In vitro antiplasmodial activity of extracts of tristaniopsis species and identification of the active constituents: Ellagic acid and 3,4,5-trimethoxyphenyl-(6'-O-galloyl)-O-/?-D-glucopyranoside

Article abstract of DOI:10.1021/np000306j

Screening of plants from New Caledonia for antiplasmodial activity against Plasmodium falciparum revealed that methanolic extracts of the leaves and bark of Tristaniopsis calobuxus, T. yateensis, and T. glauca inhibited the growth of chloroquine-sensitive and -resistant clones. Ellagic acid and the new compound 3,4,5-trimethoxyphenyl-(6'-O-galloyl)-O-β-D-glucopyranoside were identified as the active constituents (ICso 0.5 and 3.2 /

Full text of DOI:10.1021/np000306j

J . Nat. Prod. 2001, 64, 603-607
603
In Vitr o An tip la sm od ia l Activity of Extr a cts of Tr ista n iopsis Sp ecies a n d
Id en tifica tion of th e Active Con stitu en ts: Ella gic Acid a n d
3,4,5-Tr im eth oxyp h en yl-(6′-O-ga lloyl)-O-â-D-glu cop yr a n osid e
Luisella Verotta,*,† Mario Dell’Agli,‡ Andrea Giolito,† Marco Guerrini,§ Pierre Cabalion, and Enrica Bosisio*,‡
Department of Organic and Industrial Chemistry and Institute of Pharmacological Sciences, University of Milan, Italy,
Institute of Chemistry and Biochemistry “G. Ronzoni”, Milano, Italy, and Institut de Recherche pour le De´veloppement (IRD),
Noumea, New Caledonia
Received J une 19, 2000
Screening of plants from New Caledonia for antiplasmodial activity against Plasmodium falciparum
revealed that methanolic extracts of the leaves and bark of Tristaniopsis calobuxus, T. yateensis, and T.
glauca inhibited the growth of chloroquine-sensitive and -resistant clones. Ellagic acid and the new
compound 3,4,5-trimethoxyphenyl-(6′-O-galloyl)-O-â-D-glucopyranoside were identified as the active
constituents (IC50 0.5 and 3.2 µM, respectively). The growth inhibition of both clones was comparable.
The compounds showed negligible or very low cytotoxicity to human skin fibroblasts and Hep G2 cells
when tested at concentrations ranging from 0.5 to 100 µM.
Malaria remains a major problem of public health
worldwide (200 million people of which 1-2 million die each
year), mainly due to an increase in parasite resistance to
available drugs.1 The search for new antimalarial drugs is
essential and requires the identification of new biochemical
targets for drug development.2 Studies conducted in various
laboratories have led to the discovery of several new
targets, including the heme detoxification process, iso-
prenoid and phospholipid biosynthesis, nucleic acid me-
tabolism, proteases, and others.3
Screening of plants collected in New Caledonia for anti-
elastase activity led to the identification of some active
species.4 Elastase is a serine proteinase involved in a
variety of inflammatory processes, including emphysema,5
parodontosis,6 and cystic fibrosis.7 Proteinases have also
been shown to be essential to a variety of viruses and other
pathogenic microorganisms.2 In particular, Plasmodium
falciparum degrades hemoglobin by means of two aspartic
proteinases (plasmepsin I and II) and one cysteine pro-
teinase (falcipain), which can be potential targets for the
search for new drugs.2,3 Thus, we also evaluated the
extracts with anti-elastase activity for antiplasmodial
activity: D6 (chloroquine-sensitive, moderately mefloquine
resistant) and W2 (chloroquine-resistant, mefloquine sensi-
tive) clones of Plasmodium falciparum. Among the tested
plants, Tristaniopsis calobuxus Brongniart & Gris (Myr-
taceae) inhibited parasite growth in vitro. From the bark
extract, two compounds with in vitro antiplasmodial activ-
ity were isolated and identified as ellagic acid and 3,4,5-
trimethoxyphenyl-(6′-O-galloyl)-O-â-D-glucopyranoside (Fig-
ure 1).
us Brongniart & Gris gave positive results (Table 1).
Following an activity-guided procedure, the crude extract
was counterextracted and the activity was retained mainly
in the ethyl acetate fraction (Table 2), which was chro-
matographed on Sephadex LH20 eluted with methanol.
