Fitoterapia 82 (2011) 71–79
Contents lists available at ScienceDirect
Fitoterapia
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Review
The classical drug discovery approach to defining bioactive constituents
of botanicals☆
A. Douglas Kinghorn a,⁎, Hee-byung Chai a, Chung Ki Sung a,1, William J. Keller b
a
b
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
Nature's Sunshine Products, Inc., Spanish Fork, UT 84660, USA
a r t i c l e
i n f o
a b s t r a c t
Article history:
Received 20 July 2010
Accepted in revised form 20 August 2010
Available online 6 September 2010
In this review, several recently identified biologically active principles of selected botanical
dietary supplement ingredients are described, and were isolated using classical phytochemical
chromatographic methods, with various spectroscopic procedures used for their isolation and
structure elucidation. A central component of such an approach is “activity-guided
fractionation” to monitor the compound purification process. In vitro assays germane to
cancer chemoprevention were used to facilitate the work performed. Bioactive compounds,
including several new substances, were characterized from açai (Euterpe oleracea), baobab
(Adansonia digitata), licorice (Glycyrrhiza glabra), mangosteen (Garcinia mangostana), and noni
(Morinda citrifolia). Many of these compounds exhibited quite potent biological activity, but
tended to be present in their plant of origin only at low concentration levels.
© 2010 Elsevier B.V. All rights reserved.
Keywords:
Biologically active constituents
Açai (Euterpe oleracea)
Baobab (Adansonia digitata)
Licorice (Glycyrrhiza glabra)
Mangosteen (Garcinia mangostana)
Noni (Morinda citrifolia)
Contents
1.
Introduction .
2.
Açaí. . . . .
3.
Baobab . . .
4.
Licorice . . .
5.
Mangosteen .
6.
Noni . . . .
7.
Conclusions .
Acknowledgments
References . . . .
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1. Introduction
One of the scientific challenges in the investigation of
botanical dietary supplements used in the United States is
☆ Dedicated to Dr. Norman R. Farnsworth of the University of Illinois at
Chicago for his pioneering work on botanical natural products, his superb
inspiration and leadership as world authority in the field of pharmacognosy.
⁎ Corresponding author.
E-mail address: kinghorn.4@osu.edu (A.D. Kinghorn).
1
Permanent address: College of Pharmacy, Chonnam National University,
Gwangju, 500-757, Republic of Korea.
0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2010.08.015
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71
72
73
73
74
76
78
78
78
the need to determine the chemical nature of bioactive
principles present, recognizing that it is now clear that
multiple phytochemicals may be involved in mediating the
overall biological activity of a given herbal product [1,2]. In
addition, chemical constituents of a plant may serve as cofactors for already established biologically active compounds
by increasing their resultant bioavailability [2]. Another
primary reason for knowledge of the active constituents of
botanical dietary supplements to be ascertained is to develop
relevant analytical methods for product quality control [1].
However, it is our contention that searching for biologically
72
A.D. Kinghorn et al. / Fitoterapia 82 (2011) 71–79
active principles of botanical dietary supplements may also
afford legitimate new drug leads, given that many of these
same plants are used widely as herbal remedies. Such studies
may also lead to the isolation of new chemical entities, and to
previously known biologically active substances as well as to
compounds with documented toxicity.
Cancer chemoprevention refers to the use of synthetic and
naturally occurring agents to inhibit, reverse, or retard the
process of carcinogenesis, an overall process in which distinct
alterations of molecular and cellular events occur in a
multistep manner [3]. A relatively large number of phytochemical components from common foods have been shown
to be carcinogenesis blocking and/or suppressing agents, and
many of these substances exhibit inhibitory activity in vivo in
tumorigenesis experiments using mice and rats, with some
reaching clinical trials [3,4]. In fact, about 35 plant-based
foods have been identified by the U.S. National Cancer
Institute as possessing cancer chemopreventive propensities,
inclusive of cruciferous vegetables, garlic, ginger, onions,
soybeans, and turmeric, and much is now known on how
their components interact with cellular targets [3,4]. A
collaborative large-scale screening program of some 3000
taxonomically authenticated dietary and edible plants was
instituted at the College of Pharmacy, University of Illinois at
Chicago, about two decades ago, led by Dr. John M. Pezzuto,
funded through the program project mechanism by the U.S.
