Hepatotoxicity and subchronic toxicity tests of Morinda citrifolia (noni) fruit

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1 Vol.34, No.5, , Letter Hepatotoxicity and subchronic toxicity tests of Morinda citrifolia (noni) fruit Brett J. West, Chen X. Su and C. Jarakae Jensen Research and Development, Tahitian Noni International, 737 East 1180 South, American Fork, Utah 84003, USA (Received July 21, 2009; Accepted July 28, 2009) ABSTRACT Morinda citrifolia (noni) fruit juice has been approved as a safe food in many nations. A few cases of hepatitis in people who had been drinking noni juice have been reported, even though no causal link could be established between the liver injury and ingestion of the juice. To more fully evalu- in vitro - - fruit puree did not decrease HepG2 cell viability or induce neutral lipid accumulation and phospholipidosis. There were no histopathological changes or evidence of dose-responses in hematological and clinical chemistry measurements, including liver function tests. The no-observed-adverse-effect level (NOAEL) juice is unlikely to induce adverse liver effects. Key words: Morinda citrifolia INTRODUCTION Morinda citrifolia L., commonly known as Indian Mulberry or noni, is a small tree that is widely distributed across the tropics. In Okinawa, it is known as yaeyamaaoki. Noni fruit has a history of use as food and for the Asians, and those in the tropical regions of the Western Hemisphere (Morton, 1992). A commercial source of noni juice from French Polynesia was approved by the European Union as a safe novel food (European Commission, 2003). The production and use of noni fruit juice has increased greatly, with more than 80 million liters from French Polynesia alone being consumed worldwide since cern has arisen regarding a few cases of hepatitis in peo- et al., 2008). However, the association remains controversial, as no causal link could be established between the cases of liver injury and ingestion of the juice (European was previously evaluated in primary rat hepatocytes and in rat hepatoma cells (Westendorf et al., 2007). As the use of noni based products is increasing, the current investiga- potential of noni fruit in a human liver cell line. There is a large degree of variability in content, composition, and postharvest processing methods of commercial noni juice products (West et al., 2006). Therefore, a subchronic oral an important benchmark for understanding the safety of noni juice, without the potential confounding variables associated with many commercial products. MATERIALS AND METHODS Test material Noni fruits were harvested in French Polynesia and allowed to fully ripen. The fruit was then processed into a puree by mechanical removal of the seeds and skin at a good manufacturing certified fruit processing facility in Mataiea, Tahiti. For the in vitro Correspondence: Brett West ( brett_west@tni.com)

2 582 B.J. West et al. 10 test concentrations. The highest test concentration was 150 μg/ml, which is equivalent to a raw noni fruit con- ty test, the puree was freeze-dried to a 13-fold concentration. In vitro tests Human liver cells, HepG2 cell line (American Type Culture Collection), were seeded in collagen I coated microplates at 4,000 cells/well and incubated for 24 hr at 5% CO 2 and 37 C in minimum essential medium, 10% fetal bovine serum, 1% nonessential amino acids, 1% ala- al dilutions of the test samples were added to the wells, blanks and positive controls were also included. The positive controls were amitriptyline (Hall et al., 1993) and cyclosporine A (Bäckman et al., 1991). For detection of phospholipidosis detection reagent (Invitrogen, Carlsbad, controls. The HepG2 cells were incubated in triplicate in the presence of the test compounds or controls for 48 hr. For the detection of neutral lipid accumulation and cellu- - lipid stain (Invitrogen) and 4,6-diamidino-2-phenylindole, dilactate, following incubation. Fluorescence microscopy was conducted with an automated cell imager. A emission wavelengths. Cellular growth was measured at lengths for 4,6-diamidino-2-phenylindole, dilactate. Viable cell count was determined by signal intensities and was calculated as a percentage relative to the blanks. Nonlinear regression was used to determine the concentration at which inhibition of 50% cell growth (IC 50 ) occurs. Intracellular phospholipid content was monitored at 492 nm and 535 nm. Neutral lipids were measured at 575 nm tion of phospholipidosis and neutral lipid accumulation. IC 50 and induction concentrations were calculated by non- parameter sigmoidal dose response model. Subchronic oral toxicity test and 40 females, grams) were assigned to one of three dose groups or a control group. Three dose preparations of freeze-dried noni fruit puree were prepared by dissolving in distilled water at 17.2, 34.3, and 68.6 g/100 ml. The dose preparations were administered by gastric intubation at 10 ml/kg body weight, resulting in daily doses of 1.72, 3.43, and 6.86 g/kg body weight (bw). The fruit juice/kg bw, 10 ml greater than the previously established no-observed-adverse-effect-level (NOAEL) for a commercial noni juice product. The animals assigned to the control group received distilled water. Water and rodent feed were available to all groups ad libitum. Animals were weighed weekly and observed for symp- ule, blood was collected from each animal for standard laboratory analyses. Hematology and clinical chemistry measurements included red and white blood cell counts, platelet count, differential white blood cells, hemoglob- ferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen, creatinine, cholesterol, triglycerides, glucose, total protein, and albumin. Gross necropsies were also performed on the animals. The liver, spleen, heart, kidneys, adrenals, thymus, ovaries, testes, stomach, and duo- testes were also weighed and compared between groups. Means and standard deviations of continuous variables were calculated, with intergroup differences evaluated by analysis of variance and compared against historical rang- of pathological changes, compared to the control group, were evaluated by the chi-square test. Table 1. Compound Viable cell count IC 50 Neutral lipid induction Phospholipidosis induction amitriptyline cyclosporin A > 120 > 150 > 150 > 150

