The effects of Morinda citrifolia L. (noni) on the immune system:
Its molecular mechanisms of action
Afa K. Palu∗, Anne Hirazumi Kim, Brett J. West, Shixin Deng,
Jarakae Jensen, Leland White
Noni Benefits Research Department, Tahitian Noni International Research Center,
737 East 1180 South, American Fork, UT 84003, USA
The aim of this study was to investigate the mechanisms involved in the immunomodulatory effects of Morinda citrifolia L. (noni) in vitro and in
inhibit cannabinoid 1 (CB1) receptors in a concentration-dependant manner. In vivo, oral administration of TNJ ad libitum for 16 days decreased
the production of IL-4, but increased the production of IFN-?. These results suggest that noni modulates the immune system via activating of the
CB2receptors, and suppressing of the IL-4, but increasing the production of IFN-? cytokines. It may also exert beneficial immunomodulation
effects in conditions involving inadequate immune responses.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Morinda citrifolia; Rubiaceae; Immunomodulation; IL-4; IFN-?; CB2
Morinda citrifolia L. (noni) from the family Rubiaceae has
been used by Polynesians for over 2000 years for two main
purposes. First, the roots and the barks have been used as a dye
for traditional and ceremonial clothes. Second, every part of the
plant has been used medicinally in treating a variety of ailments
(Wang et al., 2002; Palu et al., 2004).
The advent of Tahitian Noni®Juice (TNJ) in 1996 fuelled
an increase in scientific research into its possible effects on
human health. In addition to traditional uses, recent research
(Hirazumi et al., 1994, 1996; Hirazumi and Furusawa, 1999) on
Abbreviations: TNJ, Tahtian Noni®Juice, a commercial noni fruit juice
brand; CB2/CB1, cannabinoid receptors 1 & 2; IFN-?, interferon gamma; IL-4,
first developed in Roswell Park Memorial Institute which utilizes a bicarbonate
buffering systems; LPS, lipopolysaccharide; Con A, concanavalin A; FCS, fetal
calf serum; DMSO, dimethyl sulfoxide.
∗Corresponding author. Tel.: +1 801 234 3624; fax: +1 801 234 1158.
E-mail address: firstname.lastname@example.org (A.K. Palu).
the effect of the polysaccharide-rich ethanol-insoluble precipi-
tate of noni (noni-ppt) in the Lewis Lung Carcinoma in mice,
has prompted studies into the possible mechanisms involved
in its anticancer activity and immunity-enhancement effects.
Hirazumi et al. (1996) reported that noni-ppt may exhibit anti-
tumor effects in mice by acting as an immunomodulator.
Concerns have recently been raised about the safety of the
noni fruit juice for consumption (Mueller et al., 2000; Carr et
al., 2004; Millonig et al., 2005; Stadlbauer et al., 2005). How-
ever, the “sketchy details and limited number of cases reported
are inadequate to draw any conclusion” (West et al., 2006). Fur-
by the European Union and found to be acceptable for human
consumption (European Commission, 2002). They concluded
that, “there is not convincing evidence for a causal relation-
ship between the acute hepatitis observed in the case studies
reported and the consumption of noni juice” (European Food
Safety Authority, 2006).
Our study reports on the effects of noni on the immune
response in vitro and in vivo in mice, and its effect on
the cannabinoid receptors as underlying mechanisms for its
2.1. Noni samples
We used noni fruit juice concentrates (NFJC, Lot# 100275)
nesia and provided by Tropical Resources Inc. The TNJ (Lot#
2004-06029) was obtained from Tahitian Noni®International
Inc. The noni-ppt was isolated from the noni fruit puree from
French Polynesia provided to us by Tropical Resources Inc.,
and the isolation was in accordance with established protocol
(Hirazumi et al., 1994). Briefly, about 19L of noni fruit puree
were partitioned into about 32 aliquots of 580g each, loaded
into four anodized-aluminium cups (Beckman, CH-3.8), and
then centrifuged (Beckman GS-6 Centrifuge). This process was
repeated until all aliquots had been centrifuged, yielding a total
of about until 4L of juice. The juice was decanted and the pulp
discarded. Two litres of the juices were mixed with 2.0L of 200
Proof Ethanol (Fisher Scientific, USA) and mixed well using
T-Line 106A Laboratory Stirrer (Talboys Engineering Corp.,
Montrose, PA, USA) for 1h, after which, the precipitates were
discarded. An equal volume of 200 Proof Ethanol was again
mixed with the supernatant and mixed well as described above
air-dried under the hood yielding about 16.27g.