Three active fractions were separated (Table 2) and sub-
mitted to further fractionation, leading to the isolation of
compounds A1-A5, B1-B4, AB1, and D1 (Table 3). From
fraction 149-185, compound D1 was crystallized and
showed an IC50 of 0.1 µg/mL against both clones. D1 was
identified as ellagic acid. From fraction 122-148 was
obtained C1, a mixture containing ellagic acid (IC50 1.6 and
1.4 µg/mL against D6 and W2 clones, respectively), which
was presumed to account for the observed biological activ-
ity.
From fraction 90-96, compound A3A was isolated as an
amorphous powder and showed an IC50 of 1.7 and 1.6 µg/
mL against D6 and W2 clones, respectively.
1
Its H NMR spectrum (500 MHz, DMSO-d6) showed the
presence of two singlets at δ 6.93 and 6.28, respectively,
each corresponding to two aromatic protons (quantitative
spectra were obtained by recording the experiments with
a recycle time of 45 s). The two shielded protons, character-
ized by an unexpectedly long relaxation time, allowed us
to propose the presence of isolated aromatic protons in
symmetrical relationship with the rest of the molecule,
positioned in an electron donor environment. A signal
consistent with a sugar anomeric proton was observed at
δ 4.88 (d, J ) 7.7 Hz). Signals for the other sugar protons
were found at δ 4.54 (dd J ) 12.0, 1.9 Hz, H-6′A) and δ
4.25 (dd, J ) 12.0, 5.8 Hz, H-6′B); the remaining protons
are part of a strong coupled system and resonate at δ 3.72
(1H, m), 3.32 (1H, m), 3.27 (1H, m), and 3.23 (1H, m). Two
more singlets, corresponding to six and three protons,
respectively, were observed at δ 3.62 and 3.55. The de-
coupled 13C NMR spectrum, together with DEPT experi-
ments, revealed the presence of five singlets: one carbonyl
group at δ 166.0 and four aromatic quaternary carbons at
δ 153.8 (1C, C-1), 138.5 (1C, C-4′′), 132.9 (1C, C-4), and
119.6 (1C, C-1′′); seven doublets at δ 108.7 (2C, C-2′′ + 6′′),
100.9 (1C, C-1′), 94.6 (2C, C-2+C-6), 76.2 (1C, C-3′), 74.0
(1C, C-5′), 73.3 (1C, C-2′), and 69.9 (1C, C-4′), respectively;
one triplet at δ 63.7 (1C, C-6′); and two methoxy groups at
Resu lts a n d Discu ssion
Among the plants evaluated for antiplasmodial activity,
the methanolic extract of the bark of Tristaniopsis calobux-
* Corresponding authors. (E.B.) Institute of Pharmacological Sciences,
Via Balzaretti, 9, 20133 Milano, Italy. Tel: 39-02-20488372. Fax: 39-02-
29404961. E-mail: enrica.bosisio@unimi.it. (L.V.) Department of Organic
and Industrial Chemistry, Via Venezian 21, 20133 Milano, Italy. Tel 39-
02-2363469. Fax: 39-02-2364369. E-mail: luisver@icil64.cilea.it.
† Department of Organic and Industrial Chemistry, University of Milan.
‡ Institute of Pharmacological Sciences, University of Milan.
§ Institute of Chemistry and Biochemistry “G. Ronzoni”.
Institut de Recherche pour le De´veloppement (IRD).
10.1021/np000306j CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/27/2001

604 J ournal of Natural Products, 2001, Vol. 64, No. 5
Verotta et al.
biogenetically from oxidative decarboxylation of 3,4,5-
trimethoxybenzoic acid. This is the first report of a 3,4,5-
trimethoxyphenol glucoside from a natural source.9
This is also the first report of the inhibition of Plasmo-
dium growth by ellagic acid. Biological properties of ellagic
acid reported previously include antimutagenic and tumor
chemopreventive activity.10-16 It has previously been shown
that in rats ellagic acid is partially absorbed, metabolized
by intestinal flora, and excreted in bile and urine as
glucuronide and glutathione conjugates.17,18 In freshly
isolated rat hepatocytes, gallic acid is converted to ellagic
acid by autoxidation, and the conversion reduced gallic acid
cytotoxicity.19 A3A is less active as compared to ellagic acid.
