Chemical Constituents of the Fruits of Morinda citrifolia (Noni) and Their
Bao-Ning Su,†,‡Alison D. Pawlus,†,‡Hyun-Ah Jung,§William J. Keller,⊥Jerry L. McLaughlin,⊥and
A. Douglas Kinghorn*,†,‡
Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy,
College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, and Nature’s Sunshine Products,
Inc., 1655 N. Main Street, Spanish Fork, Utah 84660
Received December 13, 2004
Purification of a n-BuOH-soluble partition of the MeOH extract of Morinda citrifolia (Noni) fruits led to
the isolation of two new iridoid glucosides, 6R-hydroxyadoxoside (1) and 6?,7?-epoxy-8-epi-splendoside
(2), as well as 17 known compounds, americanin A (3), narcissoside (4), asperuloside, asperulosidic acid,
borreriagenin, citrifolinin B epimer a, citrifolinin B epimer b, cytidine, deacetylasperuloside, dehy-
dromethoxygaertneroside, epi-dihydrocornin, D-glucose, D-mannitol, methyl R-D-fructofuranoside, methyl
?-D-fructofuranoside, nicotifloroside, and ?-sitosterol 3-O-?-D-glucopyranoside. The structures of the new
compounds were determined by spectroscopic data interpretation. Compound 4, borreriagenin, cytidine,
deacetylasperuloside, dehydromethoxygaertneroside, epi-dihydrocornin, methyl R-D-fructofuranoside, and
methyl ?-D-fructofuranoside were isolated for the first time from M. citrifolia. The antioxidant activity
was evaluated for all isolates in terms of both DPPH and ONOO-bioassays. The neolignan, americanin
A (3), was found to be a potent antioxidant in these assays.
Morinda citrifolia L. (Rubiaceae), commonly called Noni
or Indian mulberry, is a small evergreen tree or shrub of
Polynesian origin.1M. citrifolia bears a lumpy, green to
yellowish-white fruit, normally 5 to 10 cm in length, with
a surface covered in polygonal-shaped sections.1,2M. cit-
rifolia has a long history of use as a medicinal plant in
parts of Southeast Asia, Polynesia, and Australia and is
considered to be the second most important medicinal plant
in the Hawaiian Islands.1,3The leaves, roots, bark, and
fruits have all been used medicinally to treat a wide range
of ailments. These include, but are not limited to, diabetes,
diarrhea, hypertension, malaria, pain, and topical infec-
tions.1,4The fruits are also eaten as a food, but primarily
only in times of famine.4
Today, the use of M. citrifolia in the United States is
becoming more widespread and Noni products are com-
mercially available in health food stores, chain grocery
stores specializing in natural foods, and on the Internet.
Both the leaves and the fruits are being sold in tablet, tea,
and juice form, although the fruit as a juice is the
predominant formulation sold. This growth in popularity
in the United States may in part be attributed to claims of
Noni being a “cure-all” or aid in relieving symptoms for a
host of chronic conditions such as arthritis, cancer, diabe-
tes, and hypertension.1A number of in vitro biological
activities have been reported, such as angiogenesis inhibi-
tion,5antioxidant,6cyclooxygenases-1 and -2 inhibition,7,8
and tyrosine kinase inhibition.9Most of these studies have
involved crude extracts or fractions of M. citrifolia, and the
compound(s) responsible for the biological activities have
not been determined. In addition, an in vivo study using
the ethanol-insoluble precipitate of M. citrifolia fruits,
given intraperitoneally to mice with Lewis lung carcinoma
implanted cells, demonstrated a significant life-prolonging
effect that was increased when given in conjunction with
several chemotherapeutic agents.10Currently, a freeze-
dried Noni fruit extract is in phase I clinical trials at the
Cancer Research Center of Hawaii in cancer patients for
which there is no other standard treatment available. The
aim of this trial is to determine if Noni extract can be useful
to cancer patients for antitumor and/or symptom control.11
To date, the major chemical constituents of this plant
have been found to be anthraquinones,12flavonol glyco-
sides,13iridoid glycosides,13,14lipid glycosides,15and trit-
erpenoids.14In the present study, the n-BuOH-soluble
partition part of the MeOH extract of M. citrifolia fruits
was found to have moderate antioxidant activity in a free-
radical (DPPH) scavenging bioassay. This partition was
purified by repeated chromatography, which led to the
isolation of two new iridoid glucosides, 6R-hydroxyadoxo-
side (1) and 6?,7?-epoxy-8-epi-splendoside (2), and 17
known compounds. All isolates obtained in this study were
evaluated for their antioxidant activity, and the neolignan,
americanin A (3), demonstrated significant antioxidant
activity in two antioxidant bioassays.
