The Role of Endophytic Fungi in the Anticancer Activity of Morinda citrifolia Linn. (Noni)

Abstract

We hypothesize that the fungal endophytes of noni may possibly play a role in its overall pharmacological repertoire, especially since the perceived efficacy of the fruit in ethnomedicinal use is associated with the fermented juice. The foremost goal of this study is to explore the role of endophyte-derived secondary metabolites in the purported anticancer properties of noni. To that end, culturable endophytic fungi resident within the healthy leaves and fruit of the plant were isolated and identified by molecular sequence analysis of the 5.8S gene and internal transcribed spacers (ITS). Purified organisms were subjected to in vitro fermentation in malt extract broth for 8 weeks under anaerobic conditions at room temperature (25°C), in order to simulate the conditions under which traditional fermented noni juice is prepared. The cytotoxic potential of organic extracts derived from the fermented broths of individual endophytes was then tested against three major cancers that afflict humans. Twelve distinct endophytic fungal species were obtained from the leaves and 3 from the fruit. Three of the leaf endophytes inhibited the growth of human carcinoma cell lines LU-1 (lung), PC-3 (prostate), and MCF-7 (breast) with IC50 values of ≤10 g/mL.

Full text

Research Article
The Role of Endophytic Fungi in the Anticancer Activity of
Morinda citrifolia Linn. (Noni)
Yougen Wu,1,2 Sisay Girmay,1Vitor Martins da Silva,1Brian Perry,3,4
Xinwen Hu,2and Ghee T. Tan1
1Department of Pharmaceutical Sciences, Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo,
Hilo, HI 96720, USA
2Key Laboratory of Protection, Development and Utilization of Tropical Crop Germplasm Resources of Ministry of Education,
College of Horticulture and Landscape, Hainan University, Haikou 570228, China
3Department of Biology, University of Hawaii at Hilo, Hilo, HI 96720, USA
4CaliforniaStateUniversityEastBay,25800CarlosBeeBoulevard,Hayward,CA94542,USA
Correspondence should be addressed to Ghee T. Tan; gheetan@hawaii.edu
Received  September ; Accepted  October 
Academic Editor: Abid Hamid
Copyright ©  Yougen Wu et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
We hypothesize that the fungal endophytes of noni may possibly play a role in its overall pharmacological repertoire, especially
since the perceived ecacy of the fruit in ethnomedicinal use is associated with the fermented juice. e foremost goal of this
study is to explore the role of endophyte-derived secondary metabolites in the purported anticancer properties of noni. To that
end, culturable endophytic fungi resident within the healthy leaves and fruit of the plant were isolated and identied by molecular
sequence analysis of the .S gene and internal transcribed spacers (ITS). Puried organisms were subjected to in vitro fermentation
in malt extract broth for  weeks under anaerobic conditions at room temperature (∘C), in order to simulate the conditions under
which traditional fermented noni juice is prepared. e cytotoxic potential of organic extracts derived from the fermented broths
of individual endophytes was then tested against three major cancers that aict humans. Twelve distinct endophytic fungal species
were obtained from the leaves and  from the fruit. ree of the leaf endophytes inhibited the growth of human carcinoma cell lines
LU- (lung), PC- (prostate), and MCF- (breast) with IC50 values of ≤ 𝜇g/mL.
1. Introduction
Morinda citrifolia L. (Rubiaceae) is a medicinal plant that
has survived the test of millennia. It is best known by its
Polynesian vernacular name, noni. e small evergreen tree
is native to Southeast Asia but pantropical in its distribution.