It may be speculated that this is due to lower bioavailability
or the need to be metabolically activated. In addition, since
galloyl and glucose moieties are devoid of activity (see Table
3, gallic acid inactive vs Plasmodium growth), then it would
seem that only the trimethoxyphenol group is likely to
contribute to the observed effect.
Although the isolated compounds are less potent than
chloroquine (IC50 2.54 and 58 ng/mL for D6 and W2,
respectively) and mefloquine (3.98 and 1.36 ng/mL), it is
noteworthy that the compounds are active against the
resistant clone. In addition, interest in plant phenols as
potential complementary agents has recently arisen be-
cause they have been shown to potentiate the in vitro effect
of artemisinin.20
Three other Tristaniopsis species were also tested for
antiplasmodial activity. Antiplasmodial activity was found
in the leaves and bark of Tristaniopsis glauca Brongniart
& Gris and Tristaniopsis yateensis J . W. Dawson, and in
the leaves, branches, and root bark of T. calobuxus (Table
4).
To compare the effect on the parasite with toxicity to
mammallian cells, the antiproliferative activities of the
isolated compounds against human skin fibroblast and the
hepatoma G2 line (Hep G2) were evaluated. A3A was not
cytotoxic to either cell type at 100 µM, the highest
concentration tested, while ellagic acid, at 100 µM, showed
a slight cytotoxicity to Hep G2 and fibroblasts, with 9%
and 20% decreases in proliferation, respectively.
These results suggest a negligible cytotoxic effect of the
compounds on mammalian cells at the concentrations
found to inhibit the parasite growth (IC50 0.5 and 3.2 µM
for ellagic acid and A3A, respectively), suggesting they are
selectively toxic to the parasite.
F igu r e 1. Ellagic acid (1) and 3,4,5-trimethoxyphenyl-(6′-O-galloyl)-
O-â-D-glucopyranoside (2).
δ 60.3 (1C) and δ 55.9 (2C), respectively. Assignments were
made possible through the HSQC and HMBC experiments.
Exp er im en ta l Section
The presence of an acylated phenolic â-D-glucoside was
hypothesized. The acyl moiety is comprised of a galloyl
residue esterified with the primary alcoholic hydroxy group
of the sugar. The acylated monosaccharide is linked
through an acetal bond to a trimethoxyphenol residue, as
confirmed by HMBC experiments. NOE interactions be-
tween the aromatic protons at δ 6.28 and the anomeric
proton, and between the same aromatic protons and the
two methoxy groups at δ 3.62, indicated the presence of a
3,4,5-trimethoxyphenol glucoside.
The unambiguous structural assignment was made
possible by chemical degradation. Treatment of the natural
glucoside with NaOMe/MeOH yielded 3,4,5-trimethoxyphe-
nol â-D-glucopyranoside, which was identified by compari-
son with a synthetic sample prepared by glucosidation of
3,4,5-trimethoxyphenol. Any attempt to selectively acylate
the hydroxymethylene group of the sugar moiety in 3,4,5-
trimethoxyphenol â-D-glucopyranoside using previously
successful methods on similar phenolic glucosides failed.8
It is postulated that 3,4,5-trimethoxyphenol can be derived
P la n t Ma ter ia l. Plants were collected by Dudley Nicholls
(DN), Institut de Recherche pour le Deve´loppement (IRD),
Noumea, and dried in an oven at 40 °C. Plant identity was
verified at the herbarium of the IRD Center of Noumea, and
voucher specimens were deposited at the Laboratory of
Natural Substances of Biological Interest, Noumea, New
Caledonia: T. calobuxus Brongniart & Gris (DN no. 17,
collection J une 19th, 1996, at Rivie´re des Pirogues, direction
to Yate´); Tristaniopsis yateensis J .W. Dawson (DN no. 57,
collection J uly 17th, 1997, at the Col de Yate´); Tristaniopsis
glauca Brongniart & Gris (DN no. 58 collection J uly 17th,
1997, at Lac de Yate´).
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured on a Perkin-Elmer 241 polarimeter. Si gel 60
(70-230 mesh, Merck) was used for open-column chromatog-
raphy. Sephadex LH20 (Pharmacia Biotech) was used for gel
filtration chromatography. Mass spectra were recorded on a
VG Analytical 7070 EQ spectrometer at 70 eV in the electron
impact and chemical ionization (NH3) mode.