Compound 1, [R]23D-50.7° (c 0.28, MeOH), was obtained
as a colorless gum by repeated chromatography and finally
purified by reversed-phased HPLC. A molecular formula
of C17H26O11 was established for 1 on the basis of the
observed sodiated molecular ion peak at m/z 429.1378 [M
+ Na]+in its HRESIMS (calcd for C17H26O11Na, 429.1373).
The IR absorption bands at 3355 and 1692 cm-1, respec-
tively, indicated the presence of hydroxyl groups and a
carbonyl group in the molecule. The characteristic1H NMR
(in CD3OD) signals at δH7.46 (1H, d, J ) 1.2 Hz, H-3),
5.15 (1H, d, J ) 6.0 Hz, H-1), and 4.65 (1H, d, J ) 7.9 Hz,
Glc-1) and13C NMR (in CD3OD) signals at δC169.5 (C,
C-11), 153.1 (CH, C-3), 113.0 (C, C-4), 100.6 (CH, Glc-1),
and 99.1 (CH, C-1) suggested that compound 1 is an iridoid
glucoside.16,17The observed HMBC correlation from the
anomeric proton (Glc-1) of glucose to the acetal carbon at
δC99.1 (C-1) indicated that the location of the glucose unit
is at C-1 in the molecule of 1. The presence of a methyl
* To whom correspondence should be addressed. Tel: +1-614-247-8094.
Fax: +1-614-247-8081. E-mail: email@example.com.
†Program for Collaborative Research in the Pharmaceutical Sciences.
‡Present address: The Ohio State University.
§The Ohio State University.
⊥Nature’s Sunshine Products, Inc.
J. Nat. Prod. 2005, 68, 592-595
10.1021/np0495985 CCC: $30.25 © 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/22/2005
ester group at C-4 was readily assigned on the basis of the
chemical shifts of H-3, C-3, C-4, and C-11, as well as the
observed correlations from both H-3 (δH 7.46) and the
methoxy (δH3.69) signals to the carbonyl carbon (δC169.5)
in the HMBC spectrum of 1. Generally, the five- and six-
membered rings of iridoids are cis-fused,18although a few
trans-fused iridoids also have been reported.16,19The
chemical shifts of C-1 are about 103 ppm and 96-100 ppm
for trans- and cis-fused iridoids, respectively.16Therefore,
the rings are cis-fused in the molecule of 1, since the C-1
signal was detected at 99.1 ppm in the13C NMR spectrum
of this isolate. Further analysis of the observed 2D NMR
(1H-1H COSY, HMQC, and HMBC) correlations indicated
the gross structure of compound 1 to be 6-hydroxyadoxo-
side.16,17A literature survey revealed that 6?-hydroxya-
doxoside has been previously isolated from Bessaya plan-
taginea, and a doublet of doublets with the coupling
constants of 3.7 and 7.8 Hz was reported for H-6 of this
compound.17However, in the1H NMR spectrum of 1, the
resonance signal of H-6 was displayed as a broad singlet
at δH 4.29. This suggested that compound 1 is 6R-hy-
droxyadoxoside, which was confirmed by a ROESY NMR
correlation from H-6 to H-9.