National Cancer Institute [4,5]. A wide range of chemical
diversity was evident among the compounds found active in
one or more in vitro bioassays germane to cancer chemoprevention in this program. For example, in the five-year period
1999–2004, about 150 bioactive compounds were purified
and structurally characterized, with some 50 being new
structures [5]. Several of the in vitro-active compounds were
also active in a mouse mammary organ culture ex vivo assay
[6], used as a secondary discriminator in order to prioritize
leads for testing in subsequent full-term tumor inhibition
studies in experimental animals [5]. The results of this project
were highly supportive of the further investigation of other
types of plant samples, inclusive of those used as botanical
dietary supplements in the United States. Moreover, among
the compounds listed for evaluation for their cancer chemopreventive potential at the United States National Institutes of
Health, well-known components of dietary supplements such
as curcumin, genistein and other soy isoflavones, and
resveratrol are included, as are grape seed and green tea
extracts [7]. In the remaining parts of this review, the
bioactive constituents of five herbal products will be
described, as determined in our laboratory at the College of
Pharmacy, The Ohio State University, using in vitro bioassays
relevant to cancer chemoprevention. In addition, new
compounds from these five plants that have been determined
structurally in our work will be mentioned.
2. Açaí
The açaí palm (Euterpe oleracea Mart.; Arecaceae) is an
economically important tree that occurs in the floodplains of
the Amazon in Brazil, with both the fruits and the palm
hearts being consumed by humans [8]. In recent years, this
species has attracted considerable interest as a potential
functional food in the U.S., particularly in view of its
demonstrated antioxidant capacity [9]. The purported
health benefits from E. oleracea berries have been subjected
to a brief review [10]. When a freeze-dried extract of the
fruit pulp and skin of E. oleracea was evaluated against
various antioxidant and other biossays, very potent activity
was found in a superoxide scavenging (SOD) (peroxyl
radical) assay, as measured using an oxygen radical
absorbance capacity (ORAC) assay with a fluorescein probe
[9]. The phytochemical constituents of this plant responsible
for its antioxidant activity are still only partially resolved,
and while two major phenolic compounds were reported,
namely, cyanidin 3-glucoside and cyanidin 3-rutinoside,
these compounds accounted for only an estimated 10% of
the overall antioxidant capacities of açaí fruits [11]. Other
phenolic constituents of the fruits of this plant have been
described, and include additional anthocyanins, simple
benzenoids, ellagic acid, flavonoids, gallic acid, phenolic
acids, and the stilbenoid, resveratrol [12–16]. In addition,
amino acids, fatty acids, minerals, and sterols have been
identified [15]. In volunteer human subjects, the antioxidant
capacity of the blood plasma was increased when the pulp of
E. oleracea fruits was ingested [17].
In work carried out in our laboratory, a methanol extract
was prepared of the dried and powdered flakes of the fruit
pulp of E. oleracea, collected in Brazil. Using a hydroxyl
radical-scavenging assay to guide fractionation, 22 compounds of previously known structure were isolated, comprising benzenoids, flavonoids, lignans, monoterpenoids,
norisoprenoids, and a quinone derivative [18]. Only one of
the compounds obtained, the simple benzenoid, procatechuic
acid methyl ester (1) (Fig. 1), had been reported earlier from
E. oleracea. All of these compounds were isolated as trace
components of açaí fruits (b0.0003% w/w of the dried flakes
or less) [18]. The nine lignans isolated were the most potent
antioxidant substances isolated in our work, and representative of five different sub-types. Compounds 2–6 are an
example, in turn, of each one of these five lignan sub-types,
namely, (+)-(6R,7S,8S)-5-methoxyisolariciresinol (an aryltetretrahydronaphthalene); (+)-(7R,8S)-5-methoxy-dihydrodehydroconiferyl alcohol (a dihydrobenzofuran); (+)syringaresinol (a furofuran); threo-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxypropyl)-2-methocyphenoxy]-1,3propanediol (an 8-O-4′-neolignan); and (+)-(7R,8R,8′R)lariciresinol (a tetrahydrofuran). When evaluated in the
hydroxyl radical assay, compounds 1–6 exhibited IC50 values
of 1.1 ± 0.11, 0.56 ± 0.02, 0.98 ± 0.05, 0.40 ± 0.13, 3.5 ± 0.23,
and 0.70 ± 0.13 μg/ml, respectively; the flavonoid, quercetin,
was used as the positive control substance in this assay (IC50
value of 0.31 ± 0.02) [18]. In a further biological test
procedure, compounds active in the hydroxyl radical assay
were further tested for cytoprotective activity in cultured
MCF-7 breast cancer cells mediated by H2O2, at a concentration level of 20 μg/ml. The most active compound in this assay
was found to be procatechuic acid methyl ester (1) (74%
effect at the concentration used), and was comparable to the
positive control (quercetin; 60%) [18]. Collectively, these
lignans and other phenolic constituents would be expected to
contribute to the overall antioxidant activity of E. oleracea
fruits, in view of their high individual biological potencies,
and in spite of their low abundance in the plant, as mentioned
above.