The HSCCC experiment was carried out with a centrifugal
countercurrent chromatograph from Pharma-Tech Research

Antiplasmodial Activity of Tristaniopsis Species
J ournal of Natural Products, 2001, Vol. 64, No. 5 605
Ta ble 1. Screening of Methanolic Plant Extracts for Antiplasmodial Activity
plant
yield,a % w/w
22
parasite clone
IC50, ng/mL
Ximenia americana L. (Olacaceae) bark
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
>50000
Oncotheca balansae Baillon (Oncothecaceae) bark
Cunonia montana Schlecter (Cunonaceae) bark
Tristaniopsis calobuxus Brongniart & Gris (Myrtaceae) bark
Amyema scandens Danser(Loranthaceae) leaves
Guioa villosa Radlk (Sapindaceae) leaves
7.9
>50 000
>50 000
7.8
7.1
8970
7652
>50 000
10
12
15
22
16
>50 000
>50 000
>50 000
>50 000
Diospyros vieillardii (Hiern) Kostermans (Ebenaceae) leaves
Breynia disticha J . R. & G. Foster (Euphorbiaceae) bark
Trema orientalis L. (Ulmaceae) bark
a
Extraction recovery calculated on the dry material.
Ta ble 2. Activity-Guided Fractionation of the Bark Extract of
Ta ble 3. Antiplasmodial Activity of Isolated Compounds
T. calobuxus
compounda
A1
parasite clone
IC50, ng/mL
compound
parasite clone
IC50, ng/mL
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
>5000
methylene chloride fraction
D6
W2
D6
W2
D6
W2
7036
4297
1727
A2
2245
2651
1747
1585
>5000
ethyl acetate fraction
aqueous fraction
1112
A3A
>50 000
A4
Sephadex LH 20 fractions
20-89
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
>5000
A5
>5000
>5000
>5000
>5000
>5000
>5000
90-96
2600
1822
>5000
B1
97-105
106-121
122-148
149-185
186-end
B2
>5000
B3
1508
1254
181
113
>5000
B4
AB1
D1
145
103
2.54
58
3.98
1.36
chloroquine
mefloquine
Corp. (Baltimore, MD, model CCC 1000). It was equipped with
a SSI (Scientific Systems) liquid chromatography pump (model
300), a high-speed countercurrent chromatograph electronic
controller PTRC, and an injection valve (Rheodyne). The
HSCCC experiments were performed with a two-phase solvent
system composed of (1) CHCl3-MeOH-H2O, 5:6:4, or (2)
CHCl3-MeOH-i-PrOH-H2O, 5:6:1:4.
a
Compounds A5, B1 and AB1 were identified as p-OH benzoic
acid, gallic acid and 3,4-di-OH benzoic acid, respectively; Com-
pound A3A was identified as 3,4,5 trimethoxyphenol-(6′-O-galloyl)-
O-â-D-glucopyranoside; compound D1 is ellagic acid.
The two-phase system was prepared by thoroughly mixing
the solvents in a separatory funnel at room temperature and
leaving it to settle for 12 h. The column was entirely filled
with the stationary phase (lower phase) at a flow rate of 9
mL/min, after the mobile phase (upper phase) was pumped
into the inlet of the column at a rotation speed of 1040 rpm
and a flow rate of 1 mL/min. The elution mode was tail to head.
The equilibration volume was 90 mL for two-phase solvent
system 1 and 60 mL for two-phase solvent system 2. The
sample was filtered on a Millex HV filter (0.45 µm, Millipore).
acquired using 16 scans per series in 1K × 512W data points,
with zero filling in F1, and a sine function was applied before
Fourier transformation. 1H/13C chemical shift correlations
(HSQC) were performed using z-gradients for coherence selec-
tion. An HSQC spectrum was collected with decoupling during
the acquisition period in phase sensitivity-enhanced pure
absorption mode;21 the matrix size was 1K × 512 points, and
the experiment was zero-filled to 2K × 1K and multiplied with
shifted sine-bell-squared prior Fourier transformation. The
HMBC spectra22 were acquired using 128 scans per series in
1K × 256W data points. The HMBC spectra were optimized
Si gel 60 F254 (Merck) TLC was eluted with the organic
phase of the mixture CHCl3-MeOH-i-PrOH-H2O (5:6:1:4).