Noni has attained cross-cultural relevance as both a dietary
supplement and a complementary and alternative herbal
treatment for indications such as cancer, inammation, and
diabetes []. Noni is included in the traditional pharma-
copoeias of Polynesia, South and Southeast Asia, North-
eastern Australia, and the Caribbean, where it is embraced
for a wide variety of ailments ranging from infections to
cancer []. Its popularity in today’s herbal market may be
attributed, in part, to claims of noni being a panacea for a
vast array of chronic conditions including arthritis, diabetes,
and hypertension [, ]. While the fruit, ower, bark, and
root of Morinda citrifolia have all been employed for diverse
medicinal purposes, the leaves are associated with the most
prevalent traditional use predominantly as a topical remedy
for sores, cuts, and inammation. e current popular use
of the juice of the noni fruit bears less correlation with
traditional Polynesian practices.
e enduring ethnomedicinal uses and economic impor-
tance of noni have provided impetus for studies that aim
to verify the diverse therapeutic and nutritional claims
attributed to the plant. Despite the fact that more than 
secondary metabolites have been isolated from noni [, ], the
pharmacological signicance of these phytochemical con-
stituentsintheallegedecacyofnonijuiceinhumanshasyet
Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2015, Article ID 393960, 8 pages
http://dx.doi.org/10.1155/2015/393960

 Evidence-Based Complementary and Alternative Medicine
to be unraveled. Anecdotal evidence, personal testimonials,
and case reports of quality of life improvements and the
overall life-extending eect of noni fruit juice in individuals
aicted with cancer [] are too numerous to dismiss. Cell
culture assays demonstrated the antiproliferative [], ras-
inhibiting [], antiapoptotic [], antiangiogenic [], cancer
chemopreventive [–], and immunomodulatory [, ]
properties of extracts derived from the leaf and fruit of
noni. In vivo endpoints obtained in rodent models have, for
the most part, corroborated these in vitro observations [].
However, a Phase I clinical trial of freeze-dried noni fruit
extract has failed to establish antitumor ecacy in cancer
patientsforwhomnostandardtreatmentisavailable[,].
Cancer patients continue to consume noni juice based on its
purported usefulness despite the lack of clinical evidence to
support these claims.
Commercial and traditional preparations alike lack stan-
dardization and clarity in terms of the actual process by
which the noni juice is prepared; processing variables that
are critical to the stability and viability of the preparation
are also the primary factors that will ultimately aect the
potency of the nished product. It is oen unclear if the
noni juice is fresh or fermented and whether pasteurization
is involved. Indeed, the length of time fermentation allowed
to occur may well aect the potency of the nal product, while
pasteurization may conceivably destroy labile constituents
and/or benecial microbes. e incompletely dened nature
of “noni fruit juice” compromises the reproducibility of
studies utilizing these preparations and limiting the extent
and depth to which the results may be interpreted and
analyzed. e fact that noni fruit is commercially available
in a variety of forms, whether dried and powdered, freeze-
dried, or juiced and bottled, in a variety of concentrations
with/without other additives, further confounds the issue by
thickening the veil of obscurity that surrounds the processing
of the fruit.
Inorderforthebenetsofbotanicalstobefullyreal-
ized, they must eectively capture and translate the precise
cultural, if not ethnomedicinal, method of preparation and
useoftheplantintheeld.emostpopulartradi-
tional means of preparing noni in Hawai’i is fermentation
[]. Consensus information gathered suggests that tradi-
tional noni juice is noni juice fermented at room tem-
perature in a tightly sealed glass container for – weeks
(http://www.ctahr.hawaii.edu/noni/fruit juices.asp) with or
without the addition of a minimal amount of water. In
line with this cultural practice, and in accordance with
anecdotal evidence, fermented and unpasteurized noni juice
is the mainstay of claims and testimonials that attest to the
ecacy of noni as an alternative and/or synergistic treatment
modality for cancer.
