Nutrient and phytochemical analyses of processed noni puree

Abstract

The recent approval of noni fruit puree as a novel food ingredient, as well as the growing popularity of this fruit in health beverages, will greatly increase its use in foodstuffs, and consequently, its consumption among the general population. As such, an understanding of the nutritional profile of processed noni fruit puree is important for food technologists, nutritionists, as well as consumers. Therefore, the proximate nutritional, vitamin, mineral, and amino acid contents were determined. The phytochemical properties were evaluated, as well as an assessment made on the safety and potential efficacy of the major phytochemicals present in the puree. Processed noni fruit puree is a potential dietary source of vitamin C, vitamin A, niacin, manganese, and selenium. Vitamin C is the major nutrient present, in terms of concentration. The major phytochemicals in the puree are iridoids, especially deacetylasperulosidic acid, which were present in higher concentrations than vitamin C. The iridoids in noni did not display any oral toxicity or genotoxicity, but did possess potential anti-genotoxic activity. These findings suggest that deacetylasperulosidic acid may play an important role in the biological activities of noni fruit juice that have been observed in vitro, in vivo, and in human clinical trials.

Full text

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Nutrient and phytochemical analyses of processed noni puree
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Brett J. Westa, Shixin Dengb, C. Jarakae Jensenc
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a Research and Development, Tahitian Noni International, 737 East 1180 South, American Fork,
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Utah, 84003, U.S.A. brett_west@tni.com
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b Research and Development, Tahitian Noni International, 737 East 1180 South, American Fork,
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Utah, 84003, U.S.A. shixin_deng@tni.com
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c Research and Development, Tahitian Noni International, 737 East 1180 South, American Fork,
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Utah, 84003, U.S.A. Jarakae_jensen@tni.com
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Corresponding author:
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Brett J. West
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Research and Development, Tahitian Noni International,
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737 East 1180 South, American Fork,
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Utah, 84003, U.S.A
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Email: brett_west@tni.com
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Tel: 1 (801) 234-3621
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Fax: 1 (801) 234-1030
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Abstract
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The recent approval of noni fruit puree as a novel food ingredient, as well as the growing
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popularity of this fruit in health beverages, will greatly increase its use in foodstuffs, and
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consequently, its consumption among the general population. As such, an understanding of the
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nutritional profile of processed noni fruit puree is important for food technologists, nutritionists,
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as well as consumers. Therefore, the proximate nutritional, vitamin, mineral, and amino acid
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contents were determined. The phytochemical properties were evaluated, as well as an
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assessment made on the safety and potential efficacy of the major phytochemicals present in the
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puree. Processed noni fruit puree is a potential dietary source of vitamin C, vitamin A, niacin,
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manganese, and selenium. Vitamin C is the major nutrient present, in terms of concentration.
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The major phytochemicals in the puree are iridoids, especially deacetylasperulosidic acid, which
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were present in higher concentrations than vitamin C. The iridoids in noni did not display any
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oral toxicity or genotoxicity, but did possess potential anti-genotoxic activity. These findings
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suggest that deacetylasperulosidic acid may play an important role in the biological activities of
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noni fruit juice that have been observed in vitro, in vivo, and in human clinical trials.
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Key words: Noni, Morinda citrifolia, vitamin C, iridoids, anti-genotoxicity
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1. Introduction
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Morinda citrifolia, commonly known as noni, is a widely distributed tropical tree. It grows on
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the islands of the South Pacific, Southeast Asia, Central America, Indian subcontinent, and in the
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Caribbean. The fruit and leaves of this tree have a history of use both as food and for the
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promotion of health (Morton, 1992). The noni fruit juice industry has grown substantially in the
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paste decade, especially since the approval of noni fruit juice from French Polynesia as a novel
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food ingredient by the Commission of the European Union (European Commission, 2003).
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Since this approval, other noni fruit juice products have been approved for sale within the E.U.
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under the simplified substantial equivalence procedure (European Communities, 1997). More
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than 40 commerical sources of noni fruit juice have been granted substantial equivalence. These
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sources are from a variety of nations which include French Polyensia, Fiji, Dominican Republic,
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Panama, Costa Rica, Samoa, U.S.A. (Hawaii), Tonga, Vanuatu, Cook Islands, Palau, Solomon
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Islands, and Nauru (DG SANCO, 2010).
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Recent approval has also been given to extend the use of Polynesian noni fruit puree and fruit
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juice concentrate as novel food ingredients in a variety food categories (European Commission,
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2010). These food categories include candy, cereal products, nutritional drink mixes, ice cream,
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yogurt, baked goods, jams and jellies, carbonated beverages, food supplements, spreads, fillings
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and icings, sauces, gravies, pickles and condiments.
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The approval of noni fruit as a novel food ingredient will greatly increase the use of these
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ingredients in foodstuffs, and consequently, their consumption among the general population.