A.D. Kinghorn et al. / Fitoterapia 82 (2011) 71–79
73
Fig. 1. Structures of compounds isolated from açaí.
3. Baobab
4. Licorice
Baobab (Adansonia digitata L.; Bombacaceae) is a large tree
with an unusual shape growing in the drier parts of Africa. The
plant has a wide range of uses, not only as a food and beverage,
but also medicinally to treat fevers and dysentery [19]. In recent
years, there has been an upsurge of interest in the development
of baobab fruits as a botanical dietary supplement in the United
States. The fruit pulp affords high levels of vitamin C (range 2.8–
3 g/kg) and has also been documented as having high
antioxidant potency [20]. An aqueous extract of A. digitata
fruit pulp has shown anti-inflammatory and analgesic effects in
rat models, but at quite a high dose range (400–800 mg/kg, p.
o.) [21]. The various plant parts of baobab have been subjected
to relatively few studies phytochemically, and, for example, a
number of proanthocyanidins of previously known structure
were reported as major constituents from an 80% methanol
extract of the fruit pulp [22].
In work carried out by our group, the dried and powdered
fruit pulp of A. digitata was extracted with methanol, with the
resultant extract suspended in water, and partitioned, in turn,
with solvents of increasing polarity (hexane, chloroform, ethyl
acetate, and 1-butanol). The ethyl acetate extract was found to
exhibit potent antioxidant activity (IC50 = 0.2 μg/ml) in a
hydroxyl radical scavenging assay, carried out according to a
standard protocol [18,23]. Fractionation of this extract led to the
isolation of three compounds of previously known structure,
namely, epicatechin (7) (Fig. 2), procyanidin B2 (8), and
procyanidin B5 (9), which exhibited IC50 values in the hydroxyl
radical scavenging assay of 0.30, 0.59, and 0.05 μg/ml, respectively, compared with quercetin as positive control (IC50
0.04 μg/ml) [24]. While compounds 7–9 have all been have all
been found as constituents of A. digitata fruits earlier [22], these
substances have not previously been described as antioxidant
principles of this plant part. The isolation of these compounds
lends strong support to the use of baobab fruits as an
antioxidant botanical dietary supplement.
Licorice (Glycyrrhiza glabra L.; Fabaceae) is an extremely
well-investigated plant, used to sweeten and flavor foods,
beverages, and tobacco, and used as a medicinal plant [25]. A
major oleanane-type triterpene glycoside component, glycyrrhizin, is responsible for the sweetening effects of licorice
roots and stolons, and this compound is used commercially as
a sucrose substitute in Japan [26]. In addition to its
triterpenoid glycoside constituents, a second major group of
compounds present are present in licorice roots, namely,
phenolic substances, of which many are flavonoids substituted by isoprenoid groups [27]. There is voluminous literature
on the biological effects of the constituents of licorice,
particularly in terms of their anti-inflammatory, antimicrobial, antioxidant, antiulcer, cytoprotective, and cytotoxic activities [e.g., 28,29]. In recent years, G. glabra underground
parts have become of interest in the U.S., and are included in
products used for “detoxification” [30].