In an attempt to investigate the involvement of microor-
ganisms in the widely held medicinal properties of the
fermentedjuiceofnonifruit,culturableendophyticfungi
resident within the leaf and fruit of the plant were isolated
and identied by molecular sequence analysis of the internal
transcribed spacer (ITS) region encompassing the .S large
ribosomal subunit gene. Puried organisms were allowed
to “ferment” in regular microbiological media for  weeks
under anaerobic conditions at room temperature, in order to
simulate the conditions under which traditional fermented
noni juice is prepared. It is plausible that compounds from the
secondary metabolism of endophytic microorganisms may
contribute to the claimed anticancer activities of fermented
nonifruitjuiceinhumans,eitherviaimmunologicalor,
as yet, unidentied mechanisms in vivo. Fungal endophytes
establish stable symbiotic association with their plant host
by commensalistic, parasitic, or mutualistic means without
causing immediate overt eects. As a result of this intimate
relationship, endophytic microorganisms and plants share
metabolicpathwaysthatmediateecientinformationtrans-
fer and secondary metabolite production. Indeed, endophytic
fungiandtheirhostshavebeenknowntoproduceanalogous,
if not identical, compounds []. Although these compounds
may be synthesized in extremely minute yields in nature,
it is conceivable that amplication may be achieved with
the isolation, genetic stabilization, and scale-up of these
endophytic microorganisms.
2. Materials and Methods
2.1. Collection of Plant Tissue and Isolation of Endophytic
Fungi. Leaf and fruit samples from the ubiquitous noni tree
were collected from residential yards situated within the city
in Hilo, HI, with the permission of the owners. is collection
methodology is consistent with the process of noni juice
preparation by the general population for local consumption.
In order to ensure the true endophytic nature of isolates
obtained, healthy and mature noni leaf and fruit materials
wereprocessedforplatingwithinafewhoursofcollec-
tion. Leaves and fruits were picked from the tree, washed
thoroughly in ddH2O, and dried on a paper towel. Surface
sterilization of leaf and fruit pieces was initiated by successive
immersion in % EtOH (s) and % NaClO solution
( min). Finally, the leaf and fruit pieces were submerged in
% EtOH for  min and le to dry in sterile petri dishes. An
EtOH and ame-sterilized hole puncher was used to excise 
circular leaf discs from each leaf, while fruit pieces were diced
with a sterile scalpel to expose fresh tissue surfaces for fungal
isolation. Leaf discs and fruit pieces were transferred onto
the surface of standard % (w/v) MycoMedia malt extract
agar (MEA) supplemented with gentamycin (Fungi Perfecti)
and % potato dextrose agar (PDA) (Oxoid, UK). Plates
were sealed with Paralm and incubated in partial daylight
at room temperature (∘C) for – weeks. Morphologically
distinct mycelia emanating from the peripheral edge of all
leaf discs and fruit pieces were individually subcultured by
in vitro hyphal tip transfer and maintained on MEA and
PDA. Cultures were propagated and puried by continuous
subculture on % (w/v) MEA and % (w/v) PDA. Pure and
viable endophyte isolates from the earliest passages were
preservedforlongtermstorageonMEAslantsoverlayed
with sterilized mineral oil. Stock cultures are held at ∘Cand
maintained in the culture collection of the Daniel K. Inouye
College of Pharmacy, University of Hawaii at Hilo, Hilo, HI.
e purity of cultures was conrmed by DNA sequencing of
the ITS regions of the isolates as described below.

Evidence-Based Complementary and Alternative Medicine 
2.2. Endophyte DNA Isolation, Amplication, and Sequenc-
ing. e total genomic DNA of all isolates was extracted
from – 𝜇g of wet mycelial material using the EZNA
Fungal DNA Mini Kit (Omega Bio-Tek). e ribosomal ITS
region encompassing the .S rDNA was amplied by poly-
merase chain reaction (PCR) using the universal primer pair