Compound 2 was isolated as a colorless gum, [R]23D
-108.4° (c 0.17, MeOH). A molecular formula of C17H24O12,
indicating six degrees of unsaturation, was assigned to 2
on the basis of its HRFABMS (found m/z 443.1147, calcd
for C17H24O12Na, m/z 443.1166). Seventeen carbon signals,
including one methoxy group, two methylenes, 11 me-
thines, and three quaternary carbons, were evident from
resonance signals of two doubly oxygenated methines were
displayed at δC99.9 (Glc-1) and 93.8 (C-1) in the13C NMR
and DEPT spectra of 2. This, in combination with the
observed1H NMR signals at δH5.76 (1H, s, H-1) and 4.56
(1H, d, J ) 8.0 Hz, Glc-1), suggested that compound 2 is
also an iridoid glucoside.16-18In the same manner as for
1, the locations of the glucose unit and the methyl ester
group in 2 were also determined at C-1 and C-4 on the basis
of the observed HMBC correlations from the anomeric
proton (Glc-1) of glucose to the acetal carbon at δC93.8 (C-
13C NMR and DEPT data of 2. Similar to 1, the
1) and from both H-3 (δH7.48) and the methoxy (δH3.73)
signals to the carbonyl carbon (δC168.5), respectively. The
presence of an epoxy group between C-6 and C-7 and the
hydroxyl groups at C-8 and C-10 was assigned by further
analysis of the chemical shifts of the remaining protons
and carbons, the observed 2D NMR correlations (1H-1H
COSY, HMQC, and HMBC), and the determined unsat-
uration value of 2. In the NOESY NMR spectrum (Figure
1) of 2, correlations from H-5 to H-9 and from H-6 to H-7
were evident, but there was no correlation from H-5 to H-6
or from H-9 to H-7, indicating the ?-, R-, R-, and ?-orienta-
tions of H-5, H-6, H-7, and H-9, respectively. Another key
correlation observed in the NOESY spectrum (Figure 1) of
2 was observed between H-1 (δH5.76, 1H, s) and H-10a
(δH3.68, 1H, d, J ) 11.7 Hz), which was suggestive of an
R-orientation of the hydroxymethylene group at C-8. This
was consistent with absence of the NOESY correlation from
H-9 to both H-10a and H-10b. Accordingly, the structure
of this new iridoid glucoside was determined as 6?,7?-
In addition to compounds 1 and 2, 17 known compounds,
americanin A (3),20,21narcissoside (4),22asperuloside,23
asperulosidic acid,15borreriagenin,24citrifolinin B epimer
a,13citrifolinin B epimer b,13cytidine,25deacetylasperulo-
methyl ?-D-fructofuranoside,30nicotifloroside,31and ?-si-
tosterol 3-O-?-D-glucopyranoside, were also isolated in the
present study. The structures of these known compounds
were identified by comparing their physical and spectro-
scopic data ([R]D,1H NMR,13C NMR, DEPT, 2D NMR, and
MS) with those of published values or by comparing with
an authentic sample (?-sitosterol 3-O-?-D-glucopyranoside)
directly. Americanin A (3) was initially isolated from
Phytolacca americana in 1978,20and its structure was
revised in 1986.21The structures of americanin A (3) and
its regioisomer, isoamericannin A, were confirmed by total
neolignans are very similar.32Compound 3 obtained in the
present study was assigned as americanin A on the basis
of the observed key correlation from H-7 to C-4′ in the
HMBC spectrum acquired on a 600 MHz NMR spectrom-
eter using a cryoprobe (Supporting Information). Among
these isolates, compound 4, borreriagenin, cytidine, deacety-
lasperuloside, dehydromethoxygaertneroside, epi-dihydro-
cornin, methyl R-D-fructofuranoside, and methyl ?-D-
fructofuranoside were isolated from M. citrifolia for the first
time. One of the major compounds obtained in the present
study is an iridoid glucoside, asperuloside (∼0.08% w/w).
