Potential Health Benefits of Citrus - American Chemical Society

Chapter 6

Downloaded by UNIV OF MASSACHUSETTS AMHERST on September 15, 2015 | http://pubs.acs.org Publication Date: June 9, 2006 | doi: 10.1021/bk-2006-0936.ch006

Long-Term Screening Study on the Potential Toxicity of Limonoids 1,2

1

1

Edward G. Miller , Reed P. Gibbins , Samuel E. Taylor , Jame E. McIntosh , and Bhimanagouda S. Patil 1

2

1

Department of Biomedical Sciences, Baylor College of Dentistry, Texas A&M University Health Science Center, 3302 Gaston Avenue, Dallas, TX 75246 2Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Mail Code2119, Texas A&M University, College Station, TX 77843

Twelve pregnant rats were separated into 3 equal groups. Group 1 was fed the standardized rat chow, the AIN93G diet. Groups 2 and 3 were given the same diet supplemented with a 0.25% blend of mixed limonoid glucosides (group 2) or a 0.15% blend of mixed aglycones (group 3). At weaning, 33 pups per group were selected and placed on their mother's diet. Six weeks later 15 young female rats (5/group) were bred to males in the same group. The remaining animals were sacrificed and blood samples were collected. Multiple statistical differences were found in the blood chemistries. At weaning the pups from the 15 litters were sacrificed and autopsied. The male pups from groups 2 and 3 weighed significantly less than the male pups from group 1. For the females, the pups in group 2 weighed significantly less than the pups in group 1. The data consistently showed that at this high level of exposure, that the diets with limonoids caused problems with weight gain.

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© 2006 American Chemical Society

In Potential Health Benefits of Citrus; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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General Introduction


Limonoids - basic research Limonoids are a group of structurally related triterpene derivatives. Early research concentrated on the fact that several limonoids are extremely bitter. The primary ones are limonin, which is found in a wide variety of citrus juices, and nomilin, which primarily contributes to bitterness in grapefruit juice. The bitterness threshold for these two limonoids in citrus juice is 3-6 ppm (1,2). To minimize the economic impact of this problem a considerable amount of research has focused on the synthesis of these compounds in citrus. Through the use of radioactive tracer techniques, it was found that nomilin is synthesized from acetate and mevalonate, via farnesyl pyrophosphate in the phloem region of the stem (3). Once formed nomilin is translocated from the stem to other regions of the plant (leaves, fruit tissue, peels, and seeds) where it is metabolized into the other limonoids (4). Four different pathways, the limonin pathway, the calamin pathway, the ichangensin pathway, and the 7-acetate limonoid pathway have been identified for the biosynthesis of the other limonoids (5). Research on consumer acceptance has also shown that limonin is the major cause of delayed bitterness (6) and that bitterness decreases as fruit maturation progresses (7). Early research on limonoids in citrus tissues and juices usually started with an extraction with a non polar solvent. The aqueous phase was routinely discarded. In 1989, it was reported (8,9) that the aqueous phase contains a large number of modified limonoids. NMR analyses showed that in each of these compounds the aglycone was linked with one glucose molecule at C-17 by a pglycosidic linkage. Unlike the aglycones, which are found in low concentrations in the fruit and juice, the limonoid glucosides are found in high concentrations. Using TLC analyses (10) and HPLC (11), it was found that the concentration of mixed limonoid glucosides in orange juice averaged 320-330 ppm. The predominant limonoid glucoside accounting for over 50% of the total is limonin 17-P-D-glucopyranoside. Unlike limonin which is bitter limonin 17-P-Dglucopyranoside is essentially tasteless (8).

Limonoids - anticancer activity Starting in 1989 a number of papers have shown that limonoids can inhibit the development of carcinogen-induced tumors in animals. In most of these studies limonin and nomilin were tested for cancer chemopreventive activity.