2.2. Radioligand binding assay for CB1
The binding affinities of the noni samples (NFJC and TNJ)
for CB1receptors were investigated using membranes contain-
ing CB1receptors obtained from human recombinant HEK-293
cells that were stably transfected with a plasmid encoding the
human cannabinoid CB1 receptors in triplicate, 0.5nM [3H]
CP-55,940 as a ligand, and 10?M R(+)-WIN-55,212-2, as a
non-specific ligand. A 1% solution of DMSO was used as the
carrier vehicle with a 90min incubation time at 37◦C. All reac-
tions were carried out in an incubation buffer containing 50mM
Tris–HCl, pH 7.4, 1mM EDTA, 3mM MgCl2, and 0.5% BSA,
in accordance with established protocol (Compton et al., 1993;
Rinaldi-Carmona et al., 1996).
2.3. Radioligand binding assay for CB2
Binding affinities of noni samples for CB2receptors were
determined using membranes CB2 receptors obtained from
human recombinant CHO (Chinese Hamster Ovary) K1 cells
that were stably transfected with a plasmid encoding the human
cannabinoid CB2 receptors in triplicate, 2.4nM [3H] WIN-
55,212-2 as a ligand and 10?M R(+)-WIN-55,212-2, as a
non-specific ligand with 1% DMSO as a carrier vehicle. All
reactions were carried out in an incubation buffer containing
5mg/mL BSA in 10mM HEPES, and pH 7.0 and in accordance
with established protocol (Munro et al., 1993).
2.4. TNJ immune stimulation in C57B/6J mice
A group of 10 C57BL/6J male mice (Jackson Lab, Bar Har-
bor, ME, USA) weighing approximately 14–18g, was divided
into two groups of five each (control and treatment). Their
handling and treatment was conducted in accordance with the
US guidelines for care and use of laboratory animals (NIH
publication 85–23, 1985). Each day, for 15 days, the control
and treatment groups were provided with clean bottles con-
taining 100mL of fresh water or TNJ, respectively. On day
16, the animals were sacrificed and the spleens and peritoneal
exudate cells (PEC) were harvested as described by Delves
2.5. Collection of peritoneal exudate cells and isolation of
Peritoneal was soaked with 70% ethanol and approximately
3mL of peritoneal exudate cells was withdrawn, washed three
times with cold RPMI (Sigma, USA) and centrifuged to yield
approximately 2×106cells. The spleen was removed and
placed in a sterile Petri dish containing RPMI 1640 and 10%
FCS. It was next cut into several small pieces and carefully
massaged between two glass slides. A small portion of the cell
suspension was then aspirated for microscopic determination of
2.6. ELISA analysis of IL-4, IFN-γ and IL-12p70 in
splenocytes and peritoneal exudate cells
Splenocytes and peritoneal exudate cells (PEC) from water-
and TNJ-treated mice were cultured in the presence of LPS,
Con A and noni-ppt for 16h. They were then centrifuged and
the supernatant collected. Aliquots of the supernatant were par-
titioned for cytokine testing. The IL-4, IFN-? and IL-12p70
cytokines present in the supernatant were quantified using
ELISA (Endogen®, Rockford, IL, USA) assay kits EMIL4 (IL-
4), EM1001 (IFN-?), and EMIL12 (IL-12) in accordance with
the manufacture’s instructions without any modifications of any
of the instructions in these bioassay kits. Briefly, 50?L of Plate
Reagent was added to each well and 50?L of standards and
ered and incubated for 2h at 37◦C. After which, the plate was
washed five times then 100?L of prediluted conjugate reagent
was added to each well, the plate was covered and incubated for
1h at 37◦C. The plate was washed five times then 100?L of
premixed TMB substrate solution was added to each well and
then the plate was developed in the dark at room temperature
for 30min. A 100?L of stop solution was added to each well to
stop the reaction and the absorbance was measured and results
3.1. CB1and CB2ligand binding assays
TNJ produced inhibition of [3H] CP-55,940 binding. At
concentrations of 1 and 5mg/mL, the CB1receptors showed
inhibitions of 14 and 10%, respectively. The CB2receptors, at
the same concentrations, showed stimulations of 54 and 160%,
Fig. 1. The production of IL-4 in water- and TNJ-treated mice from their
splenocytes incubated for 72h with Con A. PEC were cultured with different
NFJC produced stimulation of [3H] WIN-55,212-2 binding.
At concentrations of 1 and 5mg/mL, the CB1receptors showed
inhibitions of 13 and 172%, respectively. The CB2receptors, at
3.2. IL-4 production from splenocytes
TNJ-treated mice showed suppressed production of IL-4
compared to water-treated mice (Fig. 1).