The presence of this compound in the n-BuOH-soluble
extract was confirmed by LC-MS analysis (Supporting
The antioxidant ability of all isolates obtained in this
study to scavenge DPPH, authentic ONOO-, and 3-mor-
pholinosydnonimine (SIN-1)-derived ONOO-was evalu-
ated. As summarized in Table 1, the neolignan, americanin
13C NMR data of these two
Figure 1. Selected NOESY correlations for 6?,7?-epoxy-8-epi-splen-
NotesJournal of Natural Products, 2005, Vol. 68, No. 4
A (3), was found to be a potent antioxidant in these
bioassays. The flavonol glycoside, narcissoside (4), exhibited
evident scavenging activity against both authentic ONOO-
and SIN-1-derived ONOO-. However, another structurally
similar flavonol glycoside, nicotifloroside (unsubstituted
instead of having a methoxy group at C-3′ as in 4), was
found to be inactive. The isolation and characterization of
3 as an antioxidant constituent of M. citrifolia fruits thus
provides a basis for the previous literature reports of the
antioxidant effects of extracts of this species.6,33Several
lignans and neolignans including compound 3 were re-
ported from M. citrifolia fruits very recently.34The copper-
induced low-density lipoprotein oxidation inhibition activi-
ties of these isolates were determined.34
General Experimental Procedures. Melting points were
determined on a Fisher-Johns melting point apparatus and
are uncorrected. Optical rotations were measured on a Perkin-
Elmer 241 automatic polarimeter. The UV spectra were
obtained with a Beckman DU-7 spectrometer, and the IR
spectra run on an ATI Mattson Genesis Series FT-IR spectro-
photometer. NMR spectroscopic data were recorded at room
temperature on a Bruker Advance DPX-300, 360, 400, or DRX-
500 MHz spectrometer with tetramethylsilane (TMS) as
internal standard. The HMBC spectrum of americanin A (3)
was acquired on a Bruker Advance DRX-600 NMR spectrom-
eter with a TXI cryoprobe. Standard pulse sequences were
employed for the measurement of 2D NMR spectra (1H-1H
COSY, HMQC, HMBC, and NOESY). FABMS was obtained
on a VG 7070E-HF sector-field mass spectrometer, while
CIMS, EIMS, and ESIMS were performed on a Finnigan/MAT
90/95 sector-field mass spectrometer. A YMC-pack ODC-AQ
column (5 µm, 25 × 2 cm i.d., YMC Co., Wilmington, NC) was
used for semipreparative HPLC, along with two Waters 515
HPLC pumps and a Waters 2487 dual λ absorbance detector
(Waters, Milford, MA). Column chromatography was carried
out with silica gel G (Merck, 70-230 or 230-400 mesh).
Analytical thin-layer chromatography (TLC) was performed
on 250 µm thickness Merck Si gel 60 F254 aluminum plates.
Plant Material. The freeze-dried fruit powder of M. citri-
folia (lot number 229) used in this study was obtained from
Nature’s Sunshine Products, Inc. A representative sample (#
N0001) was deposited in the Division of Medicinal Chemistry
and Pharmacognosy, College of Pharmacy, The Ohio State
University. LC-MS traces of the n-BuOH-soluble extract of M.
citrifolia fruits are included in the Supporting Information.
Extraction and Isolation. The freeze-dried fruit powder
(1 kg) was extracted by maceration with MeOH three times
(3 × 4.5 L) at room temperature, for 3 days each. After
filtration and evaporation of the solvent under reduced pres-
sure, the combined crude methanolic extract was suspended
in H2O (800 mL), then partitioned in turn with petroleum ether
(3 × 1000 mL), CHCl3(4 × 1000 mL), and n-BuOH (4 × 500
mL), to afford dried petroleum ether- (102 g), CHCl3- (12.5 g),
n-BuOH (41.2 g), and H2O-soluble (∼55 g) extracts. The
n-BuOH-soluble partition part of the MeOH extract was found
to be active (56.2% at 200 µg/mL) in a DPPH free-radical
Therefore, the n-BuOH-soluble extract was subjected to
chromatography over a silica gel column (9 × 45 cm), eluted
with CHCl3-MeOH (15:1 to 1:1, then pure MeOH), to give
seven fractions (F01-F07). Compound 3 (6.5 mg; 0.00065%
w/w) was obtained as a white amorphous powder from a
CHCl3-MeOH mixture (ca. 10:1) of fraction F01, eluted with
CHCl3-MeOH (15:1). Fraction F04 (9.17 g), eluted with
CHCl3-MeOH (8:1), was chromatographed over a silica gel
column (5 × 45 cm), eluted with EtOAc-MeOH (10:1 to 2:1),
and five combined subfractions were obtained (F0401-F0405).