In our work, the powdered roots and stolons of G. glabra
were extracted with chloroform, with this extract was
assessed for its antioxidant activity in the authentic peroxynitrite assay, exhibiting 88.3% scavenging activity at a
concentration of 20 μg/ml. As a result of chromatographic
work up of this extract, nine compounds were isolated,
including three phenolic compounds of previously known
structure with potent scavenging activity in this same assay,
namely, hispaglabridin B (10) (Fig. 3), isoliquiritigenin (11),
and paratocarpin B (12) (IC50 values of 3.2, 9.3, and 2.3 μg/ml,
respectively) [30]. Of these, the simple chalcone, 11, has been
found earlier to show activity related to cancer chemoprevention. For example, in our earlier work, it was isolated and
identified from the plant Dipteryx odorata Willd. (Fabaceae,
Tonka bean), and found to be active in an ex vivo mouse
mammary organ culture assay (MMOC), when used for
secondary biological evaluation [31]. Compounds showing
efficacy in the MMOC assay are regarded as good candidates
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A.D. Kinghorn et al. / Fitoterapia 82 (2011) 71–79
Fig. 2. Structures of compounds isolated from baobab.
In addition to the biologically active components of
G. glabra roots, two new isolates were obtained from this
lead, namely, a chalcone (1,2-dihydroparatocarpin A, 13), and
an inseparable group of neolignan lipid esters (14) [30].
for further evaluation as potential cancer chemopreventive
agents in full-term carcinogenesis inhibition studies in
animals [6]. Accordingly, isoliquiritigenin (11) was evaluated
in a full-term tumorigenesis study, after being synthesized by
a modification of standard method, in order to produce gram
quantities of this compound [32]. Compound 11 was
evaluated in 1,2-dimethylhydrazine-induced mouse colon
and lung tumor model, which permits the determination of
chemoprevention potential in both these organs in a single
experiment [33]. When isoliquiritigenin was tested at three
dose levels (50, 100, and 300 mg/kg in the diet), it was found
that the highest dose level, the multiplicity of both colon and
lung cancers was reduced on a statistical basis. Therefore,
phenolic constituents of licorice, inclusive of isoliquiritigenin
(11) seem to be of additional interest for their potential
cancer chemopreventive effects [30].
5. Mangosteen
The juice of the fruits of mangosteen (Garcinia mangostana
L.; Clusiaceae) has become a popular botanical dietary
supplement in the United States, owing to a perceived role
in promoting overall health [34,35]. The plant is native to
countries in South and Southeast Asia, and is known as the
“queen of fruits” in Thailand. This species has a number
of folkloric medicinal uses in various countries in Southeast
Asia, particularly to treat gastrointestinal disturbances and
for wound-healing [36]. Subsequent to the structure deter-
OH
OH
O
O
OH
O
HO
O
OH O
OH O
10
11
12
O
O
20
O
O
O
O
O
18
O
HO
OH O
13
OMe
14
Fig. 3. Structures of compounds isolated from licorice.
A.D. Kinghorn et al. / Fitoterapia 82 (2011) 71–79
mination of α-mangostin (15) (Fig. 4) in 1958, the major
xanthone constituent of G. mangostana fruits, nearly 70
additional xanthones have been isolated and characterized
from different plant parts of this species [34]. Among the
remaining constituents reported from G. mangostana are
benzophenones, flavonoids, triterpenoids, and some miscellaneous substances [34]. The purified constituents of G.
mangostana have been reported to exhibit a wide range of
effects in bioassays, such as antimicrobial (antibacterial,
antifungal, antimalarial, and anti-HIV) activities, cytotoxicity
for cancer cell lines, and action against inflammation-related
targets. In addition, many of the mangosteen xanthones have
proven to be potent antioxidants [34].
75
In our initial investigation of the biologically active
constituents of the fruits of G. mangostana, a dichoromethane
partition of a methanol extract of a freeze-dried powder of
the pericarp was investigated [37]. Altogether, 14 xanthones
were isolated, inclusive of the two major constituents, αmangostin (15) and γ-mangostin (16). These purified
compounds were then evaluated for antioxidant activity,
using both the authentic and morpholinosyndnonimine (SIN1)-derived peroxynitrite methods. Substances considered
active included a new compound, 8-hydroxycudraxanthone
G (17), along with 15 and 16 and two other known
compounds, smeathxanthone A (18) and gartanin (19) [37].