3.3. IFN-γ Production from PEC
PECS from water- and TNJ-treated mice were evaluated for
IFN-? production using ELISA. Both water- and TNJ-treated
mice showed an increase in the IFN-? production. However,
tion than from water-treated mice (Fig. 2) when culturing with
media, TNJ, and LPS. Surprisingly noni-ppt seemed to have a
suppressing effect on IFN-? production whereas LPS increased
the production of IFN-? in the TNJ-treated mice.
Fig. 2. The production of IFN-? from PEC incubated for 16h in the presence
of media, noni-ppt and LPS.
specifically CB2, are also involved in: (1) mediating the pro-
tective signals that counteract the proinflammatory responses
(Massa et al., 2004), (2) inhibiting neuropathic pain (Ibrahim
et al., 2003) without psychoactive effects (Malan et al., 2003),
(3) suppressing microbial activation (Ehrhart et al., 2005) and
(4) protecting hippocampal neurons from excitotoxicity (Shen
and Thayer, 1998). Perhaps more relevant to this study, is the
ability of herbs such as Echinacea to activate CB2receptors and
In this study, 1 and 5% TNJ and NFJC, respectively, clearly
activate CB2, but inhibit CB1receptors. The binding activity of
TNJ for CB1was basically the same at each concentration, even
when the concentration increased fivefold. However, a signifi-
cant selectivity for CB2binding/activation was observed at both
centration (1–5mg/mL) also increased the binding/activation of
CB2receptors by about threefold. However, the binding activ-
ity of NFJC was different than that of TNJ with respect to CB2
to CB1receptors by 13%, but increased the binding/activation
of the ligand ([3H] WIN-55,212-2) to CB2receptors by 132%, a
10-fold increase. Interestingly, unpublished data from fraction-
ation of the crude noni fruit juice in our lab revealed that the
activity is mostly concentrated in the petroleum ether extract,
with a CB2binding activity of 25% at 100?g/mL concentra-
tion. This is especially noteworthy, since this concentration is
only one-tenth that of TNJ, but has a corresponding activity of
approximately one-half of its effect on CB2receptors at a con-
centration of 1mg/mL. Therefore, it seems that the active noni
compound(s) that show the immunomodulatory effects may be
concentrated within the petroleum ether extract. The selective
binding/activation effects of NFJC and TNJ on the CB2recep-
tors might explain another possible mechanism whereby noni
fruit juices may modulate the immune systems, in addition to
those described here.
The activation of CB2 receptors is shown in the sci-
entific literature to be involved in immunomodulation and
anti-inflammation. It has been reported that activation of CB2
receptors is involved in mediating the protective signals that
counteract the pro-inflammatory responses (Bonhaus et al.,
1998; Thomas et al., 1998; Howlett et al., 2002; McKallip et
al., 2002; Massa et al., 2004; Raduner et al., 2006). The CB2
receptor is localized, predominantly, in peripheral tissues such
as the spleen and hemopoietic cells (Munro et al., 1993). Micro-
bial pathogens that invade tissues are subsequently recognized
by the host cells as foreign. Consequently, the host triggers
the activation of both innate and adaptive immune responses.
However, the activation of the inflammatory response to the
infection depends on the amount of pro-inflammatory cytokines
and chemokines being released. This inflammatory response is
known to be tightly regulated to avoid tissue damage in the host
being activated, in both humans and animals, have been shown
to increase the production of endocannabinoids in response to
LPS and other stimuli. Hence, it is not surprising that metabo-
are produced and released by activated immune cells. In fact,
in vitro studies have shown that stimulation with LPS increases
the production of anadamide in the immune cells, which seems
to suggest that CB2is involved in the immune cell and cytokine
biology (Di Marzo et al., 1999; Klein et al., 2000a,b; Klein,
An aberrant increase in expression of IL-4 has been shown
in the literature to be involved in allergic responses and airway
inflammation (Ricci et al., 1997; Okano et al., 2000; Bergeron
et al., 2003; Nonaka et al., 2004). Increasing expression of IL-
4 is correlated with increased severity of the allergy. However,
the opposite effect between IFN-? and IL-4 is well documented
(Nguyen and Benveniste, 2000). The treatment with agents that
down-regulate IL-4 production, since it up-regulates MHC II in
al., 2001). Our studies showed that TNJ suppressed the produc-
tion of IL-4 compared to water. This agrees with the previous
report by Hirazumi et al. (1994), that noni-ppt suppressed the
IL-4 production in LLC-infected mice. TNJ increased the pro-
duction of IFN-?, which is known to be involved in activating
from splenocytes, in conjunction with its increased production
of IFN-?, suggest that TNJ modulates the immune system.