84 These two limonoids have exhibited antineoplastic activity in a model for benz[a]pyrene-induced forestomach tumors (72) in ICR/Ha mice, in a model for 7,12-dimethylbenz[a]anthracene (DMBA) induced oral tumors in Syrian golden hamsters (75), in a model for benz[a]pyrene-induced lung tumors in A/J mice (14), in a two-stage model for DMBA-induced skin tumors in SENCAR mice (75), in a model for 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone-induced lung tumors in A/J mice (16) and in a model for azoxymethane-induced aberrant crypts in male F344 rats (77). One interesting sidelight to the data with the animal models is the apparent agreement with earlier epidemiological evidence on citrus consumption and cancer in humans (18). Approximately two dozen epidemiological studies have consistently shown that citrus consumption can significantly reduce an individual's risk for a variety of different cancers including cancers of the oral cavity, stomach, lung, colon, rectum, esophagus, larynx, and pancreas. In the model for oral carcinogenesis, 12 citrus limonoids have been tested for cancer chemopreventive activity (13,19-22). Four (limonin, limonin 17-P-Dglucopyranoside, deoxylimonin, and limonin carboxymethoxime) were found to have significant activity. Each of these limonoids lowered tumor burden by 5060%. The effect on tumor number was considerably less, a 15-30% inhibition. Three (nomilin, obacunone, and nomilin 17-p-D-glucopyranoside) were classified as having partial activity. With these chemicals there was a 20-30% reduction in tumor number. Since there was no effect on tumor volume, the overall reduction in tumor burden was similar to the reduction in tumor number. The data for the remaining limonoids (limonol, ichangensin, nomilinic acid 17P-D-glucopyranoside, deoxylimonic acid, and 17,19-didehydrolimonoic acid) showed that these chemicals were inactive. In addition to identifying new limonoids with antineoplastic activity, the data from these experiments indicated that some of the structural features in limonoids could be modified without affecting anticancer activity. One area where major modifications can be made is the D ring in the limonoid nucleus.

Limonoids - other potential health benefits Data from a number of different laboratories suggest that limonoids may have additional health-promoting properties. In one paper (23), it was reported that the substitution of grapefruit juice and orange juice for drinking water significantly lowered cholesterol levels in casein fed rabbits. Experiments with a



85 human liver cell line, HepG2 cells, showed that limonoids were primarily responsible for this effect on LDL cholesterol. In another study, it was shown that a variety of limonoids including deacetylnomilin, obacunone, nomilin, and a mixture of limonoid glucosides were potent inhibitors of proliferation of estrogen receptor-negative (MDA-MB-435 cells) and -positive (MCF-7 cells) human breast cancer cells (24). Similar cytotoxic effects were not seen with a variety of other human cancer cell lines (25) or with normal human cell lines grown in culture (26). It was also shown that limonoids could induce apoptosis in MCF-7 cells (25). Finally, in a recent paper limonin and nomilin were found to have anti-HIV activity (27). The two limonoids inhibited HIV replication in a variety of different cellular systems. Both of the compounds were also found to inhibit in vitro HIV-1 protease activity.

Limonoids - commercial interest Overall this research on the potential health benefits of citrus limonoids plus the extensive epidemiological evidence (18) has led a number of investigators and companies to start work on the production of new products. Early research on the metabolism of limonoids in the plant has led to work on the development of transgenic varieties of citrus that are free of bitter limonoids containing elevated concentrations of mixed limonoid glucosides (28,29). Commercial interest in the use of these citrus chemicals in new products has centered on the limonoid glucosides. There are several reasons for this focus. As already indicated one of the primary reasons is that the limonoid glucosides are tasteless (8). The fact that limonoid glucosides are polar also increases the potential for product development. These chemicals could easily be incorporated into a wide array of different products as food additives. Another factor is human consumption. The high concentration of mixed limonoid glucosides in citrus fruits and juices indicates that humans may be able to tolerate higher levels of exposure. Finally, research on by-products from juice processing plants has shown that mixed limonoid glucosides can be isolated in large quantities from citrus molasses and seeds (30-32). The seeds (30) are also a good source of the aglycones (limonin and nomilin). Even though the technology is in place to create a wide variety of foods containing elevated concentrations of citrus limonoids, we feel that product formulation at this time is premature. The primary reason is that very little is known about the pharmacokinetics of these chemicals in mammalian systems.