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As such, an understanding of the nutritional profile of processed noni fruit puree is important for
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food technologists, nutritionists, as well as consumers. Knowledge of the phytochemical profile
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of processed noni fruit is also important in understanding potential bioactivities, as well as in
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understanding the compounds responsible for health effects already demonstrated in human
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clinical trials. A few publications have provided some limited nutritional and phytochemical
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information on the composition of noni fruit. Proximate nutritional, fiber, sugar, partial amino
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acids, and some mineral analyses of juice pressed from raw noni fruits from Cambodia have been
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reported (Chunhieng et al., 2005). Vitamin C content, as well as that of five minerals, has been
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determined for wild noni fruit from northern Australia (Peerzada et al., 1990). Proximate
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nutritional, some minerals, vitamin A, and vitamin C contents of whole unprocessed noni fruits
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in Pohnpei have also been reported (Shovic & Whistler, 2001). Phytochemical investigations of
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raw noni fruits, and some commercial juices, have identified the presence of several different
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types of compounds (Basar & Westendorf, 2010; Potterat et al., 2007; Kamiya et al., 2005). But
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iridoids constitute the major phytochemical component of noni fruit (Deng et al., 2010 a), with a
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few other compounds, such as scopoletin, quercetin, and rutin have occurring in significant,
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although much less, quantities (Deng et al., 2010 b). These previous analyses have been limited
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in the amount of nutrient data provided. Further, they have not been representative of
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commercially processed noni fruit puree, as processing conditions do alter the nutritional and
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phytochemical profiles of fruits and vegetables (Murcia et al., 2009; Rodrigues et al., 2009).
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Therefore, the current chemical analyses were performed to provide more complete and accurate
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nutritional data. Analyses of the major phytochemicals in noni fruit were also carried out to
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provide an important reference for quality control and identity testing of these raw materials.
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As iridoids are present in significant quantities in noni fruit puree, genotoxicity and acute
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toxicity tests were performed to better understand their individual safety profiles. Noni fruit
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juice has also been shown to protect DNA against chemical mutagens in vivo and in a human
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clinical trial (Wang & Su, 2001; Wang et al., 2009). Therefore, the anti-genotoxic activities of
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the iridoids were evaluated in vitro to investigate their potential roles in this reported DNA
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protection.
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.
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2. Materials and methods
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2.1 Experimental materials
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Noni fruits were harvested in French Polynesia and allowed to fully ripen. The fruit was then
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processed into a puree by mechanical removal of the seeds and skin via micro-mesh screen in a
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commercial fruit pulper, followed by pasteurization (87°C for 3 seconds) at a good
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manufacturing certified fruit processing facility in Mataiea, Tahiti. The pasteurized puree was
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filled into aseptic containers, or totes containing 880 kg of noni fruit puree, and stored under
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refrigeration. For the chemical analyses in this study, samples were obtained from 10 different
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batches of puree.
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For the acute oral toxicity test, an iridoid enriched fruit extract was prepared. This was done by
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removal of seeds and skin from the fruit flesh, followed by size reduction with a 0.65 mm sieve.
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An aqueous extract was prepared with the remaining fruit pulp, at ambient temperature, which
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was then freeze-dried, resulting in a total iridoid concentration of 1690 mg/ 100g extract.
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Freeze-dried noni fruit powder (36 g) was extracted with 1 L of methanol by percolation to
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produce 10 g of methanol extract. Following addition of water, the methanol extract was
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partitioned with ethyl acetate (150 mL, three times) to remove non-polar impurities. The aqueous
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extract was further partitioned with n-butanol (150 mL, three times) to yield 3 g n-butanol
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extract. The extract was subjected to flash column chromatography on silica gel, eluting with a
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stepwise dichloromethane: methanol (20:1 → 1.5:1) gradient solvent system to yield sixty-two
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primary fractions. Among these, the presence of two major compounds was indicated by a
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preliminary HPLC analysis. The iridoid containing fractions were combined and subject to
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further purification by using reverse phase preparative HPLC (Symmetry PrepTM C18 column,
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Waters Corp, Milford, Massachusetts, USA), eluting with an isocratic solvent system of
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acetonitrile: water (35:65, v:v) at a flow rate of 3 mL min-1, resulting in the isolation of DAA and
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AA.
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2.2 Chemical analyses
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Proximate nutritional analyses of noni fruit puree were carried out to determine moisture, fat,
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protein, ash, and carbohydrate contents. Protein content was determined by the Kjeldahl method,
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Association of Official Analytical Chemists (AOAC) Method 979.09 (AOAC, 2000 a), with a
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Kjeltec System 1002 distilling unit (Foss Tecator, Höganäs, Sweden). Total moisture was
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determined gravimetrically by loss on drying at 100 °C in an Isotemp® 516G oven (Fisher
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Scientific, Waltham, Massachusetts, USA). Fat determination involved continuous extraction by
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petroleum ether in a Soxhlet apparatus, AOAC Method 960.39 (AOAC, 2000 b). Ash was
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determined gravimetrically following combustion in a Thermolyne® 6000 muffle furnace
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(Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 550 °C. Carbohydrate was then
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calculated by difference.