F0401 was further purified over a Sephadex LH-20 column
(2.8 × 45 cm), using pure MeOH for elution, and afforded
borreriagenin (200 mg; 0.02% w/w) and cytidine (58 mg;
0.0058% w/w), and a mixture. This mixture was purified by
semipreparative HPLC, by eluting with MeOH-H2O (40:60;
8 mL/min), to afford pure asperuloside (tR) 6.5 min, 64 mg;
0.0064% w/w) and dehydromethoxygaertneroside (tR ) 28.5
min, 1.6 mg; 0.00016% w/w). Asperuloside (750 mg; 0.075%
w/w) was obtained as a white amorphous powder from a
solution (EtOAc-MeOH, ca. 8:1) of F0402 as a major compo-
nent. Fraction F05 (4.0 g), eluted with CHCl3-MeOH (6:1),
was chromatographed over a silica gel column (5 × 40 cm),
eluted with EtOAc-MeOH (10:1 to 2:1), to give pure asperu-
losidic acid (400 mg; total yield 0.0435% w/w; an additional
35 mg was isolated from F0602) and four subfractions (F0501-
F0504). F0502 was further fractionated over a Sephadex LH-
20 column (2.8 × 45 cm), eluted using pure MeOH, and gave
three fractions (F050201-F050203). F050201 was finally
purified by preparative TLC (500 µM; 20 × 20 cm), with
CHCl3-MeOH (4:1) as developing solvent, to give epi-dihy-
drocornin (Rf) 0.54, 4.5 mg; 0.00045% w/w) and a mixture of
citrifolinin B epimer a and citrifolinin B epimer b (Rf) 0.50,
6.0 mg; 0.0006% w/w) in a ratio of 1:1 (integration of1H NMR
signals). F0503 was purified by HPLC using MeOH-H2O (40:
60; 8 mL/min) as solvent, to afford two flavonol glycosides,
nicotifloroside (tR ) 35.0 min, 16 mg; 0.0016% w/w) and
compound 4 (tR) 39 min, 11 mg; 0.0011% w/w). ?-Sitosterol
3-O-?-D-glucopyranoside (58 mg; 0.0058% w/w) was obtained
as an amorphous solid from a CHCl3-MeOH (∼6:1) solution
of F0504. Fraction F06, eluted with CHCl3-MeOH (4:1), was
separated over a Sephadex LH-20 column (2.8 × 45 cm), eluted
with MeOH, and afforded three subfractions (F0601-F0603).
F0602 was then purified by semipreparative HPLC using
MeOH-H2O (30:70; 7 mL/min) as eluent, to afford compound
1 (tR) 16.7 min, 7.5 mg; 0.00075% w/w) and asperulosidic acid
(tR) 22.0 min, 35 mg; total yield 0.0435% w/w; an additional
400 mg was isolated from F05). F0603 was chromatographed
over a silica gel column (5.0 × 40 cm) using CHCl3-MeOH-
H2O (from 8:1:0.05 to 2:1:0.1) as solvent system, to give methyl
R-D-fructofuranoside (35 mg; 0.0035% w/w) and methyl ?-D-
fructofuranoside (105 mg; 0.011% w/w), five subfractions
(F060301-F060305), and the mixture of R- and ?-D-glucopy-
ranoses (250 mg; 0.025% w/w) in a ratio of 10:3 (based on the
polarity. F060302 was finally purified by semipreparative
HPLC using MeOH-H2O (30:70; 6 mL/min) as solvent, to give
deacetylasperuloside (tR) 19.5 min, 13 mg; 0.0013% w/w) and
compound 2 (tR) 21.5 min, 1.8 mg; 0.00018% w/w). D-Mannitol
(180 mg; 0.018% w/w) was obtained as colorless needles from
a CHCl3-MeOH (ca. 2:1) solution of fraction F07.