ITSF (󸀠-CTTGGT CATTTAGAGGAAGTAA-󸀠)andITS
(󸀠-TCCTCCGCTTATTGATATGC-󸀠)[].PCRmixtures
( 𝜇L) contained approximately - 𝜇g/mL genomic DNA,
. 𝜇M of each primer, . mM of each deoxyribonucleotide
triphosphate (dNTP), . 𝜇L of x FastStart PCR buer with
 mM MgCl2,and.UofFastStartTaqDNApolymerase
(Roche Applied Science). Other components of the PCR
reaction buer (pH ., ∘C) consisted of  mM Tris/HCl,
 mM KCl, and  mM (NH4)2SO4.PCRwasperformed
in a C ermal Cycler (Bio-Rad) using the following
program:  min initial denaturation at ∘C, followed by 
cycles of  s denaturation at ∘C,  s primer annealing
at ∘C,  s extension at ∘C, and a nal  min exten-
sion at ∘C.PCRproductswereanalyzedby.%(w/v)
agarose gel electrophoresis with ethidium bromide staining
and visualization under UV light. Unused primers and
excess nucleotides were removed by adding  𝜇LExoSAP-
IT (Aymetrix) directly to the PCR reaction and incubating
at ∘C for  mins, aer which the exonuclease I/shrimp
alkaline phosphatase mixture was inactivated by heating
at ∘C for  min. e PCR mixtures were subsequently
puried using the QIAquick PCR Purication Kit (QIA-
GEN) and quantitated using the BioSpec Nanodrop (Shi-
madzu Biotech). Puried DNA (. ng/𝜇L) was sequenced
with the xl DNA Analyzer -Well Capillary Sequencer
(Applied Biosystems) using either ITSF or ITS primer,
each at a nal concentration of . 𝜇M. Cycle sequencing
was performed using uorescently labeled ddNTP (BigDye
Terminator) chemistry. All DNA electropherograms were
scrutinized, aer which sequences were manually edited and
trimmed with Sequencher (Gene Codes Corporation) to
remove predictable regions of poor quality read at the distal
ends of all sequences. Replicate sequences (–) obtained
from dierent passages of the same endophyte culture were
also aligned in order to generate high quality consensus
reads for identication purposes. In addition, forward and
reverse complementarity sequences were also matched for
base call verication. Query sequences from the endophytes
of Morinda citrifolia were used to retrieve similar sequences
from the databases of the International Nucleotide Sequence
Database (INSD) Collaboration (GenBank, ENA, and DDBJ)
through pairwise alignments facilitated by the nucleotide
Basic Local Alignment and Search Tool (BLASTn) interface
oered by the National Center for Biotechnology Information
(NCBI, http://www.ncbi.nlm.nih.gov/). Accession numbers
were obtained aer the deposition of the newly generated
sequences in the GenBank database (Table ).
2.3. Culture Fermentation and Extraction of Secondary
Metabolites. Each pure endophyte was inoculated into mL
of % (v/v) malt extract broth (Oxoid, UK) in mL
polypropylene tubes. Tubes were capped tightly simulating
the traditional method of preparation of noni juice and gently
agitatedatrpmonanorbitalplatformshakerfor–weeks
at room temperature (∘C) in partial daylight. Genomic
DNA was extracted from the mycelial growth present in each
tube for genotyping by PCR amplication of the ITS region,
followed by DNA sequencing. e tubes were centrifuged
aer which the spent culture supernatantwas extracted twice
over  hrs with equal volumes (∼ mL) of ethyl acetate
(EtOAc). Mycelial mass was also sonicated with mL EtOAc
for  mins, aer which the organic phase was combined
with the bulk EtOAc extract of the broth and evaporated to
dryness under vacuo at ∘C. e dried organic extracts were
dissolved in DMSO at  mg/mL and tested for cytotoxicity
against a panel of human cancer cell lines at the highest
concentration of  𝜇g/mL. Fresh and unfermented malt
extract broth itself was also extracted following the above
procedure in order to provide a control for the eects of broth
extractives.
2.4. Sulforhodamine (SRB) Assay for Cytotoxicity. Human
carcinoma cells of the breast (MCF-), prostate (PC-), and
lung (LU-) were cultured in complete DMEM supplemented
with % (v/v) heat-inactivated fetal bovine serum and
% penicillin/streptomycin. Cultures were incubated in a
humidied atmosphere of % CO2at ∘C.