3 583 Table 2. 0 g/kg b.w g/kg b.w g/kg b.w g/kg b.w. Female Male Female Male Female Male Female Male White blood cells (10 9 /l) 8.04 ± ± ± 2.93 * 8.37 ± ± 2.91 * 8.44 ± ± ± 3.15 Red blood cells (10 12 /l) 7.09 ± ± ± 0.40 * 6.95 ± ± 0.20 * 7.10 ± ± 0.24 * 7.04 ± 0.43 Platelets (10 12 /l) ± ± ± ± ± ± ± ± Hemoglobin (g/l) ± ± ± 6.3 * ± ± 5.3 * ± ± 4.0 * ± 7.1 Lymphocytes (%) 76.6 ± ± ± ± ± ± ± ± 9.2 Neutrophils (%) 15.4 ± ± ± ± ± ± ± ± 7.7 ALT (U/l) 31.6 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 16.5 ALP (U/l) 60.8 ± ± ± ± ± ± ± ± 33.9 Urea nitrogen (mmol/l) 6.23 ± ± ± ± 0.86 * 6.30 ± ± ± ± 0.64 Creatinine (μmol/l) 72.0 ± ± ± ± ± ± ± ± 9.5 Cholesterol (mmol/l) 1.96 ± ± ± ± 0.40 * 1.89 ± ± ± ± 0.27 Triglycerides (mmol/l) 0.53 ± ± ± ± 0.17 * 0.54 ± ± ± ± 0.29 Glucose (mmol/l) 5.56 ± ± ± ± ± ± ± ± 0.30 Total protein (g/l) 76.6 ± ± ± ± ± ± ± ± 13.5 Albumin (g/l) 40.2 ± ± ± ± ± ± ± ± 1.6 * P

4 584 B.J. West et al. RESULTS AND DISCUSSION on viable cell count, neutral lipid accumulation, and phos- icant (5-fold) induction of phospholipidosis and neutral - 50 > 150 μg/ml. from fruits, such as strawberries and plums, are known to inhibit HepG2 cell growth at > 100 ug/ml, especially with more than 4 hr incubation. This inhibition is associated with the presence of polyphenols widely distributed in fruits and vegetables, namely quercetin, chlorogenic acid, and epicatechin (Ramos et al., 2005). These com- et al., 2007; Mohd Zin et al., 2007). - organ weights or incidence of histopathological changes. There was no evidence of a dose-response in hematological and clinical chemistry measurements (Table 2) and no - but the values were typical of normal ranges in healthy ± standard deviation, white blood cell counts of ± 9 9 cells/l, respectively, 9 red blood cell counts measured in all female groups, and corresponding mean hemoglobin levels, were within the 12 cells/l and ). The only statistically significant differences group. Triglycerides were lower than the controls, while cholesterol was slightly higher, but both were still within the normal control ranges of mmol/l and ). Even though mean urea nitrogen in the male mid dose group was slightly greater than that of control group, it more closely approached the historical average of 9.46 ± 0.84 mmol/l in healthy controls (Lillie et al., 1996) and did not follow any dose-related trend. There were no ele- indicating a lack of hepatocellular and cholestatic liver ingestion of freeze-dried noni fruit puree, with a NOAEL greater than 6.86 g/kg. observed, demonstrating the usefulness of this cell line (O Brien et al et al., 2005). The results of the current investigation, in a human cell line, agree with the observations of Westendorf et al. (2007) in primary rat hepatocytes and rat hepatoma cells. Cell viability was unaffected by increased concentrations of noni induced, indicating a lack of hepatic necrosis. Liver func- - orate previous conclusions that consumption of noni fruit juice is safe, and that it is unlikely to induce adverse liv- noni fruit provides the needed bench mark for evaluating noni juice products in general. If good manufacturing and hygiene processes are followed in the production of noni juice, these should be safe for human consumption. However, this evaluation does not apply to juices that result from improper processing methods, contain microbial contaminants, or have been adulterated with other unsafe ingredients. ACKNOWLEDGMENTS The contributions of Bing-nan Zhou, Yulia Ovechkina, and Karen Marcoe to this project, as well as the contribu- - ly acknowledged. REFERENCES Brunk, U. (1991): Modulation of metabolism in HepG2 cells Mol. Pathol., 54, noni (Morinda citrifolia) collected in Tahiti. J. Nat. Prod., 70, authorising the placing on the market of noni juice (juice of the fruit of Morinda citrifolia L.) as a novel food ingredient under Regulation (EC) No 258/97 of the European Parliament and of the Council. Official Journal of the European Union, L144, 12. the Commission related to the safety of noni juice (juice of the fruits of Morinda citrifolia 376, 1-12.

5 585-79, Lillie, L.E., Temple, N.J. and Florence, L.Z. (1996): Reference val- - 15, pound from Fruit of Mengkudu (Morinda citrifolia L.). Int. J. Food Properties, 10, Morton, J.F. (1992): The ocean-going noni, or Indian Mulberry (Morinda citrifolia, Rubiaceae) and some of its colorful relatives. Econ. Bot., 46, Bernardi, P., Brain, P. and Hougham, C. (2006): High concord , , effects of food-derived polyphenols on the viability and apoptosis of a human hepatoma cell line (HepG2). J. Agric. Food Chem., 53, cells and for 60 compounds on HeLa, ECC-1 and CHO cells. 19, associated with morinda citrifolia (noni). Am. J. Gastroenterol., 103, (2006): Mineral variability among 177 commercial noni juices. 57, Morinda citrifolia) fruit juice. J. Agric. Food Chem., 55,