration of an Extract of Orange Peel in the Diet on Azoxymethane-Induced Formation of Aberrant Crypt Foci and Colon Tumor in CF-1 Mice 1

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Wei Ding , Yue Liu , Robert T. Rosen , Geetha Ghai , and Mou-Tuan Huang 1

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Department of Chemical Biology, Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020 Center for Advanced Food Technology, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, NJ 08901-8520

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Orange peel possesses antioxidant and anti-inflammatory activity and preventive cancer activity. We have evaluated the effect of an extract of orange peel on azoxymethane (AOM)-induced formation of aberrant crypt foci and colon tumor in CF-1 mice. A n extract of orange peel contains approximately 30% polymethoxyflavones. Female CF-1 (6 weeks old) that were s.c. injected with AOM (5, 10, 10, and 10 mg/Kg body weight) once every 4 days resulted in formation of an average of 5.2 aberrant crypt foci (ACF) per colon and 37 aberrant crypt (AC) per colon at 24 weeks after the first dose of AOM treatment. Feeding 0.2% orange peel extract in theAIN76A diet to mice starting at 2 weeks before the first dose of AOM until the end of the experiment inhibited AOM-induced formation of the number of A C F per colon andACper colon by 48% and 66% respectively. Feeding 0.2% norhydroxyguaiaretic acid (NDGA), an inhibitor of

© 2003 American Chemical Society In Food Factors in Health Promotion and Disease Prevention; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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lipoxygenase, in the A I N 76A diet to mice during both the initiation and promotion phases inhibited the number of AOM-induced formation of ACF per colon and AC per colon by 48% and 75% respectively. Mice developed an average of 0.52 colon tumors per mouse and 44% of mice had colon tumors after 37 weeks of the first dose of AOM injection. The number of AOM-induced colon tumors was decreased by 44% or 48%, when 0.2% orange peel in AIN 76A diet or 0.2% nordihydroxguaiaretic acid in AIN 76A diet was used to replace the control AIN 76A diet. Feeding 0.2% orange peel diet or 0.2% nordihydroxyguaiaretic acid diet also decreased colon tumor incidence by 34% or 39%, respectively.

Introduction Orange peel is a source rich in flavonoids. Monomethoxyflavones and polymethoxyflavones are the major constituents of orange peel extract. From a pharmacological point of view, the polymethoxyated flavones have a wide biological activity and cancer preventive activity. Some polymethoxyflavones have been reported to have antioxdant and anti-inflammatory activities (1,2). A polymethoxyflavone, nobiletin, extracted from orange peel, is shown to inhibit expression of inducible nitric oxide synthase and has a suppressive effect on generation of the superoxide radical (0 ") and nitric oxide (NO) (1,2). Citrus nobiletin also inhibits phorbol ester-induced inflammation, oxidative stress, and tumor promotion in mouse skin (1). Feeding citrus nobiletin in the diet to rats inhibits AOM-induced formation of A C F in colon epithelium (3). Some polymethoxyflavones have anti-mutagenic activity (4,5), anti-proliferation activity on tumor cells (6,7), anti-promotion activity in mouse epidermis and colon of epithelium (7), anti-invasion activity (8), anti-adhesive effect on platelets (9,10), and cancer preventive effect (11). Polymethoxyflavones have anti-leukemia activity (12) and differentiating activity (75). The polymethoxyflavone, nobiletin has been shown to have anti-ulcer effect (14), and protective effect on aflatoxin B-induced cytotoxicity and binding to D N A (75). Here we report that an extract of orange peel that contained approximately 30% polymethoxyflavones inhibited AOM-induced formation of A C F and colon tumors. 2

In Food Factors in Health Promotion and Disease Prevention; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Material and Methods

Materials


Azoxymethane, nordihydroxyguaiaretic acid and methylene blue were purchased from Sigma-Aldrich Fine Chemicals (St Louis, M O ) . Acetone and 10% formalin-phosphate buffer were obtained from Fisher Scientific (Springfield, NJ).

Animals Female CF-1 mice (4-6 weeks old) were purchased from Charles River Laboratories (Wilmington, M A ) . The animals had free access to food and water throughout the experiment. The mice (initially fed the control A I N 76A diet for 4 days after arrival at the laboratory) were either continued on the same diet (control) or fed a 0.2% orange peel extract in A I N 76A diet or 0.2% nordihydroxguaiaretic acid (NDGA) in A I N 76A diet until the end of the experiment. A I N 76A diet and 0.2% orange peel in A I N 76A diet and 0.2% nordihydroxyguaiaretic acid in A I N 76A diet were purchased from Research Diets, Inc. (New Brunswick, NJ).

Quantification of Aberrant Crypt Foci Female CF-1 mice (5-6 weeks old; 10-30 mice per group) were giving a s.c. injection of A O M (5, 10, 10, 10 mg/kg) once a week (total 4 injections). The test compounds in the diet were given to mice at 2 weeks before the first A O M injection, during, and continuing until the end of the experiment (initiation + post-initiation period). At 24 weeks after the first dose of A O M injection, the mice were killed and colons (from anus to caecum) were removed, opened longitudinally, rinsed with normal saline solution, was stapled on a plastic sheet. The colon samples were placed in a 10% neutral buffered formalin solution for 24 hours. The entire colon was stained with 0.2% methylene blue dissolved in phosphate buffer saline (PBS) solution for 20 minutes. The whole mount of colon samples was examined in a light microscope magnification. Only A C F meeting the criteria, with crypts of increased size with a thicker and deeply stained epithelial lining and an increased pericryptal zone compared with normal crypts, were chosen.


