J Nat Med

DOI 10.1007/s11418-009-0327-7

NOTE

Morinda citrifolia Linn. (Rubiaceae) leaf extracts mitigate

UVB-induced erythema
Brett J. West Shixin Deng Afa K. Palu
C. Jarakae Jensen

Received: 17 July 2008 / Accepted: 17 February 2009

The Japanese Society of Pharmacognosy and Springer 2009

Abstract Morinda citrifolia Linn. (Rubiaceae) leaves

have been used in tropical folk medicine to treat topical
inflammation and burns. A carbomer gel base, containing
the ethanol extract and juice pressed from the leaves, was
evaluated for potential allergenic properties in a repeatinsult patch test in 49 volunteers. To investigate the topical
photo-protective properties, the combined ethanol extract
and leaf juice were evaluated in a UVB-induced erythema
model in 25 volunteers. The crude ethanol extract of
M. citrifolia leaves was also evaluated in vitro for potential
anti-inflammatory activity in a histamine H-1 receptor
antagonism assay. There was no evidence of allergenic
potential in the repeat-insult patch test. When the combination of ethanol extract and leaf juice was applied, the
UVB dose required to induce erythema was almost 3.5
times greater than with untreated skin (P \ 0.001). In the
histamine H-1 receptor-binding assay, the crude ethanol
extract of M. citrifolia leaves inhibited receptor binding by
57%. These results suggest that M. citrifolia leaves are safe
for topical use and may be useful in mitigating UVBinduced injury to the skin.
Keywords Morinda citrifolia
Noni

UVB-induced erythema
Abbreviations
UV
Ultraviolet light
HPLC High performance liquid chromatography

Introduction
Morinda citrifolia Linn. (Rubiaceae), commonly known as
noni, is a small to medium-sized tree that grows widely in the
tropics. The fruit and leaf of this plant have a history of safe
food use in the Pacific Islands and other regions [1, 2].
Additionally, the plant has been widely used in tropical folk
medicine for a number of conditions [3]. The leaves were
found by indigenous people to be particularly useful in
treating various types of inflammation and poisonous fish and
insect stings [4, 5]. The leaves are reported to have been one
of the leading plants used in the Marquesas Islands to treat
topical inflammation, as well as in Rotuma for burns [6, 7].
UV light causes erythema by inducing cyclooxygenase2 expression, downstream histamine H-1 receptor agonism,
and free-radical generation which results from the oxidative stress induced through irradiation [811]. Further,
some compounds with antioxidant and anti-inflammatory
activities, including those derived from plant extracts, have
been found to reduce UV-induced erythema in human skin
[1214]. M. citrifolia leaves have been found to possess
antioxidant activities [15, 16], as well as to contain antiinflammatory compounds, such as E-phytol and deacetyl
asperuloside [17, 18]. As such, the current investigation
was conducted to corroborate the traditional use of the
leaves in treating topical inflammation, as well as to
determine the utility of the leaf as a photoprotective agent.

Materials and methods

B. J. West (&)
S. Deng
A. K. Palu
C. J. Jensen
Research and Development,
Tahitian Noni International, American Fork,
UT 84003, USA
e-mail: brett_west@tni.com

Ethics review
The human study protocols were reviewed by an institutional ethics review committee. Signed informed consent

123

J Nat Med

was obtained from all volunteers prior to initiation of the

studies, which were conducted in accordance with the
Declaration of Helsinki.
Plant material and sample preparation
M. citrifolia leaves were collected from the Society Islands
of French Polynesia. These were identified by local Polynesians and confirmed by a native botanist. A voucher
specimen (sample code AKP207) is deposited at the Tahitian Noni International Research Center, American Fork,
UT, USA. Then, 5 kg of fresh leaves was pressed in a cloth
copra press to extract 2.25 kg M. citrifolia leaf juice, which
was frozen for later use. Leaves were also dried and used to
prepare a crude ethanol extract by percolation of 100 g with
3 L ethanol. The ethanol was removed by evaporation under
vacuum to yield 10.6 g extract. The M. citrifolia leaf juice
extract was then thawed and combined with the crude ethanol extract (30:0.05 w/w) in a carbomer gel base to aid in
applying the extracts to the skin. The dose of the combined
extracts was 0.6 mg/cm2 skin.
Chemical profile of the combined extracts
The combined M. citrifolia leaf juice and ethanol extract
(3 g) was dissolved in 25 mL ethanol filtered with 0.2 lM
PTFE syringe filter prior to HPLC injection. Chromatographic separation was performed on a C18 column
(4.6 9 250 mm; 5 lm) with a 0.1% trifluoroacetic acid
(TFA) and MeOH gradient mobile phase at 1.0 mL/min.
The MeOH:TFA (%:%) gradient was 8:92 for 35 min,
35:65 for 2 min, 99:1 for 10 min, and then 8:92 for 3 min.
The UV spectra were monitored by a photodiode array
detector in the range of 200450 nm and quantified at
365 nm.
Repeat-insult patch test
The inclusion criteria for the repeat-insult patch test were
healthy adult volunteers with no skin conditions and limited skin pigmentation that would not interfere with
reaction readings. Those excluded were persons with
known sensitivities to cosmetics, those who were pregnant
or lactating, or those who, within 30 days prior to the test,
had used medication that might prevent or limit skin
reactions, such as antihistamines and corticosteroids. The
gel base containing M. citrifolia leaf extracts was evaluated
in 46 females and 3 males. During the induction phase,
sodium lauryl sulfate (SLS) served as an adjuvant and was
mixed with the gel base containing the leaf extracts (3%
SLS w/w).
Occlusive application of 25 lL of the mixture to the
back was accomplished with a Finn Chamber and held in