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Total dietary fiber was determined gravimetrically following enzymatic digestion with α-
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amylase (95 °C, 15 min), protease (60 °C, 30 min, pH 7.5), amyloglucosidase (60 °C, 30 min, pH
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4.0-4.7), precipitation with 95 % ethanol, and filtration through acid washed celite with ethanol
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and acetone solutions (AOAC, 2000 c). Prior to calculation of the final fiber content, the protein
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content was determined by the Kjeldahl method and subtracted. Enzymes and reagents were
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purchased from Sigma-Aldrich Corporation (St Louis, Missouri, USA)
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Fructose, glucose, and sucrose contents were determined by HPLC according to AOAC method
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982.14 (AOAC, 2000 d), using standards from Sigma-Aldrich Corp. and by separation with an
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Agilent 1100 Series LC and refractive index detector (Agilent Technologies, Santa Clara,
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California, USA). Prior to chromatographic separation, samples were diluted with ethanol:water
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(1:1, v:v) and heated to 85 °C for 25 min. Samples were then centrifuged and filtered through a
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0.45 µm nylon syringe filter and injected into a 5 µm amino column. The mobile phase was
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acetonitrile:water (80:20, v:v) with a flow rate of 1.5 mL min-1.
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Minerals were determined by inductively coupled plasma (ICP) emission spectrometry (AOAC,
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2000 e; AOAC, 2000 f). Samples were ashed and then treated with concentrated nitric and
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hydrochloric acids. The treated samples were then analyzed using an Optima 2000 DV optical
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emission spectrometer (PerkinElmer, Waltham, Massachusetts, USA). AccuTrace™ mineral
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reference standards (AccuStandard, New Haven, Connecticut, USA) were used to develop
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calibration curves at the appropriate wavelengths.
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Vitamin A, as β-carotene, was determined by a modified AOAC official method 941.15 for an
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HPLC system (AOAC, 2000 g). Briefly, samples were extracted with chloroform, followed by
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successive partitioning with n-hexane. The organic solvent was removed from the extracted
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residue by evaporation under nitrogen at 55 °C. The residue was then dissolved in propanol and
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injected into a Waters 2690 separations module coupled with a 996 Photodiode Array (PDA)
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detector, equipped with a C8 column (4.6 mm x 250 mm; 5 μm, Waters Corporation, Milford,
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Massachusetts, USA). The mobile phase was water:propanol (40:60, v:v) with a flow rate of 1
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mL min-1.
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Vitamin C was determined by titration with 2,6-dichloroindophenol, by the microfluorometric
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method, or by HPLC and UV detection of oxidized ascorbic acid (AOAC, 2000 h; AOAC, 2000
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i). Noni fruit puree was filtered, diluted with metaphosphoric acid-acetic acid solution
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(0.03%:0.08%), and filtered once more. This diluted samples were then titrated with 2,6-
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dichloroindophenol (1.1 M) and the results calculated based upon the amount consumed, having
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been previously calibrated with an ascorbic acid standard solutions. Alternately, the diluted
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samples (100 mL) were treated with 2 g acid-washed decolorizing carbon (Norit®, Norit N.V.,
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Amersfoort, Netherlands). Five mL aliquots of the Norit®-treated samples were diluted 1:1 with
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a 3% boric acid, in saturated sodium acetate, and the fluorescence read at 430 nm. For the HPLC
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analysis, the samples were filtered, diluted with 0.01 M sodium heptane sulfonate, and injected
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into a Waters 2690 separations module coupled with 996 PDA detectors, equipped with a C18
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column (4.6 mm x 150 mm; 5 μm). The pump was connected to two mobile phases: A 15 mM
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phosphate buffer (pH 3.5), and B; methanol. The elution flow rate was 0.75 mL min-1, with a
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column temperature of 10 °C. The mobile phase was programmed consecutively in linear
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gradients as follows: 0-5 min, 95% A and 5% B; 5-10 min, 90% A and 10% B; 10-15 min, 85%
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A and 15% B; 15-30 min, 80% A and 20% B. The PDA detector was monitored in the range of
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200-300 nm, and quantified at 240 nm.
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Niacin, thiamin, riboflavin, vitamin B6, vitamin B12, folic acid, biotin, and pantothenic acid
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were determined by AOAC and United States Pharmacopoeia methods (AOAC, 2000 j; AOAC,
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2000 k; AOAC, 2000 l; AOAC, 2000 m; AOAC, 2000 n; AOAC, 2000 o; AOAC, 2000 p;
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United States Pharmacopeia, 2005; Scheiner & De Ritter, 1975). Following incubation of
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samples with the appropriate inoculum and growth media solutions, as per AOAC methods,
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turbidity was measured with the Autoturb3 microbiological assay system (Shaefer Technologies,
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Indianapolis, Indiana, USA) to determine niacin, vitamin B12, biotin, and folic acid content.