possible mechanism by which noni may modulate the immune
of the immune cytokines. Our results show that both TNJ and
NFJC potently activate CB2, but inhibit CB1receptors. Also,
TNJ suppressed IL-4, but increased the production of IFN-?.
mechanism described warrants further clinical research into the
effects of noni on the cannabinoid systems in vivo. Such studies
could thus clarify the dosage and actual mode of action on this
system and CNS in general for its possible effects on the signal
transduction pathways in humans.
Grateful appreciation is extended to my ancestors, and my
mother Silivia Latai Tulaki Palu, who as a Tongan traditional
healer, provided supplemental details on the current usages of
noni, as well as additional historical information on noni’s myr-
iad of medicinal applications.
a possible role in airway remodelling in asthma. Clinical and Experimental
Allergy 33, 1389–1397.
of adenylyl cyclise by cannabinoid receptor agonists: evidence for agonist-
specific trafficking of intracellular responses. The Journal of Pharmacology
and Experimental Therapeutics 287, 884–888.
Carr, M.E., Klotz, J., Bergeron, M., 2004. Coumadin resistance and the vitamin
supplement “Noni”. American Journal of Hematology 77, 103.
Compton, D.R., Rice, K.C., De Costa, B.R., Razdan, R.K., Melvin, L.S., John-
son, M.R., Martin, B.R., 1993. Cannabinoid structure-activity relationships:
correlation of receptor binding and in vivo activities. Journal of Pharmacol-
ogy and Experimental Therapeutics 265, 218–226.
Delves, P.J., 1994. Chapter 12: cellular activation. In: Hames, B.D., Rickwood,
Oxford, UK, pp. 229–254.
Di Marzo, V., Bisogno, T., De Petrocellis, L., Melck, D., Orlando, P., Wagner,
J.A., Unos, G., 1999. Biosynthesis and inactivation of the endocannabinoid
2-arachidonoylglycerol in circulating and tumoral macrophages. European
Journal of Biochemistry 264, 258–267.
European Food Safety Authority, 2006. Opinion on a request from the Com-
mission related to the safety of noni juice (juice of the fruits of Morinda
citrifolia). European Safety Authority Journal 376, 1–12.
F., Tan, J., Shytle, R.D., 2005. Stimulation of cannabinoid receptor 2 (CB2)
suppresses microglial activation. Journal of Neuroinflammation 2, 29.
European Commission, 2002. Scientific Committee on Food. Opin-
SCF/CS/NF/DOS/18 ADD 2 Final. December 11, 2002, available
from http://europa.eu.int/comm/food/fs/sc/scf/out151 en.pdf.
Hirazumi, A., Furusawa, E., 1999. An immunomodulatory polysaccharide-rich
substance from the fruit juice of Morinda citrifolia (noni) with antitumour
activity. Phytotherapy Researches 13, 380–387.
Hirazumi, A., Furusawa, E., Chou, S.C., Hokama, Y., 1994. Anticancer activity
of Morinda citrifolia (noni) on intraperitoneally implanted Lewis lung car-
cinoma in syngeneic mice. In: Proceedings of the Western Pharmacology
Society 37, pp. 145–146.
contributes to the anticancer activity of Morinda citrifolia (noni) fruit juice.
In: Proceedings of the Western Pharmacology Society 39, pp. 7–9.
Howlett, A.C., Barth, F., Bonner, T.I., Cabral, G., Casellas, P., Devane, W.A.,
twee, R.G., 2002. Proceedings of the International Union of Pharmacology
XXVII. Classification of Cannabinoid Receptors, 54, pp. 161–202.
Ibrahim, M.M., Deng, H., Zvonok, A., Cockayne, D.A., Kwan, J., Mata, H.P.,
Vandera, T.W., Lai, J., Porreca, F., Makriyannis, A., Malan, T.P., 2003.
Activation of CB2cannbinoid receptors by AM1241 inhibits experimen-
tal neuropathic pain: pain inhibition by receptors not present in the CNS.
In: Proceedings of the National Academy of Sciences 100, pp. 10529–
pression of the effect of interleukin (IL)-4 and IL-5. European Respiratory
Journal 17, 499–506.
Klein, T.W., 2005. Cannabinoid-based drugs as anti-inflammatory therapeutics.