123

place for 48 h with Scanpor tape. For each volunteer,

application of the mixture occurred five times at the same
site on the back, with 48 h rest between applications. After
the induction phase, the skin of the volunteers was not
exposed for at least 7 days. Following this rest period, the
skin of the volunteers was exposed to the gel base containing the leaf extracts, without SLS, by occlusive
application for 48 h at a site different from the one used
during induction. For 72 h following application, subjects
were evaluated for possible delayed reaction, including
redness, itching, and irritation. The skin of the volunteers
was scored for reactions according to the International
Contact Dermatitis Research Group and under the supervision of a consulting dermatologist [23]. Reactions were
scored on a scale of 0 (no reaction) to 4 (erythema, induration, and bullae).
UVB-induced erythema model
The efficacy of the leaf extracts in mitigating UVB-induced
erythema was evaluated in 25 adult human volunteers.
Volunteers included 3 males and 22 females, aged 21
58 years. All volunteers had Fitzpatrick skin type 2,
defined as skin that always burns easily and tans minimally
within the first 3045 min of sun exposure after a winter
season of no sun exposure. Excluded were those who were
currently under a physicians care or taking medication that
may interfere with the trial; those with a history of disease
(e.g., skin cancer, diabetes, lupus) that might increase the
risk associated with UV exposure or of adverse effects
from sun exposure; those who were pregnant or lactating;
those with a history of hypersensitivities to skin-care
products, or with blemishes, sunburn, suntan, scars, dermal
lesions, or pigmentation at the test sites.
Within the infrascapular area of the back, both left and
right of the midline, 50-cm2 test sites were outlined with a
gentian violet marker. Five test sites on one side of the
midline were exposed in series, with a 25% increase in
exposure time at each site, to determine the minimal erythemal dose (MED). The MED is the smallest dose of UVB
radiation, in J/m2, required to produce minimally discernable erythema, or redness of the skin, 2224 h after
exposure. A 150-W xenon arc solar simulator with a continuous emission spectrum range of 290320 nm was used
as the UVB light source (Solar Light Company, Philadelphia, PA, USA). The UVB radiation emitted was
monitored and controlled during exposure with a UV dose
control system, model DCS-1 UV meter/dose controller
system (Solar Light Company).
After the MED of the untreated skin was determined for
each subject, the gel base containing the extracts was
evenly applied with a volumetric syringe to the 50-cm2 test
sites within the infrascapular area opposite the one

J Nat Med

previously exposed. An 8% homosalate emulsion was also

applied as a reference standard. A Woods lamp was used
to verify evenness of the applications. Fifteen minutes after
applying the extracts, a series of test sites in this area were
exposed to increasing doses of UVB radiation, beginning
with the MED for untreated skin. Evaluation of the leafextract test sites was made 2224 h after exposure. The
MED with the leaf extracts was determined based on the
same criteria as for the untreated skin. The visual-discrimination proficiency of the person evaluating the test
sites had been demonstrated by the FarnsworthMunsell
100 Hue Test, administered by an ophthalmologist [24].

50 mM TrisHCl buffer, pH 7.4, 2 mM MgCl2, 100 mM

NaCl, and 250 mM sucrose. Nonspecific binding was
determined by incubation in the presence of 1 lM pyrilamine. The receptor/[3H]pyrilamine complexes were
collected by vacuum filtration through Whatman GF/B
filters and washed with buffer. Radioactivity was measured
by scintillation counting and was used to determine
[3H]pyrilamine-specific binding. Inhibition of histamine
H-1 receptor binding was expressed as a percentage based
on the differences observed in the presence and absence of
the extract.