86 Research is needed to determine how effectively limonoids are absorbed in the GI tract. Once absorbed, how are these chemicals distributed throughout the body? How rapidly are the chemicals metabolized and do any of these metabolites have health-promoting properties? Finally, at higher levels of consumption are there any side effects or toxicity? To start to answer these questions this laboratory has shifted its focus from basic studies on carcinogenesis to research on uptake, distribution, turnover, and toxicity in mammalian systems. The primary objective of this experiment was to provide data on any potential toxicity that might be produced over several generations of rats through long-term systemic exposure.

Experimental Design

First generation For the experiment, 12 Sprague Dawley rats 10 days into their pregnancies were purchased from Harlan (Indianapolis, IN). At the time of arrival, the rats were separated into 3 equal groups (4 rats/group) and immediately placed on one of the three test diets. The animals in the control group, group 1, were fed the AIN93G diet prepared by Bio-Serv in Frenchtown, NJ. The pregnant rats in groups 2 and 3 were fed the same diet supplemented with a 0.25% mixture of limonoid glucosides (group 2) or a 0.15% mixture of limonoid aglycones. In an earlier short-term feeding trial with hamsters (35), 2 groups of animals were fed diets supplemented with mixed limonoid glucosides at a concentration of 0.05% or 0.50%. For this experiment, the concentrations were set between these limits (0.05-0.50%). The percent composition of the limonoid glucoside mixture isolated from citrus molasses was 35.9% limonin 17-P-D-glucopyranoside, 32.0% nomilinic acid 17-p-D-glucopyranoside, 12.7% nomilin 17-p-D-glucopyranoside, 8.7% deacetylnomilinic acid 17-p-D-glucopyranoside, 6.6% deacetylnomilin 17-p-Dglucopyranoside, and 4.1% obacunone 17-P-D-glucopyranoside (55). The aglycone mixture prepared in this laboratory was 90% limonin and 10% nomilin. Limited availability of the two chemicals dictated the reduced concentration (0.15%) and the 9/1 ratio. The limonin and nomilin were prepared from citrus seeds (30). During this time and throughout the rest of the experiment the food in powdered form and water were provided ad libitum. The animals were housed in solid bottomed plastic cages containing litter and nesting material in a room reserved for breeding. The room was maintained at 22°C with a 14:10 h light-dark cycle.


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Second generation At the time of weaning (32 days later), 33 pups per group were randomly selected and placed in individual stainless steel cages. The pups were moved to a separate room reserved for rats maintained at 22°C with a 12:12 h light-dark cycle. The pups were placed on their mother's diet. Approximately 6 weeks later, 15 female rats (5/group) weighing at least 200 grams were selected and bred to males in the same group but from different litters. The average weights for the females used in this part of the study were 210 g (group 1), 213 g (group 2), and 221 g (group 3). During the six week period weight gain and food intake were monitored. After the pregnancies were established, the remaining animals were sacrificed and blood samples were collected and processed. Serum samples were sent to an outside laboratory (Texas Veterinary Medical Diagnostic Laboratory System in College Station, Texas), where a complete series of blood chemistries were run. The technicians at this facility had no prior knowledge about how the rats had been treated.

Third generation At day 21, the pups from the 15 litters were sacrificed (carbon dioxide exposure). The sex and weight of the pups were determined. Following an autopsy the heart, a kidney, and the small lobe of the liver were taken. The samples were fixed in 10% neutral-buffered formalin, embedded in paraffin, processed by routine histological techniques, and stained with hematoxylin and eosin. The histologist who examined the slides for abnormalities had no prior knowledge about the treatment assignments..