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For folic acid determination, the puree samples were hydrolyzed in potassium phosphate buffer,
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treated with folate conjugase (Difco Laboratories, Detroit, Michigan, USA), followed by
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incubation with Lactobacillus casei (ATCC, Manassas, Virginia, USA) at 37°C for 22 hours.
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For biotin determination, samples were filtered and incubated with Lactobacillus plantarum
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(ATCC). For niacin determination, samples were also incubated with L. plantarum, but were
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first hydrolyzed with sulfuric acid. Puree samples were diluted with 0.1 M sodium phosphate
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buffer and autoclaved at 123 °C for 10 min then incubated with Lactobacillus delbrueckii
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(ATCC) to determine vitamin B12 content. For riboflavin, thiamin, pantothenic acid, and
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vitamin B6 determinations, samples were extracted with 5 % acetonitrile, containing 0.6 N acetic
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acid, and then filtered. The extracts were loaded into an Agilent 1100 Series HPLC system, with
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a C18 column, and eluted with an isocratic mobile phase of methanol:glacial acetic acid: water
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(27:1:73, v:v:v) at a flow rate of 1 mL min-1. Analytes were detected and quantified at 270 nm,
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with the exception of pantothenic acid, which was quantified at 210 nm.
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Vitamin E was determined by HPLC similar to a previously reported method (Omale and
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Omajali, 2010), with modifications. The samples were filtered and extracted directly with n-
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hexane. The hexane was removed by evaporation and the sample redissolved in propanol.
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Separation was carried out with a Waters 2690 separations module coupled with a 996
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Photodiode Array (PDA) detector and a C8 column (4.6 mm x 250 mm; 5 μm, Waters Corp),
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with detection at 210 nm. The mobile phase was 2-propanol:H20 (60:20, %:%), with a flow
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rate of 1 mL min-1.
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Vitamin K was determined according to AOAC method 992.27 (AOAC, 2000 p), using an
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Agilent 1100 Series LC with a UV detector, following extraction of samples with
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dichloromethane and isooctane (2:1, v:v). The mobile phase was 30% dichloromethane and
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0.02% isopropanol in isooctane, with a flow rate of 1 ml min-1. Vitamin K concentration was
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determined at 254 nm. Amino acids were determined with a Beckman 7300 automated amino
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acid analyzer (Beckman Coulter, Inc. Fullerton, California), following hydrolysis with 6 M
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hydrochloric acid for 24 hr at 110 °C. For methionine and cystine, the samples (0.1 g) were first
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treated with 2 mL 88% performic acid overnight at 5 °C. The tryptophan analysis involved
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hydrolysis with 4.2 M sodium hydroxide for 22 hr at 110 °C (AOAC, 2000 q). Amino acid
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standards were obtained from Beckman Coulter, Inc. Biotin, folic acid, vitamin B12, niacin, and
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vitamin K standards were obtained from the United States Pharmacopeia (Rockville, Maryland).
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The remaining vitamin standards were obtained from Sigma-Aldrich Corporation.
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The iridoid content, inclusive of deacetylasperulosidic acid (DAA) and asperulosidic acid (AA),
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was determined by HPLC, according to a previously reported method (Deng et al., 2010 b).
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Briefly, puree samples were diluted with water:methanol (1:1), mixed thoroughly, and filtered.
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Filtered samples were collected in 5 mL volumetric flasks for HPLC analysis. Chromatographic
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separation was performed on a Waters 2690 separations module coupled with a 996 PDA
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detectors, equipped with a C18 column (4.6 mm x 250 mm; 5 μm). The pump was connected to
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two mobile phases: A; acetonitrile, and B; 0.1% formic acid in water (v:v), and eluted at a flow
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rate of 0.8 mL min-1. The mobile phase was programmed consecutively in linear gradients as
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follows: 0-5 min, 0% A; and 40 min, 30% A. The PDA detector was monitored in the range of
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210-400 nm. The injection volume was 10 µL for each of the sample solutions. The column
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temperature was maintained at 25 °C.
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Other significant secondary metabolites, such as scopoletin, rutin, and quercetin, were also
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determined by HPLC (Deng et al., 2010 a). Noni fruit puree sample preparation was the same as
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that for iridoid analyses. Chromatographic separation was performed on a Waters 2690
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separations module coupled with a 996 PDA detector, and equipped with an Atlantis® C18
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column (4.6 mm x 250 mm; 5 μm). The pump was connected to a mobile phase system
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composed of three solvents: A; acetonitrile, B; methanol, and C; 0.1 % trifluoroacetic acid in
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water. The mobile phase was programmed consecutively in linear gradients as follows: 0 min,
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10% A, 10% B, and 80% C; 15 min, 20% A, 20% B, and 60% C; 26 min, 40% A, 40% B, and
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20% C; 28-39 min, 50% A, 50% B, and 0% C; and 40-45 min, 10% A, 10% B, and 80% C. The
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elution was run at a flow rate of 1.0 mL min-1. The UV spectra were monitored at 210 nm, 450
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nm, and 365 nm for quantitative analysis. The injection volume was 50 µL for each of the
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sample solutions. The column temperature was maintained at 25 °C. Reagents for the
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phytochemical analyses were obtained from Sigma-Aldrich Corporation and Fisher Scientific.