Data analysis

Results and discussion

Summary statistics were calculated, and the MEDs at test

sites of untreated skin, with combined leaf extracts, and
with 8% homosalate were compared using the Mann
Whitney U test [25], after the ShapiroWilk test [26]
revealed that the data are not normally distributed.

During the challenge of the repeat-insult patch test of a

carbomer gel containing crude M. citrifolia leaf juice and
ethanol extracts, there were no reactions in any of the
volunteers. Therefore, the M. citrifolia leaf extracts are
apparently suitable for use in skin applications. The average MED at untreated skin sites and those protected by
application of crude M. citrifolia leaf extracts or 8%
homosalate are compared in Table 1. The UVB dose
required to induce erythema at the protected sites was
almost 3.5 times greater than the MED of untreated skin
(P \ 0.001). The level of protection was similar for both
M. citrifolia extract-treated and 8% homosalate-treated
sites (P [ 0.01). UV-induced erythema has been used
previously as an experimental model for studying the
topical anti-inflammatory properties of drugs [19, 20]. In
this investigation, the increased UV dose necessary to
produce erythema indicates that M. citrifolia leaf extracts
mitigate the effects of UV light and provide some measure
of defense against localized inflammation (topical microswelling).
The chemical profile, according to HPLC (Fig. 1), of the
combined M. citrifolia leaf juice and ethanol extract
included rutin, kaempferol 3-O-rutinoside, quercetin,
kaempferol, and pyropheophorbide a concentrations of
0.692, 0.165, 0.148, 0.0507, and 0.0651 mg/g, respectively.
Components of the leaves, such as quercetin, have been
observed to inhibit histamine H-1 receptor binding

Histamine H-1 receptor antagonism assay

The crude ethanol extract of M. citrifolia leaves was
evaluated in vitro for potential anti-inflammatory activity
in a histamine H-1 receptor antagonism assay, following a
previously described method [27], with some modification.
The extract (100 lg/mL) was incubated for 180 min at
25C with 1.2 nM of [3H]pyrilamine in culture with
Chinese hamster ovary cells expressing recombinant
human H-1 receptors. The incubation buffer contained
Table 1 Minimal erythemal doses (J/m2) in human volunteers

Mean SD

No treatment

Combined
M. citrifolia leaf
extracts

8% Homosalate

12.46 3.18

42.98 7.82*

53.21 13.68*

Minimum

5.42

30.33

23.85

Maximum

18.45

60.89

73.80

SD Standard deviation
* P \ 0.001, compared to no treatment

Fig. 1 HPLC profile of

M. citrifolia leaf juice and
ethanol extract mixture (30:0.05
w/w). Rutin (1), kaempferol
3-O-rutinoside (2), quercetin
(3), kaempferol (4), and
pyropheophorbide a (5) are
identified

123

J Nat Med

[21, 22]. Previous experiments have also revealed that

histamine H-1 receptor antagonists inhibit UV-induced
erythema [10], revealing that histamine production might
be involved in its development. In the histamine H-1
receptor-binding assay, the crude ethanol extract of
M. citrifolia leaves inhibited receptor binding by 57%. As
binding to the H-1 receptor is necessary for histamine to
exert its inflammatory effect, these results suggest one
possible mechanism by which M. citrifolia leaves reduce
inflammation; this is consistent with the reported traditional use of these in treating poisonous fish and insect
stings.
The observed histamine H-1 receptor antagonism supports the assumption that the protective effect involves
anti-inflammatory activity. Additionally, the previously
demonstrated antioxidant activity of the leaf also suggests
that the scavenging of free radicals is another mechanism
whereby the UV damage is mitigated.

12.

13.

14.

15.

16.

17.

18.

References
1. West BJ, Jensen CJ, Westendorf J (2006) Noni juice not hepatotoxic. World J Gastroenterol 12:36163619
2. West BJ, Tani H, Palu AK, Toslon CB, Jensen CJ (2007) Safety
tests and antinutrient analyses of noni (Morinda citrifolia L.) leaf.
J Sci Food Agric 87:25832588
3. Morton JF (1992) The ocean-going noni, or Indian Mulberry
(Morinda citrifolia, Rubiaceae) and some of its colorful relatives. Econ Bot 46:241256
4. Cambie RC, Ash J (1994) Fijian medicinal plants. CSIRO,
Canberra, p 356
5. Dittmar A (1993) Morinda citrifolia L.uses in indigenous
Samoan medicine. J Herbs Spices Med Plants 1(3):7792
6. Brown FBH (1935) Flora of southeastern Polynesia. III. Dicotyledons. Bernice P. Bishop Museum Bulletin 130, Honolulu, pp
1386
7. McClatchey WC (1993) Studies on the ethnobotany of the island
of Rotuma. Masters Thesis, Brigham Young University, Provo
8. Buckman SAY, Gresham A, Hale P, Hruza G, Anast J, Masferrer
J, Pentland AP (1998) COX-2 expression is induced by UVB
exposure in human skin: implications for the development of skin
cancer. Carcinogenesis 19:723729
9. Malaviya R, Malaviya R, Uckun FM (2000) Anti-inflammatory
activity of 2,4,6-trihydroxy-p-methoxyphenyl-acetophenone
(compound D-58). Dermatology 201:337342
10. Grundmann JU, Bockelmann R, Bonnekoh B, Gollnick HP
(2001) UV erythema reducing capacity of mizolastine compared
to acetylsalicylic acid or both combined in comparison to indomethacin. Photochem Photobiol 74(4):587592
11. Spagna G, Tomaino A, Cimino F, Barbagallo RN, Ventura D,
Bonina F, Saija A (2002) Chemical analysis and photoprotective

