Food Contaminants - ACS Publications - American Chemical Society

Chapter 26

Evaluation of the Allergenic Potential of Morinda citrifolia L. (Noni) Leaf

Downloaded by STANFORD UNIV GREEN LIBR on July 1, 2012 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch026

Brett J. West and 'Afa K. Palu Research and Development, Tahitian Noni International, 737 East 1180 South, American Fork, UT 84003

The allergenic potential of Morinda citrifolia L. (noni) leaf was evaluated in a pepsin resistance assay. Proteins extracted from the leaf were subjected to simulated gastric fluid at various incubation times, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), to determine their resistance to digestion by pepsin, Noni leaf proteins were readily digested at all incubation times. The results indicate noni leaf is not allergenic, as a lack of resistance to pepsin is a common characteristic of non-allergenic proteins. These data corroborate earlier studies of the leaf, demonstrating the potential for safe food use. This assay method may also be a useful tool in evaluating the safety of newly introduced tropical foods.

Morinda citrifolia L., more commonly known as noni, is distributed across the tropics and has large, evergreen, dark, glossy, prominently veined, elliptical to oblong leaves (1). Noni leaves have long been recognized as food among several cultures. They are included in the United Nations (UN), World Health Organization (WHO), and Food and Agriculture Organization (FAO) food composition tables for East Asia and the Islands of the Pacific (2, 3). Polynesians wrapped fish in noni leaves for cooking, and then ate them together (4). Indonesians also ate the leaves with fish, as a raw vegetable, or fruit side dish with rice known as "lalab", as a steamed vegetable, and as a vegetable© 2008 American Chemical Society

In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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412 coconut-sambal dish called "oorab" (5). The consumption of noni leaves was so widespread in the Samut Prakarn province of Thailand that contamination of this plant by heavy metal emission from a local factory was considered a public health problem (6). Noni leaves provide certain nutritional benefits. They have been investigated as a potential source of dietary protein in tropical areas where other sources may not be readily available (7, 8). The leaves are an excellent source of (3-carotene, with a total carotene content higher than Chinese cabbage, taro, or any other green leaf produce commonly consumed in the Pacific. It was this property that resulted in the successful treatment of night blindness of children in the island nation of Kiribati (P). In addition to these nutritive properties, the phytochemicals have been found which exhibit significant antioxidant activity (/ft / / ) . Toxicity tests on the leaves began as early as 1965, when the L D of the methanol extract was found to be greater than 1000 mg/kg when injected intraperitoneally in male mice (12). The extract did not cause any symptoms of toxicity, including convulsions, diarrhea, tail erection, or exophthalmos. Acute oral toxicity tests were also performed for hot water (MCW) and 50% ethanol (MCE) extracts in separate groups of 10 rats. All animals were observed for 14 days. No deaths nor adverse effects observed. Therefore, the L D for these extracts was greater than 2000 mg/kg (73, 14). A second acute oral toxicity test of MCE and MCW was performed in separate groups of 50 mice (25 males and 25 females) at a dose of 2000 mg/kg. No deaths occurred, nor were other adverse effects seen. These results substantiated the L D of >2000 mg/kg in the rat studies (75). Additionally, a subacute (28 day) oral toxicity test of MCE and MCW in mice at 200 mg/kg (25 males and 25 females/test group) produced no evidence of toxicity (16). A subchronic (90-day) oral toxicity study of MCW and MCE was also performed in mice. The extracts were examined in separate groups of 50 mice (25 males, 25 females) at 20 mg/animal (approx. 1000 mg/kg). Animals were observed 90 days for clinical symptoms, death, and body weight. No evidence of toxicity was observed (77). The no-observable-adverse-effect-level (NOAEL) of the extracts in rodents is greater than 2000 mg/kg. While these toxicity tests reveal no inherent toxicity, they do not address the issue of allergenicity. With the ever increasing popularity of noni among populations where it is not part of the traditional diet, an assessment of the allergenic potential of the leaf is important. However, making this assessment presents several challenges. The currently accepted and standard methods for investigating the potential for Types I (immediate) and IV (delayed) hypersensitivity reactions are not designed for ingested and chemically complex substances, such as foods. These assays involve sensitization and challenge, either via the cutaneous and inhalation routes, or by intravenous injection. Such methods involve non50

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413 physiological routes of exposure, in which digestion is not accounted for. Only very recently has there been some development using the brown Norway rat as a model for assessing the allergenicity of individual novel proteins (75). There also seems to be no generally accepted standard methods for assessing Types II (cytotoxic) and III (immune complex) hypersensitivities. With new food sources, there is the disadvantage that epidemiological data to determine the sensitization rate is lacking. Further, there is often no previously sensitized subpopulation from which sera may be provided to conduct relevant Ig-E binding component studies. Some alternative methods for assessing the ability of a substance to sensitize susceptible individuals have been developed for recombinant proteins in genetically modified foods. However, these are only feasible when a small number of proteins are being investigated. These methods, such as protein profiling and comparison against databases containing amino acid sequences of allergens, are not an economically reasonable approach for new natural foods, where very large numbers of individual proteins may be involved. With these limitations in mind, a modified active systemic anaphylaxis test in guinea pigs was performed, where the intravenous challenge of the test group was replaced with an oral challenge. None of the animals in the test group showed sings of acute systemic anaphylaxis (19). Injection of whole leaf, or even an aqueous extract, to sensitize the animals is inappropriate. These are either not readily absorbed (due to insoluble carbohydrates, such as cellulose and lignin) or may cause inflammatory reactions that are unrelated to oral sensitization (due to many high molecular weight soluble carbohydrates, of which carrageenan is an excellent example). Therefore, afreezedried 80% ethanol extract of the leaf, with a protein content of 11.9%, was used as the test material, This allowed the extraction of ethanol-soluble proteins, similar to extraction of allergenic wheat gliadin peptides and storage proteins in rye and maize, etc. However, ethanol extraction may not include some proteins at the higher end of the molecular weight range of food allergens (20). Consequently, additional data are needed,which may be obtained from examination of a property common to food allergens. Allergenic proteins in food are resistant to digestion by the gastrointestinal tract. Conversely, proteins that are readily digested are not allergenic (21). The property of resistance to digestion may be evaluated in vitro and is useful in estimating the allergenicity of protein in novel foods (22, 23). Following the basic protocol of Astwood et al. (21), the resistance of M. citrifolia leaf proteins to digestion was evaluated in simulated gastric fluid (32% pepsin).