Nature 5, 400–411.
tem and cytokine network. In: Proceedings of the Society for Experimental
Biology and Medicine, 225, pp. 1–8.
Klein, T.W., Newton, C.A., Nakachi, N., Friedman, H., 2000b. Delta-9-
Tetrahydrocannabinol treatment suppresses immunity and early IFN-
gamma, IL-12, and IL-12 receptor beta-2 responses to Legionella
pneumophila infection. The Journal of Immunology 164, 6461–6466.
2003. CB2 cannabinoid receptor agonists: pain relief without psychoactice
effects? Current Opinion in Pharmacology 3, 62–67.
Massa, F., Marsicano, G., Hermann, H., Cannich, A., Monory, K., Cravatt, B.F.,
Ferri, G.-L., Sibaev, A., Storr, M., Lutz, B., 2004. The endogenous cannabi-
noid system protects against colonic inflammation. The Journal of Clinical
Investigation 113, 1202–1209.
McKallip, R.J., Lombard, C., Fisher, M., Martin, B.R., Ryu, S., Grant, S.,
Nagarkatti, P.S., Nagarkatti, M., 2002. Targeting CB2 cannabinoid recep-
tors as a novel therapy to treat malignant lymphoblastic disease. Blood 100, Download full-text
Millonig, G., Sadlmann, S., Vogel, W., 2005. Herbal hepatotoxicity: acute hep-
atitis caused by a noni preparation (Morinda citrifolia). European Journal of
Gastroenterology and Hepatology 17, 445–447.
Mueller, B.A., Scott, M.K., Sowinski, K.M., Prag, K.A., 2000. Noni juice
(Morinda citrifolia): hidden potential for hyperkalemia? American Journal
of Kidney Diseases 35, 310–312.
Munro, S., Thomas, K.L., Abu-Shaar, M., 1993. Molecular characterization of
a peripheral receptor for cannabinoids. Nature 365, 61–65.
Nguyen, V.T., Benveniste, E.N., 2000. IL-4 Activated STAT-6 inhibits IFN-
gamma-induced cd40 gene expression in macrophages/microglia. The
Journal of Immunology 165, 6235–6243.
Nonaka, M., Pawankar, R., Fukumoto, A., Ogihara, N., Sakanushi, A., Yagi, T.,
2004. Induction of eotaxin production by interleukin-4, interleukin-13 and
lipopolysaccharide by nasal fibroblasts. Clinical and Experimental Allergy
Okano, M., Satoskar, A.R., Abe, M., Harn Jr., D.A., Nishizaki, K., Takeda,
Y., Yoshino, T., Brombacher, F., Satoskar, A.A., 2000. Interleukin-4-
independent production of Th2 cytokines by nasal lymphocytes and nasal
eosinophilia in murine allergic rhinitis. Allergy 55, 723–731.
Palu, A.K., Su, C., Zhou, B.N., Jensen, J., 2004. Morinda Citrifolia L.: a dual
inhibitor of COX-2 and 5-LOX enzymes. In: WorldNutra 5th International
Conference and Exhibition on Nutraceuticals and Functional Foods, p. 470.
Gertsch, J., 2006. Alkylamides from Echinacea are a new class of cannabi-
nomimetics. The Journal of Biological Chemistry 281, 14192–14206.
Ricci, M., Matucci, A., Rossi, O., 1997. Source of IL-4 able to induce the devel-
opment of TH2-like cells. Clinical and Experimental Allergy 27, 488–500.
F., Casellas, P., Ferrara, P., Le Fur, G., 1996. Characterization of two cloned
human CB1 cannabinoid receptor isoforms. Journal of Pharmacology and
Experimental Therapeutics 278, 871–878.
Shen, M., Thayer, S.A., 1998. Cannabinoid receptor agonists protect cultured
rat hippocampal neurons from excitotoxicity. Molecular Pharmacology 54,
Stadlbauer, V., Fickert, P., Lackner, C., Schmerlaib, J., Krisper, P., Trauner, M.,
Journal of Gastroenterology 11, 4758–4760.
Thomas, B.F., Gilliam, A.F., Burch, D.F., Roche, J.M., Seltzman, H.H., 1998.
Comparative receptor binding analyses of cannabinoid agonists and antag-
onists. The Journal of Pharmacology and Experimental Therapeutics 285,
G., 2002. Morinda citrifolia (Noni): a literature review and recent advances
in Noni research. Acta Pharmacological Sinica 12, 1127–1141.
West, B.J., Jensen, C.J., Westendorf, J., White, L.D., 2006. A safety review of
noni fruit juice. Journal of Food Science 71, R100–R106.