123

19.

20.

21.

22.

23.
24.

25.

26.
27.

effect of an extract of wine from Jacquez grapes. J Sci Food Agric

82:18671874
Montenegro L, Bonina F, Rigano L, Giogilli S, Sirigu S (1995)
Protective effect evaluation of free radical scavengers on UVB
induced human cutaneous erythema by skin reflectance spectrophotometry. J Cosmet Sci 17:91103
Elmets CA, Singh D, Tubesing K, Matsui M, Katiyar S, Mukhtar
H (2001) Protective effect evaluation of free radical scavengers
on UVB induced human cutaneous erythema by skin reflectance
spectrophotometry. J Am Acad Dermatol 44:425432
Tanaka S, Sato T, Akimoto N, Yano M, Ito A (2004) Prevention
of UVB-induced photoinflammation and photoaging by a polymethoxy flavonoid, nobiletin, in human keratinocytes in vivo and
in vitro. Biochem Pharmacol 68:433439
Sang S, Chenk X, Zhu N, Stark RW, Badmaev V, Ghai G, Rosen
RT, Ho CT (2001) Flavonol glycosides and novel iridoid glycoside from the leaves of Morinda citrifolia. J Agric Food Chem
49:44784481
Mohd-Zin Z, Hamid AA, Osman A, Saari N (2006) Antioxidative
activities of chromatographic fractions obtained from root, fruit
and leaf of mengkudu (Morinda citrifolia L.). Food Chem
94:169178
Saludes JP, Garson MJ, Franzblau SG, Aguinaldo AM (2002)
Antitubercular constituents from the hexane fraction of Morinda
citrifolia Linn. (Rubiaceae). Phytother Res 16:683685
Takashima J, Ikeda Y, Komiyama K, Hayashi M, Kishida A,
Ohsaki A (2007) New constituents from the leaves of Morinda
citrifolia. Chem Pharm Bull 55:343345
Abeyama K, Eng W, Jester JV, Vink AA, Edelbaum D, Cockerell
CJ, Bergstresser PR, Takashima A (2000) A role for NF-kBdependent gene transactivation in sunburn. J Clin Invest
105:17511759
Bickel A, Dorfs S, Schmelz C, Forster C, Uhl W, Handwerker
HO (1998) Effects of antihyperalgesic drugs on experimentally
induced hyperalgesia in man. Pain 76:317325
Shimizu M, Tomoo T (1994) Anti-inflammatory constituents of
topically applied crude drugs. V. Constituents and anti-inflammatory effect of Aoki, Aucuba japonica Thunb. Biol Pharm Bull
17:665667
Melzig MF, Pertz HH, Krenn L (2001) Anti-inflammatory and
spasmolytic activity of extracts from Droserae Herba. Phytomedicine 8(3):225229
Rietschel RL, Fowler JF (1995) Fishers contact dermatitis, 4th
edn. Williams and Wilkins, Baltimore, p 836
Farnsworth D (1957) The FarnsworthMunsell 100 Hue test for
the examination of colour discrimination: manual. Munsell Colour, Baltimore
Mann HB, Whitney DR (1947) On a test of whether one of two
random variables is stochastically larger than the other. Ann Math
Stat 18:5060
Shapiro SS, Wilk MB (1965) An analysis of variance test for
normality (complete samples). Biometrika 52:591611
Kelly JX, Smilkstein MJ, Cooper RA, Lane KD, Johnson RA,
Janowaky A, Dodean RA, Hinrichs DJ, Winter R, Riscoe M
(2007) Design, synthesis, and evaluation of 10-N-substituted acridones as novel chemosensitizers in Plasmodium falciparum.
Antimicrob Agents Chemother 51:41334140