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2.3 Acute toxicity test of iridoids
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Twenty healthy Sprague–Dawley rats (10 males, 10 females, body weight 181-205g) were
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selected for the tests. An iridoid enriched fruit extract was dissolved in water to produce a total
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iridoid concentration of 8.5 mg/mL. A dose of 340 mg total iridoids/kg body weight (bw) was
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given to each animal by gastric intubation (20 mL/kg bw twice per day). For 14 days following
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the administration of the iridoid solution, animals were observed daily for occurrences of death
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and symptoms of toxicity, including convulsions, irregular breathing, piloerection, and paralysis.
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As decreased weight is a typical symptom of toxicity, body weights were recorded for each
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animal on days 0 and 14. The acute toxicity test was carried out in accordance with EC
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Directive 86/609/EEC (European Communities, 1986).
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2.4 Primary DNA damage test in E. coli PQ37
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The SOS-chromotest in E. coli PQ37 was used to determine the potential for DAA and AA to
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induce primary DNA damage. This test was carried out according to the previously developed
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method (Fish et al., 1987). DAA and AA were isolated from noni fruits from Tahiti and purified
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to > 98%. E. coli PQ37 was incubated in LB medium for 12 hours at 37 °C to reach the
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exponential growth phase. An aliquot of this culture was diluted with fresh LB medium to OD600
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= 0.05. Aliquots of the diluted E. coli PQ37 suspension were incubated in a 96-well plate at 37
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°C in the presence of DAA or AA for 2 hours. The DAA and AA concentrations tested were
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7.81, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 µg mL-1. Samples were evaluated in triplicate.
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Following incubation with the samples, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside was
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added to the wells to detect β-galactosidase enzyme activity, which is induced during SOS repair
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of damaged DNA. Nitrophenyl phosphate is also added to the wells to measure alkaline
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phosphatase activity, an indicator of cell viability. The samples were again incubated for 90
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minutes and the absorbances of the samples, blanks and controls were measured at 410 and 620
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nm with a microplate reader. Vehicle blanks and positive controls, 1.25 µg mL-1 4-
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nitroquinoline 1-oxide (4NQO), were included in this test. The induction factor of each material
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was calculated by dividing the absorbance of the sample at 620 nm by that of the blank, while
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also correcting for cell viability. Induction factors less than two indicate an absence of genotoxic
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activity.
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2.5 Anti-genotoxicity test in E. coli PQ37
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The primary DNA damage test was performed again, similar to the method described above.
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However, the method was modified to include incubation of E. coli PQ37 in the presence of both
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1.25 µg mL-1 4NQO and 250 µg mL-1 DAA or AA. 250 µg mL-1 represented the lower mid-
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range of concentrations evaluated in the genotoxicity test. Therefore, it was selected for
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screening of anti-genotoxic activity. Induction factors were calculated in the same manner as
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described above. The percent reduction in genotoxicity was determined by dividing the
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difference between the induction factor of 4NQO and the blank (induction factor of 1) by the
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difference between the induction factor of 4NQO plus DAA or AA and the blank. In this test, as
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well as in the primary DNA damage tests, comparisons were made with Student’s t-test.
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2.6 Statistical analyses
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Means and standard deviations were calculated for each set of analytical results obtained from
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the different batches. In both the primary DNA damage test and the anti-genotoxicity test,
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intergroup comparisons were made with Student’s t-test.
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3. Results and discussion
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The nutrient composition of processed noni fruit puree is summarized in Table 1. Proximate
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nutritional parameters are within the typical ranges for fruits in general. Processed noni fruit
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puree contains 2 g 100g-1 dietary fiber. Noni fruit does not contain a significant quantity of
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protein or fat. However, all but one essential amino acid, tryptophan, as well as histidine,
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essential for infants, were detected in the puree (Table 2). Aspartic acid was the most
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predominant amino acid. The protein content of the processed puree from French Polynesia is
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within the same range as that reported for raw noni fruit from Pohnpei, but is much less than that
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reported for pressed juice from Cambodian noni fruit (Shovic & Whistler, 2001; Chunhieng et
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al., 2005). Cambodian noni juice is reported to contain 2.5 % protein, about five times greater
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than that reported in processed puree or raw Pohnpei fruit. However, this is an unusually high
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quantity for a fruit juice. The protein content of purple passion fruit (2.2 %) is one of the highest
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known among fruits, and, by comparison, the juice pressed from passion fruit contains 0.39 g
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100 g -1 (USDA, 2009). Therefore, it is possible that the high protein content in Cambodian noni
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juice is reported in error.