Spots were revealed by UV absorption (254 nm) and by
spraying with FeCl3 or H2SO4 followed by heating. NMR: the
sample was prepared by dissolving 8 mg in 0.6 mL of DMSO-
d6 (99.96% D); 500 MHz spectra were recorded on a Bruker
AMX500 spectrometer, equipped with a 5 mm inverse broad-
n
for a J C-H of 8 Hz, with n ) 2-4. The experiment was zero-
filled and multiplied with sine-bell prior to Fourier transfor-
mation. The recycle delay for all 2D spectra was about 3 s.
E xt r a ct ion a n d Isola t ion . For initial assays, the plant
material was powdered, delipidized with petroleum ether at
40-60 °C, and extracted twice with methanol (5 g/30 mL). The
extracts were filtered, concentrated in vacuo, and weighed for
the determination of the w/w yield (Table 1). For purification
and product isolation, 300 g of T. calobuxus bark was delip-
1
band probe with a shielded z-axis gradient, at 313 K. H and
13C chemical shifts are referenced to the solvent signal (2.49
ppm 1H and 49.5 ppm 13C). The DQF-COSY spectrum was

606 J ournal of Natural Products, 2001, Vol. 64, No. 5
Verotta et al.
Ta ble 4. Antiplasmodial Activity of Methanolic Extracts of
Tristaniopsis spp.
Dowex 50 W resin (15 mg), filtered, and evaporated to dryness.
One milligram of 3,4,5-trimethoxyphenol-â-D-glucopyranoside
was obtained.
parasite
clone
IC50
ng/mL
,
Syn th esis of 3,4,5-Tr im eth oxyp h en ol. A 3.5 g sample of
3,4,5-trimethoxyanilin was added to a solution of 50% H2SO4
(74 mL), under nitrogen, with stirring. At 0 °C, a solution of
sodium nitrite (1.58 g/28 mL of water) was added dropwise,
and then a solution of CuSO4‚3H2O (9.8 g/53 mL of water) was
added. The mixture was stirred at room temperature for 30
min and then warmed at 60 °C for 45 min. The solution was
extracted with CH2Cl2 (4 × 100 mL). The organic phases were
dried and evaporated to dryness (2.4 g). The crude material
was purified by Si gel (125 g) eluted with CHCl3-EtOAc, 9:1
(3.1 L). A total of 140 fractions (20 mL each) were collected.
Fractions 65-130 (1.7 g) were crystallized (i-Pr)2O, to yield
0.95 g of 3,4,5-trimethoxyphenol, mp 142 °C [lit.23 mp 145-6
°C]; 1H NMR (CDCl3, 200 MHz) δ 6.18 (2H, s, H-2+H-6), 4.80
(s, OH), 3.83 (9H, s, OCH3); CIMS m/z 184 [M]+ (68), 169 (100).
Syn th esis of 3,4,5-Tr im eth oxyp h en ol-â-D-glu cop yr a -
n osid e. 3,4,5-Trimethoxyphenol (564 mg) in 1.25 M NaOH (4.1
mL) was added to a solution of triethylbenzylammonium
bromide (TEBA) (39 mg) and R-bromotetraacetylglucose (1.4
g) in CHCl3 (8 mL). The mixture was stirred at 70 °C for 20 h.
Water was added (40 mL), and the mixture was extracted with
CHCl3 (3 × 40 mL) and dried with Na2SO4. After evaporation
under vacuum, 1.53 g of an oily material was obtained and
purified over silica gel (230 g) eluted with petrol-EtOAc 7:3
(1 L). A total of 200 fractions of 5 mL each were collected.
Fractions 141-181 were pooled and taken to dryness, to afford
596 mg of 3,4,5-trimethoxyphenoltetraacetyl-â-D-glucopyra-
noside (38%).
plant extract
T. calobuxus Brongniart & Gris leaves
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
D6
W2
7605
4058
15 960
7895
15 284
7945
15 682
8024
7453
4221
14 800
7529
7864
branches
root bark
bark
T. yateensis J .W. Dawson
T. glauca Brongniart & Gris
leaves
bark
leaves
4476
idized and extracted twice with 300 mL of methanol (recovery
11%). The crude extract was further fractionated in methylene
chloride (yield 11%), ethyl acetate (yield 5%), and water-soluble
phases. Extracts were kept at -20 °C until use and dissolved
in DMSO for the biological assays.