Experimental Results Food intake & weight gain The data for food intake during the six-week period showed that there were some problems for the rats in group 2. The primary problem was seen in the male rats given the diet containing the mixed limonoid glucosides. During three of the



88 first four weeks daily food intake was significantly reduced (two-tailed Student's t-test) by 1.0-2.0 g when compared to the data for the male rats in group 1. During week two a significant reduction (1.5 g) was also seen for the female rats in group 2. Throughout the six-week period the weight gain profile for the male rats in group 2 was significantly less than the weight gain profile for the male rats in group 1. At the end of the six-week period, the average weight for the male rats in the three groups was 349 g (group 1), 318 g (group 2), and 342 g (group 3). For the females, the averages were 219 g (group 1), 209 (group 2), and 214 g (group 3).

Blood chemistries For the blood assays, the male rats in groups 2 and 3 were compared to the male rats in group 1 and the females in groups 2 and 3 were compared to the females in group 1. For 14 out of the 17 assays, one or more significant differences (two-tailed Student's t-test) were found in these four comparisons. Multiple differences (2-4) were found in 9 of the 17 assays. The data for these nine assays are given in Table I. Overall the results were highly consistent. Significant differences when they occurred were in almost every case in the same direction. For most of the assays, the non-significant results for groups 2 and 3 trended in the same direction as the significant results. For example, if the significant changes for groups 2 and 3 were higher than the corresponding data for group 1, then the non-significant results usually were elevated. This can be seen in the results for creatine kinase, globulins, alanine transaminase, amylase, creatinine, total serum protein, and alkaline phosphatase. The primary exception was the data for blood calcium. A significant decrease in blood calcium levels was seen in the male rats in groups 2 and 3; however, the levels of blood calcium were significantly increased in the female rats in group 3 and slightly elevated in the female rats in group 2. Even though a large number of numerically significant differences were found the results usually fell within the normal range of values for rats. This was also found to be true for the assays (phosphorus, albumin globulin ratio, glucose, aspartate transaminase, albumin) in which a single significant difference was seen. The exceptions were creatine kinase and amylase. Both sets of values were routinely high in the male and female rats in the three groups. The way in which the animals were sacrificed may have contributed to these two sets of results. To get adequate amounts of blood, the animals were stunned for a limited period of time in C 0 and decapitated. Muscular movement during this time could have led to the release of creatine kinase. The reason for the extremely high means for the male and female rats in group 1 is not known. The manner in which the blood was collected might have also led to contamination with saliva and the high values for amylase. The values for the female rats in groups 2 and 3 were especially high. 2



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Table I. Blood Assays (Multiple Differences)" Test Creatine kinase Calcium Globulins Alanine transaminase cholesterol Amylase creatinine serum protein Alkaline phosphatase a

b

Group 1 Males Females 3440 8060

Group 2 Males 393 d

b

Females 716

d

Group 3 Females Males 290 201 e

b

11.1 2.18 37.3

10.5 1.74 48.0

10.4 2.22 34.1

10.7 1.93 36.4"

10.1 2.34 27.0

104 3300 0.221 6.53 196

94.1 1770 0.286 5.99 194

91.6 3610 0.269 6.54 171

d

92.7 2380 0.323 6.23 176

88.9 3930 0.295 6.85° 153

d

b

b

b

e

e

6

e

c

e

11.2 2.00 35.3" b

95.9 3120 0.300 6.48 144 d

b

b

Units for the different assays were mg/dl for calcium, cholesterol, and creatinine; g/dl for total serum protein and globulins; U/l for amylase, alanine transaminase, alkaline phosphatase, and creatine kinase. " Indicate that these values are significant (two-tailed Student's t-test) when compared to the corresponding values for group 1 ( p

Potential Health Benefits of Citrus - American Chemical Society

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