Of these xanthones, α-mangostin (16) was chosen for
OH O
O
OH
O
MeO
OH
O
OH
OMe
O
HO
HO
OH
OH
HO
O
15
OH
17
16
OH O
OH
HO
OH O
OH
OH
O
OH
O
OH
O
OH
OH
O
OH
O
18
O
19
OH
O
HO
OH
20
O
OH
OH
H3CO
HO
OH
OH
O
O
OH
O
21
HO
O
O
23
22
O
OH
OH
HO
O
OH
HO
O
OH
MeO
HO
O
24
OH
O
MeO
O
HO
O
25
Fig. 4. Structures of compounds isolated from mangosteen.
OH
OH
26
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A.D. Kinghorn et al. / Fitoterapia 82 (2011) 71–79
evaluation in a follow up mouse mammary organ culture
(MMOC) assay [6], in view of its considerable potency in the
SIN-1 derived peroxynitrite test (IC50 b0.49 μM). This
compound was found to inhibit 7,12-dimethylbenz[α]anthracene-induced preneoplastic alveolar lesions in the MMOC
assay, exhibiting an IC50 value of 1.0 μg/ml (2.4 μM) [37].
In another approach to investigating the constituents of
G. mangostana fruits, the dichloromethane-soluble extract,
obtained as mentioned above, was subjected to activityguided fractionation using, as an in vitro screening monitor, a
quinone reductase (QR) induction assay in cultured Hepa
1c1c7 murine hepatoma cells. This led to the isolation, as QR
inhibitors, of two new compounds, 1,2-dihydro-1,8,10-trihydroxy-2-(2-hydroxypropan-2-yl)-9-(3-methylbut-2-enyl)
furo[3,2-b]xanthen-11-one (20) and 6-deoxy-7-demethylmangostanin (21), along with two known compounds, 1,3,7trihydroxy-2,8-di(3-methylbut-2-enyl)xanthone (22) and
mangostanin (23) [38]. The known compound, α-mangostin
(15) was found to be inactive in the QR induction assay. These
four compounds (20–23) showed CD values (concentration to
double QR activity) in the range 1.8–5.6 μM (0.68–2.2 μg/ml),
with toxicity (IC50) for the host hepa 1c1c7 cells from 17.6 to
N 48.5 μM [6.7– N 20 μg/ml)] [38]. When a library containing
16 purified mangosteen xanthones was evaluated in a
hydroxyl radical scavenging assay, only γ-mangostin (16)
was found to be active (IC50 0.20 μg/ml), and this showed
comparable activity to the three positive controls used,
namely, gallic acid, quercetin, and vitamin C, with IC50
values of 1.0, 0.38, and 0.40 μg/ml, respectively [38]. Indeed,
α-mangostin (15) and γ-mangostin (16) would seem to be
appropriate for use as positive controls themselves in the
peroxinitrite (SIN-1) and hydroxyl radical scavenging assays,
respectively [34,35]. It is relevant to note that in a recent study
in which volunteer human subjects ingested a mangosteencontaining product, α-mangostin (15) was deemed as being
bioavailable [39].
An extensive in vitro screening procedure was carried on
both natural product extracts and pure compounds using a
non-cellular, enzyme-based microsomal assay. Aromatase
catalyzes the biosynthesis of estrogen from androgens, and
inhibition of this enzyme reduces bodily estrogen production,
which, in turn, may have an effect in inhibiting the development and progress of hormone-responsive breast cancer
[40]. Aromatase inhibitors are of interest as both cancer
chemotherapeutic agents and cancer chemopreventives [40].
When 12 mangosteen xanthones were tested in this assay, αmangostin (15), γ-mangostin (16), garcinone D (23), and
garcinone E (24) each showed dose-dependent inhibitory
activity, with IC50 values of 20.7, 6.9, 5.2, and 25.2 μM,
respectively [41]. It is of interest to note that all four of these
compounds have a C-2 unsubstituted prenyl group, an
unsubstituted prenyl group or a hydroxylated prenyl group
at C-8, and three hydroxy groups at C-1, C-3, and C-6 on the
xanthone nucleus. A follow-up cell-based assay was used
with SK-BR-3 cells, which express high levels of aromatase,
and, of these four compounds, the most potent was found to
be γ-mangostin, which exhibited IC50 values of 5.0 and
26.0 μM, respectively, in aromatase inhibition and cytotoxicity evaluations [41].