The ethyl acetate extract (1.7 g) was purified on Sephadex
LH20 (2.5 × 88 cm) eluted with MeOH at a flow rate of 14
mL/h. A total of 214 fractions (6 mL each) were collected. Based
on the TLC similarities, identical fractions were combined to
give a total of seven fractions: 20-29 ) 240 mg; 90-96 ) 79
mg; 97-105 ) 194 mg; 106-121 ) 113 mg; 122-148 ) 205
mg; 149-185 ) 337 mg; and 186-214 ) 478 mg.
Fraction 90-96 (79 mg) was submitted to countercurrent
chromatography using the solvent system CHCl3-MeOH-
H2O, 5:6:4 (mobile phase ) upper phase). Six fractions were
obtained: A1 (8 mg), A2 (12.5 mg), A3 (15 mg), A4 (2.1 mg),
A5 (11.9 mg), and a fraction that was further purified by silica
gel (CHCl3-MeOH 85:15) chromatography to afford AB1 (5
mg).
A5 and AB1 were identified as p-hydroxybenzoic acid and
3,4-dihydroxybenzoic acid, respectively, by comparison of their
spectral data (NMR and MS spectra) with commercial samples.
A3 was purified on silica gel (CHCl3-MeOH, 85:15) to yield
6 mg of compound A3A (0.002%), 3,4,5-trimethoxyphenyl-(6′-
O-galloyl)-O-â-D-glucopyranoside, as a white amorphous pow-
der: [R]25D -23.4° (c 0.19, MeOH); 1H NMR (300 MHz, DMSO-
d6) δ 6.93 (2H, bs, H-2′′, H-6′′), 6.28 (2H, bs, H-2, H-6), 4.88
(1H, d, J ) 7.7 Hz, H-1′), 4.45 (1H, dd, J ) 12.0, 1.9 Hz, H-6′A),
4.29 (1H, dd, J ) 12.0, 5.8 Hz, H-6′B), 3.72 (1H, m, H-5′), 3.62
(6H, s, 2 × OCH3), 3.55 (3H, s, OCH3), 3.32 (1H, m, H-3′), 3.27
(1H, m, H-4′), 3.23 (1H, m, H-2′); 13C NMR (75.5 MHz, DMSO-
d6) δ 166.0 (s, C-7′′), 153.8 (s, C-1), 153.3 (s, C-3, C-5), 145.6
(s, C-3′′, C-5′′), 138.5 (s, C-4′′), 132.9 (s, C-4), 119.6 (s, C-1′′),
108.7 (d, C-2′′,6′′), 100.9 (d, C-1′), 94.6 (d, C-2, C-6), 76.2 (d,
C-3′), 74.0 (d, C-5′), 73.3 (d, C-2′), 69.9 (d, C-4′), 63.7 (t, C-6′),
60.3 (q, OCH3), 55.9 (q, OCH3); CIMS (NH3, positive) m/z 516
[MNH4]+, 364 [MNH4 - C7H5O4]+, 347 [M-C7H5O4]+, 202 [364
- C5H10O5]+, 185 [347 - C5H10O5]+; CIMS (NH3, negative) m/z
497 [M - H]-, 169 [C8H9O4]-; EIMS (70 eV) m/z 346 (5), 184
(100), 169 (49).
Fraction 97-105 (215 mg) was submitted to CCC using the
solvent system CHCl3-MeOH-i-PrOH-H2O, 5:6:1:4 (mobile
phase ) upper phase). Eight fractions were obtained, of which
B1 (150 mg, 0.04% based on dry material) was identified as
gallic acid by comparison of its spectral data with those of a
commercial sample.
Fraction 149-185 (337 mg) precipitated a residue (D1 ) 125
mg) that was identified as ellagic acid. Additional ellagic acid
was also isolated from the mother liquor (D2 ) 195 mg).
Fraction 122-148 (295 mg) was counterextracted between
BuOH-n-PrOH-H2O, 2:1:3, and the organic phase (C1 ) 142
mg) contained additional ellagic acid.