A standard protocol for the assessment of cellular toxicity
measures the ability of cultured cells to proliferate in the
presence of a test extract and subsequently quantitates total
protein content with sulforhodamine B dye as a measure of
thepercentageofsurvivingcells[].Cells(×4/mL in
 𝜇L of media) were added to the wells of a microplate
containing test samples ( 𝜇L).Attheendoftheassay(d),
cells were xed to the plastic substratum by the addition of
cold % (v/v) aqueous trichloroacetic acid (TCA). Plates
were then incubated at ∘C for  min, washed with tap water
(x), and air-dried. e TCA-xed cells were stained with
.% (w/v) sulforhodamine B (SRB) dissolved in % (v/v)
aqueous acetic acid for  min. Free SRB dye was removed by
washing with % (v/v) aqueous acetic acid (x). Plates were
then air-dried. Bound dye was solubilized by the addition of
 mM unbuered Tris base, pH  ( 𝜇L). Plates were then
placed on an orbital shaker for  min, aer which absorption
(Abs) data was determined at nm using the VICTOR
X Multilabel Plate Reader equipped with the WorkOut .
soware for data acquisition and analysis. In each case, a zero-
day control was performed by adding an equivalent number
of cells to several wells of the microplate and incubating
at ∘Cformin.ecellswerethenxedwithTCA
and processed as described above. Abs values generated
by each dose-response treatment procedure were averaged
over triplicate wells, and the average Abs (Absav)valuewas
obtained by subtracting the zero day control. e resulting
Absav values were then normalized as a percentage relative
to the solvent-treated control incubations. e nal DMSO
concentration in all test wells was .% (v/v). IC50 (median
inhibitory concentration) values were then calculated using
nonlinear regression analysis of plots of % survival versus
concentration. e standard cytotoxic agent, ellipticine, was
used as the toxicity control. All endophyte extracts were
evaluated at the highest concentration of  𝜇g/mL, with

 Evidence-Based Complementary and Alternative Medicine
T : Culturable fungal endophytes from Morinda citrifolia Linn. (noni).
Code Accession number Closest (published) Genbank match
Published
reference sequence
Accession number
(GenBank)
Max identity
Noni leaf endophytes
MRL-A KF Stemphylium solani AF %
MRL- KF Fusarium sp.
(anamorphic Gibberella)EU %
MRL-- KF Acremonium sp. GU %
MRL- KF Didymellaceae sp.HM %
MRL- KF Hypocreales sp. — —
IXLP KF Guignardia sp. AB %
VIIILP- KF Colletotrichum gloeosporioides
(anamorphic Glomerella cingulata)DQ %
XILP- KF Colletotrichum boninense complex JQ %
VIIILP KF Leptosphaerulina australis JN %
XILP KF Mycosphaerella sp. HM %
XILP KF Xylariaceae sp. JX %
XIILP- KF Aspergillus pseudodeectus
(anamorphic Emericella)EF %
Noni fruit endophytes
XSM KF Phlebiopsis sp. JQ %
VIIIfP KF Paraconiothyrium sporulosum AB %
IIfP KF Phomopsis sp.
(anamorphic Diaporthe)GQ %
follow-up dose response determinations over ve -fold serial
dilutions where necessary. All IC50 values represent the
average of at least three independent experiments performed
in triplicate.
3. Results and Discussion
3.1. Isolation of Endophytic Fungi from Morinda citrifolia
Linn. Six separate attempts to isolate noni endophytes were
carried out over a span of more than a year. A total of thirty
isolates of culturable endophytic fungi were obtained on malt
extract agar (MEA) and potato dextrose agar (PDA). Each
isolate was subcultured and puried by successive hyphal
tip transfer over - passages, aer which PCR amplication
andITSsequencingwereappliedtoconrmthepurityof
all cultures. Genomic DNA was extracted from pure cultures
and sequenced to enable identication of the endophytes
based on the sequence of the ribosomal ITS region. As a
quality control measure, the ITS genotype of all fungi in broth
cultures was conrmed to be identical to that of endophytes
originally isolated on solid nutrient media. A total of 
unique endophytic fungal species were obtained:  from noni
fruits and  from noni leaves (Table ). e survey revealed
thepresenceofdistinctgenerabelongingtotheAscomy-
cota in the healthy mature leaves of noni. ree additional
genera occurred in noni fruits inclusive of a genus from the
Basidiomycota (Phlebiopsis sp.). In addition to saprobes, all
genera encountered contain economically important species
that are pathogenic in association with other plant hosts, but
there have been no reports of virulence to the commercially
signicant M. citrifolia per se, except for certain species of
Colletotrichum and Guignardia which are established noni
pathogens (http://www.ctahr.hawaii.edu/noni/gallery); their
presence in healthy plants is possibly representing a latent
and asymptomatic infection. A total of  genera belonging
to  dierent families are, therefore, represented. Phlebiopsis
sp. (Polyporales) is the only Basidiomycete isolated in this
study. e remaining endophytes belong to  dierent classes
(Sordariomycetes, Dothideomycetes, and Eurotiomycetes) in
the Ascomycota (Table ).