The major xanthones, α- and γ-mangostin (15 and 16) are
consumed in quite large amounts when mangosteen fruit
juice is taken regularly as a botanical dietary supplement [34].
Thus, as a result of work carried out in our laboratory and in
others, these compounds have been shown to be active in
several bioassays germane to cancer chemoprevention
[34,35,37,38,41]. However, further testing of these compounds needs to be conducted using appropriate in vivo
models. It is worth noting that a crude preparation of compound 16 was found to prevent the induction and/or the
development of aberrant crypt foci, dysplastic foci, and the
accumulation of β-catenin on preneoplastic lesions induced
by the carcinogen 1,2-dimethylhydrazine in the rat colon
[42].
One other new compound, mangostingone (26) was isolated
and structurally determined in our work on G. mangostana, for
which no in vitro biological activity was attributed [37]. This
compound was assigned with a somewhat unusual 2-oxo-3methylbut-3-enyl group, and was not tested biologically owing
to the very limited quantity obtained [37].
6. Noni
Noni (Morinda citrifolia L.; Rubiaceae) is a medicinal plant
of south Asian origin, with traditional uses that have spread
across the tropics to Australia, the Caribbean, Polynesia, and
southeast Asia. The powdered fruits and fruit juice of this
plant have become well established in the United States and
elsewhere as a popular dietary supplement, for potential
effects on arthritis, cancer, cardiovascular disease, inflammation, and as a general tonic [43–45]. The ethnobotanical uses
of the various M. citrifolia plant parts include both external
and internal applications [45]. Approximately 200 compounds have been isolated from M. citrifolia, with the most
prominent representatives being anthraquinones, fatty acid
derivatives, flavonoids, iridoids, lignans, phenylpropanoids,
saccharide derivatives, and triterpenoids [43–45]. Several
noni preparations and purified constituents have been evaluated using in vitro and in vivo bioassays germane to cardiovascular disease, cancer, fertility, inflammation, and
infectious diseases [45]. A phase I clinical trial of a freezedried noni fruit extract has been conducted on cancer patients
in Hawaii [45].
Our recent work included the chromatographic purification of a dried 1-butanol-soluble partition of a crude methanol extract of freeze-dried M. citrifolia (noni) fruits, which
led to the isolation of ten iridoids, three saccharides, two
flavonoids, a lignan, a nucleoside, a polyol, and a sterol [46].
Among these compounds, the neolignan, americanin A (27)
(Fig. 5) and the flavonol glycoside, narcissoside (28) were
found to be potent antioxidants in the various in vitro assays
used [46]. Americanin A (27) was reported earlier at Seoul
National University in Korea to possess in vivo anti-inflammatory activity in rats, inclusive of the inhibition of the induction
of both edema and arthritis [47]. In a further study by our group,
carried out on freeze-dried noni fruits, and using a larger
sample than previously available, five anthraquinones were
isolated from a chloroform-soluble partition of the crude
methanol extract, using a quinone reductase (QR) induction
assay carried out using hepa 1c1c7 cells [48]. A new
anthraquinone, 2-methoxy-1,3,6-trihydroxyanthraquinone
(29), was found to exhibit very promising biological activity,
and represents an excellent new lead compound for cancer
A.D. Kinghorn et al. / Fitoterapia 82 (2011) 71–79
77
OMe
OH
HO
OH
O
O
O
OH O
O
H
OH
O
OH
OMe
O
OH
O OH
HO
HO
OH
O
HO
OH
HO
O
OH
27
O
O
28
29
O
OH
HO
OH
H
COOMe
Me
O
HO
H
OGlc
OH
OH
O
O
30
O
H
31
COOMe
OH O
O
32
O
OH O
O
O
OH
MeO
O
HO
OH
OH
OH
CH2OH
OGlc
33
34
HO
HO
35
Fig. 5. Structures of compounds isolated from noni.