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Study on AOM-induced Formation of Colonic Tumor in CF-1 Mice


Female CF-1 mice were received s.c, injection with A O M (5, 10, 10, and 10 mg of A O M / K g ) at 6 weeks of age once every 4 days (total 4 injections and total 35 mg of A O M ) . The mice were given A IN 76 A diet or test compound in A I N 76A diet at 2 weeks before the first dose of A O M , and continuing until the end of the experiment. Colon samples were removed, opened longitudinally, rinsed with normal saline solution, was stapled on a plastic sheet. The colon samples were stored in a 10% neutral buffered formalin. Colon tumors were counted and recorded.

Results

Chemical Composition of an Extract of Orange Peel Orange peel extract is a crude product containing approximately 30% polymethoxylated flavones. The chemical structures of major compounds are shown in Figure 1. They include SjoJ^^'^'-hexamethoxyflavone (nobiletin), 5,6,7,8,4'-penta-methoxyflavone (tangeretin), S^J^'^'-penta-methoxyflavone (sinensetin), 5,7,8,4'-tetra-methoxyflavone, 3,5,6,7,8,3',4-heptamethoxyflavone, 5,7,8,3',4 -pentamethoxyflavone. Other bioactive compounds include the monohydroxylated analogs of the polymethoxylated flavones. Also present in large quantity are peel waxes. Analysis was accomplished using a gradient reversed-phase system (Zorbax C-18 column, MacMod Analytical, Chadds Ford, PA), versus a standard of tangeretin (5,6,7,8,4'-pentamethoxyflavone) obtained from Apin Chemicals Ltd (Abingdon, Oxon, U K ) . The wavelength used for detection was 302 nm. Response factors for all compounds versus tangeretin were assumed to be equal. Orange peel extract was enriched from crude cold-pressed peel oil solids, a byproduct of the orange juice industry. f

Inhibitory Effect of an Extract of Orange Peel on AOM-induced Formation of A C F in CF-1 Mice The possible pathway of chemically-induced formation of aberrant crypt foci and subsequently developing to colon adenoma and adenocarcinoma in CF1 mouse is shown in Figure 2. Female CF-1 mice were injected s.c. with A O M developed an average of 5.2 ACFs per colon and 37 ACs per colon at 24 weeks



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S^^J^^^'-Heptamethoxyflavone

5,7,8,3T-Pentamethoxyflavone

Figure L Chemical structures of some polymethoxyflavones in orange peel extract.


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Mice

\

Azoxymethane ( A O M ) Normal mice

1AC


A C : Aberrant crypt

A C F : Aberrant / / crypt foci

It Adenoma

if Adenocarcinoma Figure 2. The possible pathway for azoxymethane-inducedformation of aberrant crypt foci and colon tumor in CF-1 mice.


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after the first dose of A O M injection (Table I). Feeding with 0.2% orange peel extract in A I N 76A diet to mice starting at 2 weeks before the first dose of A O M and continuing until the end of the experiment inhibited the number of A O M induced ACFs per colon by 48%, and the number of AOM-induced A C s per colon by 66% (Table I). Feeding 0.2% nordihydroxyguaiaretic acid in A I N 76A diet to mice at 2 weeks before the first dose of A O M injection and continuing until the end of the experiment inhibited the number of ACFs per colon by 48%, and the number of AOM-induced A C s per colon by 75% (Table I). It is interesting that both feeding 0.2% orange peel extract diet and 0.2% nordihydroxyguaiaretic acid diet to mice had the smaller A C F size in comparison with the A C F size in the control group diet (Table I).

Inhibitory Effect of an Extract of Orange Peel on AOM-induced Formation of Colon Tumors in CF-1 Mice Female CF-1 mice were s.c. injected with A O M (5, 10, 10, and 10 mg/Kg body weight) starting at 6 weeks of age once every 4 days resulted in formation of an average of 0.52 colon tumors per mouse and 44% of mice had colon tumor at 37 weeks after the first dose of A O M injection. Feeding 0.2% orange peel extract or 0.2% nordihydroxyguaiaretic acid in the diet to mice inhibited the number of AOM-induced colon tumor per mouse by 44% or 48%, respectively. Colon tumor incidence was reduced by 34% or 39%, respectively (Table II).