Several endophytes were isolated on more than one occa-
sion, with an average of - unique endophytes obtained per
isolation attempt using both MEA and PDA. Colletotrichum
gloeosporioides and C. boninense complex were reproducibly
isolated on multiple occasions possibly reecting the high
concentration and/or wide occurrence of this endophyte.
In addition, all endophytes obtained in this study have
previously been reported to exist in endophytic relationships
with other host plants.
3.2. Molecular Identication of Endophytes. e paucity of
discriminatory morphological and physiological character-
istics for reliable species identication renders genotypic
methods superior to phenotypic and biochemical techniques.
e internal transcribed spacers, ITS and ITS, are non-
functional RNA sequences situated between structural rRNA

Evidence-Based Complementary and Alternative Medicine 
T : Cytotoxic activities (IC50) of fungal endophyte extracts against three human cancer cell lines.
Code Genbank match Order >family IC50 (𝜇g/mL)[a]
LU-[b]MCF-[c]PC-[d]
Noni leaf
endophytes
MRL-A Stemphylium solani Pleosporales >Pleosporaceae 425
MRL- Fusarium sp.
(anamorphic Gibberella)Hypocreales >Nectriaceae > > >
MRL-- Acremonium sp. Hypocreales >Hypocreaceae > > >
MRL- Didymellaceae sp.Pleosporales >Didymellaceae > > >
MRL- Hypocreales sp. Hypocreales > > >
IXLP Guignardia sp. Botryosphaeriales >Botryosphaeriaceae > > >
VIIILP- Colletotrichum gloeosporioides
(anamorphic Glomerella cingulata)Glomerellales >Glomerellaceae > > >
XILP- Colletotrichum boninense complex Glomerellales >Glomerellaceae > > >
VIIILP Leptosphaerulina australis Pleosporales >Didymellaceae 4 0.6 0.4
XILP Mycosphaerella sp. Capnodiales >Mycosphaerellaceae > > >
XILP Xylariaceae sp. Xylariales >Xylariaceae 51010
XIILP- Aspergillus pseudodeectus
(anamorphic Emericella)Eurotiales >Trichocomaceae > > >
Noni fruit
endophytes
XSM Phlebiopsis sp. Polyporales >Phanerochaetaceae > > >
VIIIfP Paraconiothyrium sporulosum Pleosporales >Montagnulaceae > > >
IIfP Phomopsis sp.
(anamorphic Diaporthe)Diaporthales >Valsaceae > > >
Positive control
(ellipticine) . ±. . ±. . ±.
[a]e IC50 value is dened as the concentration of extract required to produce %reduction in viability compared to the standard cytotoxic agent, ellipticine.
All fungal extracts were tested in triplicate, and IC50 values were calculated from the mean dose-response curves. IC50 values represent the average over
triplicate experiments performed with three successive passages of the same endophyte.
[b]LU-, human lung adenocarcinoma.
[c]MCF-, human mammary adenocarcinoma.