chemoprevention. This anthraquinone showed a CD value
(concentration to double QR activity) of 9 nM (2.7 μg/ml), with
no toxicity for the host hepa 1c1c7 cells up to the highest dose
tested [IC50 N20 μM (N69.9 μg/ml)], and was some 40 more
potent as an enzyme inducer than the positive control used, Lsulforaphane, a constituent of broccoli flowers and sprouts [QR
CD 1.34 μM (0.61 μg/ml)] [48]. Unfortunately, compound 29
was only obtained as a trace constituent of noni fruits
(0.17 ppm yield), so its further biological evaluation as a
potential cancer chemopreventive agent, which seems advisable, will probably require chemical synthesis. This compound
was not present in a sample of noni roots later investigated in
our laboratory, among nearly 15 anthraquinones isolated and
identified. However, the previously known compound and very
closely structurally related substance, 1,3,6-trihydroxy-2methylanthraquinone (30), was found to be present. Compound 30 was demonstrated as being only moderately potent
as a QR inducer [QR CD 0.56 μM (0.15 μg/ml); CD], with
somewhat more toxicity evident for the hepa 1c1c7 cells than
compound 29 [IC50 N 47 μM (12.8 μg/ml)] [49]. Therefore, minor
modification of the C-2 substituent affects not only the QR
enzyme inducing potency but also the cellular toxicity among
this group of anthraquinones [48,49]. A number of other
anthraquinones from noni fruits and roots were also evaluated
in the QR in vitro assay, but showed either less potent inducing
activity when compound with both compared 29 and 30, or else
were regarded as inactive in this bioassay [48,49]. Kamiya and
co-workers have also described anthraquinones in noni fruits
[50,51].
Anthraquinones have been speculated as being the
causative agents of two cases of hepatotoxicity related to
the ingestion of noni fruit juice documented in Austria, but
without any analytical evidence for their presence [52]. In
2006, West and associates concluded that the concentration
levels (b1 ppm) of the anthraquinones reported from
M. citrifolia fruits [48,50] would be too low to have any
toxicological consequence, and also surmised that the
anthraquinone structural types represented could not be
reduced to anthrone radicals able to cause tissue damage [53].
However, the known compound 1-hydroxyanthraquinone
(31) was found in our laboratory work to be a constituent of
the roots of M. citrifolia, albeit in quite low yield (2 ppm) [49].
This compound has also been isolated from a chloroformsoluble extract of the roots of the related species, Morinda
officinalis F.C. How [54], a plant which is used in traditional
Chinese medicine as an analgesic and tonic [55]. 1-
78
A.D. Kinghorn et al. / Fitoterapia 82 (2011) 71–79
Hydroxyanthraquinone (31) was shown to be carcinogenic to
male ACI/IN rats, when fed at a 1% dietary dose for 480 days,
and produced adenomas or adenocarcinomas in the cecum or
colon in 25 out of 29 rats in the dosed group, in addition to
liver neoplasms and benign stomach tumors in a smaller
number of animals [56]. In 2002, 1-hydroxyanthraquinone
was classified as a Group 2B carcinogen (“possibly carcinogenic to humans”) by the International Agency for Research
on Cancer, World Health Organization, Lyon, France. Accordingly, it is not recommended that the roots of
either M. citrifolia or M. officinalis are ingested by humans
[55]. 1-Hydroxyanthraquinone (31) has not been detected as
a constituent of M. citrifolia fruits thus far [42,48,50,51].
As a result of our various phytochemical investigations on
noni fruits and roots, two new iridoid glucosides (32 and 33)
were isolated in our laboratory by extraction into 1-butanol
from the fruits [48], and two new benzophenones (34 and 35)
from a chloroform partition of the methanol-soluble extract
of the roots [49].
7. Conclusions
In this review, a research program has been described on
the investigation of the isolation of biologically active
constituents of selected botanical dietary supplements,
choosing a number of in vitro bioassays germane to cancer
chemoprevention to monitor chromatographic fractionation.