Discussion The results of the present study demonstrate that feeding 0.2% orange peel in the diet to mice starting at 2 weeks before carcinogen injection, during, and continuing to end of the experiment (initiation period + promotion period) inhibited the number of AOM-induced formation of ACFs per colon, colon tumor per mouse and colon tumor incidence. The mechanism of the inhibitory action of orange peel on AOM-induced formation of A C F , colon tumor and colon tumor incidence is unknown. However, it is believed that antioxidant and anti-inflammatory property of polymethoxyflavones may play an important role on the carcinogenic pathways. Among polymethoxyflavones, nobiletin has been studied in more detail. Nobiletin may contribute the important role on anticarcinogenic and anti-tumor promoting activity of orange peel. Topical application of citrus nobiletin to the back of mice inhibits phorbol ester-induced skin inflammation, oxidative stress, and tumor promotion in mice (/). Citrus nobiletin suppresses free radical generation and has anti-inflammatory activity (2). Feeding citrus nobiletin in the diet to rats inhibits azoxymethane induced


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Table I. Inhibitory Effect of Oral Administration of Orange Peel Extract (OPE) on Azoxymethane (AOM)-induced Formation of Aberrant Crypt Foci in CF-1 Mice


Formation of ACF ACF/colon (% inhibition) AC/colon (% inhibition) AC/ACF (% inhibition) lAC-ACF/colon 2AC-ACF/colon 3AC-ACF/colon 4AC-ACF/colon 5AC-ACF/colon 6AC-ACF/colon 7AC-ACF/colon

Control diet

Control diet

+

+

0.2% OPE diet

0.2% NDGAdiet

+

+

-

AOM 2.7±0.9 (48%) 12.6±2.8 (66%) 4.7 (34%)

AOM 2.7±0.9 (48%) 9.4±2.2 (75%) 3.5 (51%)

15.0±2.5 5.5±1.2 1.0±0.4 1.0±0.4 0.2±0.2 0.3±0.3 0.2±0.2

6.4±1.4 2.0±0.3 0.2±0.2 0.2±0.2 0 0.2±0.2 0

6.8±1.5 1.0±0.3 0.2±0.2 0 0 0 0

Vehicle 0

AOM 5.2±1.2

-

-

0

37±5.9

-

-

0

7.1

0 0 0 0 0 0 0

Female CF-1 mice were given AIN 76A diet, 0.2% orange peel extract in AIN 76A diet (0.2% OPE diet), or 0.2% nordihydroguaiaretic acid in AIN 76A diet (0.2% NDGA diet) starting at 2 weeks before the first dose of AOM. The mice received s.c. injection with A O M (5, 10, 10, 10 mg of AOM per kg of body weight) in saline once every 4 days starting at 2 weeks after 0.2% OPE diet or 0.2% NDGA diet. The mice were killed at 24 weeks after the first dose of AOM treatment. Colon samples were removed and stained with methylene blue and AACF were counted under microscope as described in the Materials and Methods section.


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Table II. Inhibitory Effect of Oral Administration of Orange Peel Extract (OPE) on Azoxymethane (AOM)-induced Formation of Colon Tumors in CF-1 Mice Control diet + Vehicle

Control diet + AOM

0.2% OPE diet + AOM

15

27

17

AOM 11

51.3±1.9

46,7±1.9

46.7±2.2

45.8±2.1

Colon tumors per mouse (% inhibition)

0

0.52±0.12

0.29±0.11 (-44%)

0.27±0.14 (-48%)

Colon tumor incidence (% inhibition)

0

44%

29% (-34%)

27% (-39%)


Measurement

Number of mice per group Body weight

0.2% NDGA diet

+

Female CF-1 mice were injected s.c. with saline or A O M in saline (5, 10, 10 and 10 mg of AOM/Kg body weight) starting at 6 weeks of age once every 4 days (total 4 injections and total 35 mg of AOM). The mice were given AIN 76A diet in the control groups during the whole period. Both 0.2% orange peel extract in AIN 76A diet or 0.2% nordihydroxyguaiaretic acid in AIN 76 A diet were given to mice at 2 weeks before the first dose of AOM, and continuing until the end of the experiment. The mice were killed at 37 weeks after the first dose of AOM injection. The colon samples were removed and stored in a 10% formalin phosphate buffer. The colon tumors were counted and recorded.



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formation of aberrant crypt foci in colon epithelium of rats (3). Citrus flavones have anti-mutagenic activity (4,5) and anti-proliferation activity (6,7). Feeding 0.2% orange peel in the diet to mice inhibits the proliferation of A C F (see Table I). Our results suggest that orange peel may inhibit proliferation of A C F . In addition, citrus flavones also have several other biological activities, anti-invasion activity (8), anti-adhesive effect on platelets (9,10), cancer preventive activity (1,3,11), anti-leukemia activity (12) and cell differential activity (13). It is believed that the ortho position of 3',4'-dimethoxy in C ring and the ortho position of 3-methoxy and 4 position of keto group in Β ring are important for antioxidant and anti-inflammatory activity. Thus, sinensetin and nobiletin may be expect to have potent antioxidant and anti-inflammatory activity and cancer chemopreventive activity. Further study is need to confirm this hypothesis. Orange peel contains several polymethoxyflavones and its activity may be result from a combination of several polymethoxyflavones. Orange peel has been widely used as a food additive and food flavor. Intake more orange peel in daily food may be to have health benefit. Human chronic trial on the effect of orange peel on human colon pre-neoplastic tumors is under investigating.

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Food Factors in Health Promotion and Disease Prevention - American

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