3,4,5-Trimethoxyphenoltetraacetyl-â-D-glucopyranoside (500
mg) in MeOH (40 mL) was treated with 0.2 M NaOMe-MeOH
(29 mL) for 3 h at room temperature. The mixture was
neutralized with Dowex 50 W (5 g) and filtered. After solvent
evaporation, 315 mg of crude material was obtained, which,
after crystallization (i-PrOH-MeOH, 1:1), afforded 290 mg of
3,4,5-trimethoxyphenol-â-D-glucopyranoside: mp 204 °C; [R]25
D
-78° (c 1, MeOH); 1H NMR (DMSO-d6, 200 MHz) δ 6.40 (2H,
bs, H-2+H-6), 4.81 (1H, d, H1′), 3.74 (6H, s, OCH3), 3.64 (3H,
s, OCH3), 3.32 (1H, m), 3.27 (1H, m), 3.23 (1H, m); 13C NMR
(25.4 MHz, DMSO-d6) δ 154.0 (s, C-1), 153.3 (s, C-3, C-5), 132.5
(s, C-4), 101.1 (d, C-1′), 94.4 (d, C-2, C-6), 77.4 (d, C-5′), 76.9
(d, C-3′), 73.3 (d, C-2′), 70.2 (d, C-4′), 61.0 (t, C-6′), 60.2 (q,
OCH3), 55.8 (q, OCH3); CIMS m/z 346 [M]+ (5), 184 (100), 169
(63).
In Vitr o Test for An tip la sm od ia l Activity. Material
obtained at each purification step was tested for antiplasmo-
dial activity against P. falciparum clones CDC/Sierra Leone I
(D6: chloroquine sensitive, moderately mefloquine resistant)
and CDC/Indochina III (W2: chloroquine resistant, mefloquine
sensitive). Tests were performed at the Walter Reed Army
Institute of Research (WRAIR) under a grant from the UNDP/
World Bank/WHO Special Programme for Research and
Training for Tropical Diseases (TDR). The method used was
a modification of the procedures described previously.24-26
The
system is limited to the assessment of intrinsic activity against
erythrocytic asexual blood stages. Chloroquine and mefloquine
were used as reference compounds. IC50 for chloroquine was
2.54 and 58 ng/mL against D6 and W2, respectively. IC50 for
mefloquine was 3.98 and 1.36 ng/mL against D6 and W2,
respectively.
Cytotoxicity Assa y. Cytotoxicity was evaluated in human
dermal fibroblasts from child skin biopsies and Hep G2 cell
(ATCC, Rockville, MD) lines. Cell proliferation was followed
by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazo-
lium bromide) test.27 Fibroblasts (2 × 104/ml) were grown in
DMEM (Dulbecco’s modified Eagle’s medium) containing 10%
fetal calf serum, 1% penicillin/streptomycin, and 1% L-
glutamine. Hep G2 (25 × 104/mL) were cultivated in DMEM
containing 10% fetal calf serum, 1% penicillin/streptomycin,
1% L-glutamine, and 1% nonessential amino acids. The
compounds to be tested were added to culture medium dis-
solved in DMSO or methanol, 1 day after plating. The final
concentration of the vehicle in control and test medium was
Hyd r olysis of A3A. A 1.5 mg sample of A3A in MeOH (0.2
mL) was treated with NaOMe-MeOH (0.3 mL) for 3 h at room
temperature. The reaction mixture was then stirred with a

Antiplasmodial Activity of Tristaniopsis Species
J ournal of Natural Products, 2001, Vol. 64, No. 5 607
al. Phytochemistry 1987, 26 (4), 1147-1152). This communication
again refers to another paper (Nonaka, G. I.; et al. Chem Pharm.
Bull. 1982, 30 (6), 2061-2067), where the authors describe the
isolation and structural elucidation of a different compound, namely,
2,4,6-trimethoxyphenol 1-O-â-D-glucopyranoside.
(10) Wood, A. W.; Huang, M. T.; Chang, R. L.; Newmark, H. L.; Lehr, R.
E.; Yagi, H.; Sayer, J . M.; J erina, D. M.; Conney, A. H. Proc. Natl.
Acad. Sci. U.S.A. 1982, 79, 5513-7
1% of the incubation volume. The effect of compounds on cell
proliferation was assayed at concentrations ranging from 0.5
to 100 µM, at 24 and 48 h after treatment. Each concentration
was tested in quadruplicate in two separate experiments.
Ack n ow led gm en t. The authors are indebted to Dr. P.
Olliaro (TDR, WHO) for providing facilities for the biological
assays and for helpful discussion. Fibroblasts were a generous
gift of Prof. Abatangelo, University of Padova, Italy. This work
was supported by funds from the Ministry of the University
and Scientific and Technological Research (MURST 40% and
60%).
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