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The fructose, glucose and sucrose content ranges of processed noni fruit puree are inclusive of
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the amounts reported for Cambodian noni fruit juice, but the dietary fiber content is slightly less
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(Chunhieng et al., 2005). The fat content is essentially the same in both the puree and the
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Cambodian juice, but is reportedly twice as much (0.3 g 100 g -1) in Pohnpei noni fruit (Shovic &
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Whistler, 2001).
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Vitamin C is the most prominent vitamin in noni fruit puree, with a mean content of 1.13 mg-1 g.
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At this concentration, 100 g of puree provides 251% of the recommended daily vitamin C
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requirement for adults (FAO/WHO, 2001). The reported vitamin C contents of Australian and
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Pohnpei noni fruits fall within the range observed in the noni fruit puree (Shovic & Whistler,
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2001; Chunhieng et al., 2005). Vitamin A in raw Pohnpei noni fruit is reported as undetectable, <
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3.5 retinol equivalents (RE) 100 g-1 (Shovic & Whistler, 2001). However, noni fruit puree from
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French Polynesia is found to contain appreciable quantities of β-carotene. As calculated from β-
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carotene concentration, the mean vitamin A content per 100 g of puree is 318.17 RE. The joint
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FAO/WHO recommendation for average vitamin A daily intake by adults is 270 RE for females
336
and 300 RE for males (FAO/WHO, 1998). As such, noni fruit puree appears to have the
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potential to be a significant dietary source of vitamin A. The niacin content of processed noni
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fruit is great enough to have some nutritional impact, but will only be significant when larger
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quantities are consumed. At 100 g, the puree provides 18 to 21 % of the recommended niacin
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intake for adults (FAO/WHO, 2001). Thiamin, riboflavin, vitamin B6, vitamin B12, folic acid,
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pantothenic acid, and vitamin K were below detection limits. Processed noni fruit puree
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contains, but is not a significant source of, vitamin E and biotin.
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Potassium appears to be the most abundant mineral in processed noni fruit puree. This is also
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consistent with mineral analyses performed for Cambodian noni fruit juice and raw noni fruit
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from Pohnpei and Australia (Shovic & Whistler, 2001; Chunhieng et al., 2005; Peerzada et al.,
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19900. In the processed puree, it is more than four times the concentration of calcium, the next
348
most abundant mineral, although neither is present in nutritionally significant quantities. The
349
mean calcium content of the puree is greater than the amounts reported for the other sources of
350
noni fruit, with the exception of a minimal difference between Pohnpei fruit (48.2 mg 100 g-1 vs.
351
41.7 mg 100 g-1). Only two minerals are present in nutritionally significant amounts. In 100 g of
352
noni puree, manganese and selenium contents would meet approximately 18 to 26 % of the
353
recommended daily allowance for adults (Institute of Medicine, 2000; Institute of Medicine,
354
2001). The average manganese content of the processed puree, 0.47 mg 100 g-1, is five times
355
greater than the amount reported in raw Pohnpei noni fruit, 0.094 mg 100 g-1 (Shovic & Whistler,
356
2001). Selenium concentration has only been reported previously for Cambodian noni fruit
357
juice, with an amount equivalent to what is observed in the processed puree (Chunhieng et al.,
358

17
2005). The differences in mineral contents between the different sources of noni could be due
359
to stage of ripeness at harvest, climate, soil conditions, and genetic variability between the
360
sources (Cunningham et al., 2001; Razafimandimbison et al., 2010).
361
The phytochemical analyses reveal that iridoids are the major secondary metabolites produced by
362
noni fruit and are present in significant quantities following processing (Table 3). The DAA
363
content of the puree is similar to that of juice pressed from raw noni fruits from French
364
Polynesia, but the mean AA content is 1.8 times greater than that of the pressed juice, 38.79 mg
365
100 g-1 vs. 21.80 mg 100 g-1 (Deng et al., 2010 a). The average DAA content, dry weight basis,
366
of the processed puree is approximately 6.5 times higher than that reported in commercial
367
ground noni fruit powder from Hawaii, and 2.3 times greater than ripe noni fruit grown in a
368
botanical garden in Switzerland (Potterat et al., 2007). Comparing against the same samples, the
369
mean AA concentration in the puree is 2.9 and 5.5 times greater than in ground noni fruit powder
370
and the botanical garden fruit, respectively. The differences observed between the botanical
371
garden sample and the puree are likely due to differences in growing environments. The lower
372
iridoid levels in the commercial fruit powder from Hawaii might be due to aggressive drying
373
methods at elevated temperatures, but additional research would be required to confirm this.