[d]PC-, human prostate adenocarcinoma.
that are ultimately excised during maturation of the poly-
cistronic pre-rRNA transcript. e ribosomal DNA region
spanning ITS and ITS and the intercalary .S rRNA has
been employed in the molecular systematics of fungi at the
species level, and even within species, because of the optimal
degree of variation and resolution that it oers compared to
other genic regions of rDNA. Indeed, the ITS region of the
ribosomalrepeatunithasbecometheprimarygeneticmarker
for molecular identication in many groups of fungi [, ].
e culturable endophytes of noni were discriminated
to species level where possible through DNA sequencing of
the nuclear ribosomal ITS region coupled with BLAST-based
similarity searches against existing INSDC databases. BLAST
hitswereexaminedcriticallytoisolatepublishedannotations
of equivalent read length and uncompromised technical
quality. Both BLAST Query coverage and Maximum Identity
values of ≥% were adopted for identication purposes. In
addition, best matches in similarity sequences were associ-
ated with high BLAST quality scores (– bits) and
Expect values of zero. Published sequences were assigned
greater weight as reference sequences. We consistently obtain
agreement among the top ranking BLAST matches for each
endophyte; this served to increase the condence level of the
taxonomic assignment.
BLAST queries with ITS sequences of the noni leaf and
fruit isolates revealed highest similarities to the endophytic
fungi listed in Table . Anamorphic and teleomorphic forms
of the endophytes, in addition to synonyms, are frequently
representedintheBLASThitlists.Despitetherigorous
and systematic approach adopted, a few isolates could not
be assigned to species level due to the absence of relevant
and denitive Genbank records. Furthermore, assignments
to species level employing a heuristic search function such
as BLAST and a database of nominally curated public
sequences cannot be expected to be unambiguous. Species
names were avoided except in cases where an overwhelming
number of published records suggest a particular fungal
individual. Noni endophytes that retrieved Genbank matches
with Query cover and Identity values of <% (but with
Expect values of ) were assigned to taxons higher than
the Genus level (Didymellaceae sp., Xylariaceae sp., and
Hypocreales sp.).

 Evidence-Based Complementary and Alternative Medicine
e use of primers ITSF and ITS in fungal genomic
PCR amplication typically produces amplicons –bp
in length containing the variable ITS (∼ bp) and ITS
(∼ bp) regions of the rDNA gene cluster anking the
highly conserved .S rDNA (∼ bp). Amplications of the
ITS region using this universal primer pair resulted in a
single PCR product of approximately  bp for all the noni
endophytes isolated except Paraconiothyrium sporulosum
(VIIIfP). e length increase to ∼, bp of the amplicon
forthisfruitendophytemaybeattributedtothepresenceof
an intragenic insertion in the S rDNA of ∼ nucleotides.
iswasveriedbysequencealignmentswiththeSSUrDNA
of the model organism Saccharomyces cerevisiae and the ITS
regions of other reference intron-containing and non-intron-
containing Paraconiothyrium sporulosum isolates occurring
among the top  BLAST matches for VIIIfP. Indeed, the
SSU rDNA from lichen-forming and other ascomycetous
fungi frequently contain Group I introns in conserved regions
[].eseinsertionsvaryinoccurrencewithingroupsand
species,andevenwithinanindividualfungus[].
3.3. Cytotoxicity of Noni Juice and Endophytic Fungal Extracts.
Fermented noni juice, with or without pasteurization, exhib-
ited only weak in vitro cytotoxic eects against the cancer cell
linesusedinthisstudy.IC
50 values obtained were well below
the activity threshold dened by the National Cancer Institute
of the US (viz.  𝜇g/mL for a crude extract). Fresh noni juice
itself was also found to be noncytotoxic. Noni preparations
that were not laboratory-processed were deemed to be too
dilute to enable the in vitro detection of cytotoxic constituents
by cell culture methods. In eect, sugars and other nutritional
components in these juice preparations were observed to
promote the growth of cultured cancer cells.
Fermented malt extract broths ( mL) typically yielded
- mg of dried EtOAc extract aer – weeks of endophyte
growth, ∼mgofwhichmaybeattributedtothesolvent-
extractable constituents of the broth itself. e latter was
shown to be noncytotoxic to all the cancer cell lines tested
at a concentration of  𝜇g/mL. e EtOAc extracts of
all endophytic fungi isolated were noncytotoxic (IC50 >
 𝜇g/mL) to the cancer cell lines examined except for
the leaf endophytes Stemphylium solani,Leptosphaerulina
australis, and Xyl a r ia sp. (Table ). IC50 values obtained were
reproducible over three successive passages of the fungi. e
fermentation eects of all endophytes isolated on fresh noni
juice itself, or on culture media/noni juice mixtures, remain to
beelucidated,despitethefactthatthefruitendophytesfailed
to yield cytotoxic constituents upon fermentation in regular
microbiological media.