6r-Hydroxyadoxoside (1): colorless gum; [R]23D-50.7° (c
0.28, MeOH); UV (MeOH) λmax(log ?) 231 (3.55) nm; IR (dried
film) νmax 3355, 1692, 1635, 1294, 1077, 807 cm-1;1H NMR
(360 MHz, in CD3OD, TMS) δ 7.46 (1H, d, J ) 1.2 Hz, H-3),
5.15 (1H, d, J ) 6.0 Hz, H-1), 4.65 (1H, d, J ) 7.9 Hz, Glc-1),
4.29 (1H, brs, H-6), 3.87 (1H, dd, J ) 12.2, 1.5 Hz, Glc-6a),
3.79 (2H, brd, J ) 5.4 Hz, H2-10), 3.69 (3H, s, -COOMe), 3.66
(1H, dd, J ) 12.2, 5.6 Hz, Glc-6b), 3.35 (1H, m, Glc-5), 3.27-
3.31 (Glc-3 and Glc-4, overlapped with solvent signal), 3.19
(1H, dd, J ) 9.0, 7.9 Hz, Glc-2), 3.13 (1H, m, H-8), 2.26 (1H,
1H NMR spectra in DMSO-d6), in order of
Table 1. Antioxidant Activity of Compounds Isolated from M.
aAll compounds obtained in this study were evaluated in both
the DPPH and peroxynitrite free-radical scavenging assays. Except
for compounds 3 and 4, all other isolates were indicated to be
inactive (IC50 values over 30 µΜ).bNot determined, since com-
pound 4 was not active in the DPPH assay.cPositive controls used.
Journal of Natural Products, 2005, Vol. 68, No. 4 Notes
m, H-7a), 2.04-2.09 (2H, m, H-5, and H-9), 1.54 (1H, m, H-7b);
13C NMR (90 MHz, in CD3OD, TMS) δ 169.5 (C, C-11), 153.1
(CH, C-3), 113.0 (C, C-4), 100.6 (CH, Glc-1), 99.1 (CH, C-1),
78.3 (CH, Glc-3), 78.0 (CH, Glc-5), 74.7 (CH, Glc-2), 73.2 (CH,
C-6), 71.5 (CH, Glc-4), 62.7 (CH2, Glc-6), 62.3 (CH2, C-10), 51.7
(CH3, -COOMe), 49.9 (CH, C-9), 43.1 (CH2, C-7), 42.4 (CH,
C-8), 35.5 (CH, C-5); LRESIMS m/z 835 (100) [2M + Na]+, 429
(55) [M + Na]+, 360 (15), 339 (13); HRESIMS m/z 429.1378
[M + Na]+(calcd for C17H26O11Na, 429.1373).
6?,7?-Epoxy-8-epi-splendoside (2): colorless gum; [R]23D
-108.4° (c 0.17, MeOH); UV (MeOH) λmax (log ?) 232 (4.00),
280 (3.05) nm; IR (dried film) νmax 3397, 1700, 1639, 1440,
1293, 1178 cm-1;1H NMR (400 MHz, in CD3OD, TMS) δ 7.48
(1H, d, J ) 1.5 Hz, H-3), 5.76 (1H, s, H-1), 4.56 (1H, d, J ) 8.0
Hz, Glc-1), 3.87 (1H, dd, J ) 11.9, 1.8 Hz, Glc-6a), 3.80 (1H, d,
J ) 2.5 Hz, H-6), 3.73 (3H, s, -COOMe), 3.68 (1H, d, J ) 11.7
Hz, H-10a), 3.65 (1H, dd, J ) 11.9, 5.8 Hz, Glc-6b), 3.50 (1H,
d, J ) 2.6 Hz, H-7), 3.47 (1H, d, J ) 11.7 Hz, H-10b), 3.23-
3.33 (H-5, Glc-3, Glc-4, and Glc-5, overlapped with solvent
signal), 3.13 (1H, dd, J ) 8.9, 8.1 Hz, Glc-2), 2.32 (1H, d, J )
8.8 Hz, H-9);13C NMR (100 MHz, in CD3OD, TMS) δ 168.5
(C, C-11), 154.2 (CH, C-3), 107.7 (C, C-4), 99.9 (CH, Glc-1),
93.8 (CH, C-1), 80.7 (C, C-8), 78.4 (CH, Glc-3), 78.0 (CH, Glc-
5), 74.6 (CH, Glc-2), 71.6 (CH, Glc-4), 65.2 (CH2, C-10), 62.8
(CH2, Glc-6), 60.8 (CH, C-7), 57.9 (CH, C-6), 51.9 (CH3,
-COOMe), 46.4 (CH, C-9), 33.3 (CH, C-5); LRCIMS m/z 438
(80) [M + NH4]+, 258 (55), 241 (15), 198 (35), 180 (40), 75 (100);
HRFABMS m/z 443.1147 [M + Na]+(calcd for C17H24O12Na,
Measurement of DPPH Free-Radical Scavenging Ac-
tivity. This assay used the stable free radical 1,1-diphenyl-
2-picrylhydrazyl (DPPH) to determine the potential scavenging
activity of extracts and pure compounds, as previously de-
scribed.35Briefly, extracts and pure compounds, in DMSO,
were plated in triplicate to give a final concentration of 200
µg/mL and incubated in 200 µM DPPH in EtOH at 37 °C for
30 min in the dark. DMSO was used as the negative control
and gallic acid as the positive control. Absorbance was
measured at 515 nm, and the percent scavenging activity was
determined by comparison with the DMSO negative control.