Material and Methods Proteins were extracted from recently harvested M. citrifolia leaves with the Plant Total Protein Extraction Kit (Sigma®). The total protein content of the



414 extract was determined using the Quick Start™ Bradford Protein Assay, with a bovine serum albumin standard (Bio-Rad Laboratories). Soybean lipoxidase and trypsin inhibitor (both from Fluka) were used as reference controls. The total protein contents of these preparations were also determined by the Quick Start™ Bradford Protein Assay. A lipoxidase test solution was made with 15.39 mg in 2 mL Tris/NaCl buffer. Trypsin inhibitor solution was made with 26.55 mg in 2 mL Tris/NaCl buffer. A 0.32% pepsin solution was prepared with 32.96 mg pepsin (2,580 units/mg) from porcine gastric mucosa (Sigma®) and 27.66 mg NaCl in 10 mL deionized water. This solution was adjusted to pH 2.07 with 0.1 N HC1. The pepsin resistance test was carried out as follows: 50 \iL aliquots of M citrifolia leaf protein extract solution, lipoxidase solution, and trypsin inhibitor solution were each incubated in 200 nL of 0.32% pepsin solution at 37° C for 0, 15, 30, and 60 seconds. Immediately following incubation, each sample was neutralized with 75 nL of 160 mM Na C0 then heated at 96° C for 5 min. All samples were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using precast 10-20% Tris-Tricine/Peptide Ready Gels (Bio-Rad Laboratories) and Tris/Tricine/SDS buffer (Bio-Rad Laboratories). Polypeptide molecular weight standards, as well as biotinylated broad range molecular weight standards (both from Bio-Rad Laboratories) were run with each gel, as well as the pepsin solution and sample solutions alone. Proteins were visualized by Coomassie brilliant blue staining. 2

3

Results and Discussion The M citrifolia leaf extract contained 1.91 mg protein/mL. The lipoxidase and trypsin inhibitor solutions contained 2.47 and 1.96 mg protein/mL, respectively. Therefore, the reference controls were appropriate for evaluation of the extracted leaf protein solution. The SDS-PAGE results are presented in Figures 1-3. Trypsin inhibitor is a known allergen and is resistant to digestion. Soybean lipoxidase is a nonallergenic protein and is sensitive to pepsin digestion. Figures 1 and 2 demonstrate that our test system was suitable, as the trypsin inhibitor was stable throughout the incubation period, while the lipoxidase was degraded rapidly. Figure 3 displays SDS-PAGE results for M. citrifolia leaf proteins, alone and in pepsin solution. Proteins from this leaf are very sensitive to the effects of pepsin. The proteins were degraded immediately, at 0 seconds, even with neutralization of the pepsin solution. Only protein from the simulated gastric fluid (0.32% pepsin solution) remained after any incubation time.



Figure 1. SDS-PAGE results for soybean trypsin inhibitor in 0.32% pepsin solution.



Figure 2. SDS-PAGE results for soybean lipoxidase in 0.32% pepsin solution.



Figure 3. SDS-PAGE results for M . citrifolia leafprotein in 0.32% pepsin solution.


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Conclusion M. citrifolia belongs to the Rubiaceae family, which is not one of the botanical families that contain common allergenic food plants (24). Recorded human use and toxicity tests reveal that noni leaves are safe for consumption. Further, proteins in M. citrifolia leaf are readily digested by pepsin in vitro. Thus, these proteins will be easily digested when the leaf is eaten by humans. As pepsin resistance is a typical characteristic of food allergens, a lack of resistance corresponds to a lack of allergenic potential. These findings corroborate the modified acute systemic anaphylaxis test results, indicating that M. citrifolia leaf poses little or no allergenic risk. The apparent lack of toxicity and allergenicity, and the nutritive value of M. citrifolia leaf demonstrate that it is a healthy and viable ingredient in global food markets. It is also a food resource in the tropics, particularly in areas where conditions do not permit the sustained growth of other crop foods. We have observed that M. citrifolia is a hardy plant that grows in many soil types and requires very little, if any, care. These properties make it an ideal plant for further agricultural development. This is the first reported use of the pepsin resistance test in the evaluation of a whole natural food, newly introduced from the tropics. Much attention has been focused on methods to evaluate novel proteins in genetically modified food crops, which methods are not very feasible when applied to natural plant foods. On the other hand, very little attention has been paid to developing possible methods for evaluating underutilized natural plant foods from underdeveloped nations. Pepsin resistance is a useful property that should be examined as an initial screen for potential allergenicity, when foods are introduced from one population or region to another.

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