374
375
Scopoletin, rutin, and quercetin were also present after processing (Table 3). The scopoletin
376
content of the puree falls within the range reported for juice pressed from raw noni fruits from 14
377
different islands in the Pacific and Indian Oceans (Basar & Westendorf, 2010). The rutin content
378
of the puree falls within the lower range reported for some commercial noni juice samples, but
379
the quercetin content is almost double that of the highest amount reported in any of these
380
samples (Deng et al., 2010 b). Quercetin is an aglycone of rutin, and its increased concentration
381

18
in the puree is likely the result of rutin glycolysis during processing and pasteurization (Rohn et
382
al., 2007).
383
384
In the puree, the total iridoid content was 20 times greater than the combined concentrations of
385
the other three phytochemicals. Deacetylasperulosidic acid accounted for 78% of the total
386
iridoid content. Due to their prevalence in noni fruit, both iridoids may be used as markers for
387
identification of products containing authentic noni ingredients. The reported bioactivities of
388
iridoids correlate well to several of the in vitro and in vivo bioactivities reported for noni fruit
389
juice and noni fruit extracts, including antioxidant, anti-inflammatory, immunomodulatory,
390
hepatoprotective, and hypolipidemic activities (Tundis et al., 2008; Wang et al., 2002). As the
391
major phytochemical in processed noni fruit, deacetylasperulosidic acid should be investigated
392
further for its role in the potential health benefits observed in human clinical trials of noni fruit
393
juice (Wang et al., 2009 a; Wang et al., 2009 b; Palu et al., 2008; Akinbo et al., 2006; Langford
394
et al., 2004; Ma et al., 2008).
395
396
No deaths or symptoms of toxicity were observed in the acute toxicity test. Animals also gained
397
appropriate weight (Table 4). The LD50 of noni iridoids was determined to be >340 mg/kg bw.
398
In the primary DNA damage test in E. coli PQ37 (Table 5), the mean induction factors for DAA
399
and AA, at 1000 µg mL-1, were 1.07 and 1.09, respectively. At all concentrations tested, DAA
400
and AA did not induce any SOS repair at a frequency significantly above that of the blank.
401
Statistically, induction factors were no different than that of the blank, and all results remained
402
well below the two-fold criteria for genotoxicity. SOS-chromotest results have a high level of
403
agreement (86%) with those from the reverse mutation assay (Legault et al. 1994). Therefore,
404

19
the SOS-chromotest has some utility in predicting potential mutagenicity, in addition to primary
405
DNA damage. The lack of DAA and AA toxicity in these tests is consistent with the results of
406
toxicity tests of noni fruit juice (West et al., 2009 a; West et al., 2009 b; Westendorf et al., 2007).
407
408
In the anti-genotoxicity test, 4NQO, exhibited obvious genotoxicity, inducing SOS repair more
409
than 8-fold above that of the vehicle blank. But the induction factors of 4NQO plus DAA or AA,
410
were the same as those of DAA or AA alone (Table 6), with no statistical difference from that of
411
the vehicle blank. The reductions in genotoxicity from 250 µg mL-1 DAA and AA were 98.96
412
and 99.22 %, respectively. Therefore, the genotoxic activity of 4NQO was almost entirely
413
abolished by the addition of either iridoid.
414
415
A double-blind human clinical trial revealed that ingestion of noni fruit juice reduced the amount
416
of aromatic DNA-adduct formation in the lymphocytes of current heavy cigarette smokers
417
(Wang et al.; 2009 b). 4NQO exhibits genotoxic activity in E. coli through the formation of
418
4NQO-guanine and 4NQO-adenine adducts (Ikenaga et al., 1975; Thomas et al., 1991). These
419
DNA lesions lead to the induction of the SOS repair mechanism. As such, the reduction in
420
4NQO genotoxicity by DAA and AA equates to a reduction in DNA adduct formation.
421
Therefore, the results of the current anti-genotoxicity test suggest the possible involvement of
422
these iridoids in noni juice’s DNA protective effects.
423
424
4. Conclusion
425
426

20
Processed noni fruit puree is a potential dietary source of vitamin C, vitamin A, niacin,
427
manganese, and selenium. Vitamin C is the major nutrient present, in terms of concentration.
428
The major phytochemicals in the puree are iridoids, especially DAA. The iridoids in noni did
429
not display any toxicity. On the other hand, these iridoids did display potential anti-genotoxic
430
activity. Even though processed noni fruit puree contained an appreciable quantity of vitamin C,
431
the average DAA content was approximately 22 % greater than that of vitamin C. These
432
findings suggest that DAA may play an important role in the biological activities of noni fruit
433
juice that have been observed in vitro, in vivo, and in human clinical trials.
434
435
Acknowledgements
436
This research was supported financially by Morinda Holdings, Inc., a manufacturer of noni fruit
437
puree. Some nutrient analyses were performed by Charles Tolson.
438
439
440

21
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441
442
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623
624
625

30
Table 1. Nutrient content of processed noni fruit puree
626
Assay
Mean
S.D.