Active compounds were tested in triplicate in the same manner
at final concentrations of 2.5, 5, 10, and 20 µg/mL. The IC50
values were obtained through extrapolation from linear re-
Measurement of ONOO--Scavenging Activity. ONOO-
used in this study was purchased from Cayman Chemical Co.
or derived by 3-morpholinosydnonimine (SIN-1). ONOO--
scavenging ability was measured by monitoring the oxidation
of DHR 123 using the modified method of Kooy et al.36Briefly,
a stock solution of DHR 123 (5 mM) purged with nitrogen was
prepared in advance and stored at -20 °C. A working solution
of DHR 123 (5 µM) diluted from the stock solution was placed
on ice in the dark immediately prior to each experiment. The
rhodamine buffer (sodium phosphate dibasic, 50 mM; sodium
phosphate monobasic, 50 mM; sodium chloride, 90 mM;
potassium chloride, 5 mM) including diethylenetriaminepen-
taacetic acid (DTPA, 5 mM) was purged with nitrogen and
placed on ice before use. The ONOO--scavenging ability was
determined at room temperature by a microplate fluorescence
spectrophotometer FL500 (Bio-Tek Instruments) with excita-
tion and emission wavelengths of 485 and 530 nm, respec-
tively. The background and final fluorescent intensities were
measured 5 min after treatment with or without native
ONOO-(10 µM) in 0.3 N sodium hydroxide or SIN-1 (10 µM).
Oxidation of DHR 123 by decomposition of SIN-1 gradually
increased, whereas native ONOO-rapidly oxidized DHR 123
with its final fluorescent intensity being stable over time on
the inhibition of DHR 123 oxidation by ONOO-. Penicillamine
was used as a positive control.
Resources Center, University of Illinois at Chicago (UIC), and
J. Fowble, College of Pharmacy, The Ohio State University
(OSU), for facilitating the running of the 360, 400, and 500
MHz NMR spectra, and Drs. J. A. Anderson and Y. Wang,
We thank Dr. R. Kleps, Research
Research Resources Center, UIC, for the MS data. We also
thank Dr. C. Cottrell, The Campus Chemical Instrument
Center, OSU, for running the HMBC spectrum for americanin
A. We are very grateful to Dr. J. T. Dalton, Division of
Pharmaceutics and Pharmaceutical Chemistry, College of
Pharmacy, OSU, for providing the LC-MS instrument, and Mr.
W. P. Jones, Division of Medicinal Chemistry and Pharma-
cognosy, College of Pharmacy, OSU, for conducting the LC-
Supporting Information Available:
of compounds 1-3, partial HMBC NMR spectrum of compound 3
acquired at 600 MHz with a TXI cryoprobe, and the LC-MS analysis
profiles of asperuloside and a n-BuOH-soluble extract of M. citrifolia.
This material is available free of charge via the Internet at http://
1H and13C NMR spectra
References and Notes
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Notes Journal of Natural Products, 2005, Vol. 68, No. 4