3.4. Morphological Observations of Extract-Treated Can-
cer Cells. e changes in morphology of cancer cells in
response to treatment were examined daily using a Zeiss
Axiovert inverted microscope (x). LU-, MCF-, and PC-
responded similarly to the endophyte extracts when viewed
daily over the course of the -day cytotoxicity assay. e
magnitude of the global cellular changes increased with the
concentration of extract present, and with time of expo-
sure. Cell shrinkage with increased cytoplasmic granularity
was observed  hours aer plating. Nuclear condensation
(pyknosis) and apoptotic bodies were clearly visible  hours
aer treatment with pronounced lysis occurring on day
. It is common for cytotoxic natural product extracts to
induce apoptotic changes in cultured cells. e morphologi-
calchangesobservedwerelikelyduetothecompositeeectof
uncharacterized metabolites present in each crude endophyte
extract on cellular pathways that ultimately led to cell death
and disintegration. While apoptosis is a denitive marker for
the presence of cytotoxic constituents, valuable insight into
the eect of discrete metabolites on the death pathways can
only be achieved with the isolation and characterization of
pure fungal constituents.
4. Conclusions
Surveys have indicated that plants with ethnomedical sig-
nicance have a greater likelihood of hosting endophytes
that produce pharmacologically interesting natural products
[, ]. erefore, it stands to reason that the medicinal
properties of plants could be due, in part, to their resident
endophytes []. Since the recognition that medicinal plants
constitute a repository of endophytic fungi that produce novel
and pharmaceutically important metabolites, studies on the
applied aspects of these relatively cryptic associations have
focused on medicinal plants [, ].
Nonihasbecomepervasiveinthethrivingmarketfor
nutraceuticals. Its multifaceted use is fueled predominantly
bythepublic’sdesireforsafeandeectivealternativesto
drugs. Unfortunately, the public’s faith has been undermined
by much misconception and marketing hype. Noni fruit juice
is also an established ingredient in CAM approaches for
cancer. Despite its popularity, noni’s reputed ecacy in cancer
patients remains to be substantiated by unequivocal scientic
evidence. Our present work will provide the foundation
for further inquiry into the plausible role of microbes in
the purported pharmacological eects of noni. A survey
of culturable fungal endophytes resident within the noni
plant of Hawaii revealed the presence of  distinct genera
belonging to the Ascomycota in the healthy mature leaves of
noni (Table ). ree additional genera occur in noni fruits
inclusive of a genus from the Basidiomycota. e small-scale
fermentation of malt extract broth by Stemphylium solani,
Leptosphaerulina australis, and Xy laria sp., utilizing condi-
tions that simulate the traditional method of preparation
of fermented noni fruit juice, resulted in the expression of
solvent-extractable cytotoxic component(s) into the growth
medium. In addition to the phytochemical isolation of these
cytotoxic constituents from large-scale fermentation cultures,
the ensuing phase of this study will interrogate the chemical
prole of endophyte-fermented noni juice by a reductionist
approach.Freshnonijuicewillbelter-sterilizedbeforebeing
inoculated by individual fungus endophyte and subjected to
anaerobic fermentation.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.

Evidence-Based Complementary and Alternative Medicine 
Acknowledgments
e authors wish to thank the Genomics Core Facility at
theUniversityofHawaiiJohnA.BurnsCollegeofMedicine
(JABSOM) for providing capillary DNA sequencing ser-
vices in support of this study. Yougen Wu was supported
by a postdoctoral scholarship from the China Scholarship
Council-University of Hainan Joint Scholarships program.
Vitor Martins da Silva was supported by the Coordination
for Enhancement of Higher Education Personnel (Capes)
program of Brazil.
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