With the experimental approach taken, several highlights
may be briefly summarized. Thus, a minor chalcone constituent of the roots and stolons of licorice (Glycyrrhiza glabra),
isoquiritigenin (11), has been shown to prevent the incidence
of colon and lung tumors in mice induced by the carcinogen,
1,2-dimethylhydrazine, when administered at a dose of
300 mg/kg according to a standard protocol [30]. Also,
aromatase inhibition has been described for the first time
from an extract of mangosteen fruits (G. mangostana), and
four xanthone constituents (15, 16, 23, 24) with several
structural features in common have been isolated and shown
to be responsible for this type of activity [41]. Moreover, it has
been possible to purify and structural characterize a structurally new anthraquinone (29) as a promising lead for
further evaluation as a chemopreventive agent, from the
fruits of noni (M. citrifolia) [48]. From a phytochemical point
of view, various classes of lignans and neolignans (e.g. 2–6) have
been isolated for the first time from the fruits of açaí (Euterpe
oleracea), and these would be expected to contribute to the
overall potent antioxidant activity demonstrated for this
botanical [18]. Also, although the underground parts of licorice
are extremely well studied by others in terms of the chemical
constituents present, a novel group of neolignan lipids esters
(14) was characterized structurally, a compound class not
previously reported for this plant [30]. Finally, a significant
observation is the isolation of the suspected human carcinogen,
1-hydroxyanthraquinone (31) in the roots of noni, although this
compound was not detected in the much more widely consumed
M. citrifolia fruits [48–51]. Accordingly, it is not recommended
that noni roots be ingested by humans.
The overall relevance of the work described in this review
on açaí and noni fruits to cancer chemoprevention has been
substantiated by some very recent work in the laboratory of
Dr. Gary D. Stoner, College of Medicine, The Ohio State
University. In previous work, this group has demonstrated
the usefulness of a rat esophageal model for evaluating the
potential cancer chemopreventive effects of berries such as
black raspberry (Rubus occidentalis L.; Rosaceae), and their
anthocyanin and ellagitannin constituents [57]. Thus, in a
direct comparison with black raspberries and four other berry
types, powdered preparations of açaí and noni fruits were fed
to male F344 rats that had been previously treated with the
carcinogen N-nitrosomethylbenzylamine (NMBA), and were
then evaluated for their effects in reducing the resultant
tumorigenesis in the esophagus. The powdered fruits were
tested at a single dose of 5% of the diet of the test animals,
with the experiment conducted for a total of 35 weeks,
according to a well-established post-initiation protocol [58].
It was found that both açaí and noni fruits inhibited tumor
incidence and multiplicity in a comparable manner to
powdered black raspberry and the other four fruits evaluated.
Moreover, the fruits both reduced levels of interleukin-5 and
GRO/KC in the plasma of the carcinogen-treated rats, in a
similar manner to black raspberry [56]. Clearly, follow-up
studies are needed, including the determination of more
detailed phytochemical profiles of the fruits of E. oleifera and
M. citrifolia than are presently available. It is pertinent to
point out that when 13 constituents of noni fruits (three
anthraquinones, eight saccharide fatty acid esters, an iridoid
glycoside, and a flavonol glycoside) were evaluated in an
Epstein–Barr virus early antigen activation assay induced by
12-O-tetradecanoylphorbol 13-acetate, all of these compounds were perceived as being moderately inhibitory [51].
Accordingly, it is hoped that the studies described in this
review will stimulate others to continue to elucidate the biologically active principles of botanical dietary supplements. It is
important that our present-day knowledge in this regard
continues to be supplemented with the passage of time.
Acknowledgments
We wish to thank Dr. Norman R. Farnsworth, on the occasion
of his 80th birthday, for his superb inspiration over many years of
pioneering work on bioactive natural products, and for his
leadership as a world authority in the field of pharmacognosy.
We are grateful to the collaborators who have supported the
work described, as mentioned throughout this review, as well as
to several outstanding postdoctoral and graduate student
participants in the research, whose names are mentioned in
the bibliography. Phytochemical and in vitro biological work
carried out at the College of Pharmacy at The Ohio State
University described in this review was aided by faculty start-up
funding from the Molecular Carcinogenesis and Chemoprevention Program of The Ohio State University Comprehensive
Cancer Center. S. K. C. was supported while on a sabbatical year
at The Ohio State University by an award from the Chonnam
National University.
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