Protein (g/100 g)
0.55
0.11
Fat (g/100 g)
0.10
0.12
Moisture (g/100 g)
91.63
1.98
Ash (g/100 g)
0.54
0.19
Carbohydrate (g/100 g)
7.21
1.81
Fructose (g/100 g)
1.07
0.39
Glucose (g/100 g)
1.30
0.36
Sucrose (g/100 g)
< 0.1
-
kilojoules/100g
135.56
31.73
Dietary Fiber (g/100 g)
2.01
0.27
Ca (mg/100 g)
48.20
16.04
K (mg/100 g)
214.34
56.91
Na (mg/100 g)
16.99
5.98
Mg (mg/100 g)
26.10
8.33
P (mg/100 g)
20.35
6.78
Fe (mg/100 g)
0.74
0.06
Cu (mg/100 g)
0.08
0.07
Mn (mg/100 g)
0.47
0.62
Se (mg/100 g)
0.01
0.01
Zn (mg/100 g)
0.06
0.07
β-carotene (µg/g)
19.09
12.15
Niacin (mg/g)
0.03
0.01
Vitamin C (mg/g)
1.13
0.77
Thiamin (mg/g)
< 0.018
-
Riboflavin (mg/g)
< 0.018
-
Vitamin B6 (mg/g)
< 0.018
-
Vitamin B12 (µg/g)
< 0.0012
-
Vitamin E (µg/g)
10.96
6.62
Folic acid (µg/g)
< 0.06
-
Biotin (µg/g)
0.02
0.00
Pantothenic acid (mg/g)
< 0.018
-
Vitamin K (µg/g)
< 0.10
-
S.D. = standard deviation
627
628

31
Table 2. Amino acid profile of processed noni fruit puree
629
Amino acid
Mean
S.D.
Alanine (mg/g)
0.45
0.04
Arginine (mg/g)
0.32
0.04
Aspartic Acid (mg/g)
0.80
0.08
Cystine (mg/g)
0.23
0.03
Glutamic Acid (mg/g)
0.64
0.05
Glycine (mg/g)
0.36
0.04
Histidine (mg/g)
< 0.1
-
Isoleucine (mg/g)
0.29
0.01
Leucine (mg/g)
0.38
0.02
Lysine (mg/g)
0.25
0.04
Methionine (mg/g)
< 0.1
-
Phenylalanine (mg/g)
0.21
0.05
Proline (mg/g)
0.26
0.03
Serine (mg/g)
0.27
0.02
Threonine (mg/g)
0.27
0.03
Tryptophan (mg/g)
< 0.1
0.00
Tyrosine (mg/g)
0.25
0.03
Valine (mg/g)
0.36
0.03
630
631
Table 3. Phytochemical content of processed noni fruit puree
632
Assay
Mean
S.D.
Deacetylasperulosidic acid (mg/100 g)
137.61
13.69
Asperulosidic acid (mg/100 g)
38.79
9.18
Scopoletin (mg/100 g)
5.68
1.58
Rutin (mg/100 g)
1.42
0.84
Quercetin (mg/100 g)
1.59
0.71
633
634
Table 4. Acute toxicity test of noni iridoids
635
Animal
Sex
Animal
Number
Body Weight (g)
LD50
(mg iridoids/kg bw)
Before
After
S.D. Rat
Male
10
191.2 ± 5.9
216.1 ± 8.3
> 340.0
Female
10
192.8 ± 12.3
289.4 ± 12.3
> 340.0

32
636
637
Table 5. Primary DNA damage assay in E. coli PQ37
638
Compound
Concentration
(µg mL-1)
Induction factor
Deacetylasperulosidic acid
1000
1.07 ± 0.14
500
1.03 ± 0.02
250
1.06 ± 0.06
125
1.00 ± 0.08
62.5
1.05 ± 0.07
31.2
1.04 ± 0.08
15.6
1.03 ± 0.16
7.81
0.93 ± 0.13
Asperulosidic acid
1000
1.09 ± 0.03
500
1.07 ± 0.04
250
1.11 ± 0.16
125
1.02 ± 0.08
62.5
1.04 ± 0.13
31.2
0.99 ± 0.06
15.6
1.04 ± 0.11
7.81
1.01 ± 0.05
4NQO
1.25
8.69 ± 3.69*
* P < 0.05, compared to vehicle blank
639
640
641
Table 6. Anti-genotoxicity test in E. coli PQ37
642
Compound
Concentration
(µg mL-1)
Induction factor
Positive control (4NQO)
1.25
8.69 ± 3.69**
4NQO + deacetylasperulosidic acid
250*
1.08 ± 0.12
4NQO + asperulosidic acid
250*
1.06 ± 0.03
* DAA or AA concentration; 4NQO concentration is 1.25 µg mL-1
643
** P < 0.05, compared to vehicle blank
644
645