J. Nat. Prod.

2003, 66, 1501-1504 1501

New Bioactive Polyphenols from Theobroma grandiflorum (“Cupuaçu”)

Hui Yang,† Petr Protiva,‡ Baoliang Cui,† Cuiying Ma,† Scott Baggett,† Vanessa Hequet,§ Scott Mori,§
I. Bernard Weinstein,‡ and Edward J. Kennelly*,†
Department of Biological Sciences, Lehman College and The Graduate Center, The City University of New York,
250 Bedford Park Boulevard West, Bronx, New York 10468, Herbert Irving Comprehensive Cancer Center,
College of Physicians and Surgeons of Columbia University, 701 West 168th Street, New York, New York 10032, and
The New York Botanical Garden, 200th Street and Southern Boulevard, Bronx, New York 10458

Received August 5, 2003

Activity-guided fractionation of Theobroma grandiflorum (“cupuaçu”) seeds resulted in the identification

of two new sulfated flavonoid glycosides, theograndins I (1) and II (2). In addition, nine known flavonoid
antioxidants, (+)-catechin, (-)-epicatechin, isoscutellarein 8-O-β-D-glucuronide, hypolaetin 8-O-β-D-
glucuronide, quercetin 3-O-β-D-glucuronide, quercetin 3-O-β-D-glucuronide 6′′-methyl ester, quercetin,
kaempferol, and isoscutellarein 8-O-β-D-glucuronide 6′′-methyl ester, were identified. Theograndin II (2)
displayed antioxidant activity (IC50 ) 120.2 µM) in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free-radical
assay, as well as weak cytotoxicity in the HCT-116 and SW-480 human colon cancer cell lines with IC50
values of 143 and 125 µM, respectively. While 1 was less active as an antioxidant than 2, the known
compounds were more potent in the DPPH assay (IC50 range 39.7-89.7 µM).
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Publication Date (Web): October 23, 2003 | doi: 10.1021/np034002j

Theobroma (Sterculiaceae) is a tropical American genus products. This pulp is used to prepare drinks (e.g., “vinho
of 22 species of understory trees that grow in evergreen do cupuaçu” and “suco de cupuaçu”), ice cream, liquors,
rainforests between latitudes 18° N and 15° S.1 The genus jellies, preserves, and candy. In the United States, “cu-
is noteworthy because it includes the economically impor- puaçu” has been used in certain popular mixed fruit juice
tant “cacao” or chocolate tree (Theobroma cacao L.). The drinks. Moreover, the seeds of T. grandiflorum have
uses and cultivation of T. cacao were developed by the received attention because of their potential for being used
Mayans in Central America before the arrival of Europe- as a chocolate substitute.10 The composition of T. grandi-
ans.2 Within this genus, Theobroma grandiflorum (Willd. florum seed oil has been studied, and various lipids and
ex Spreng.) K. Schum., known commonly as “cupuaçu”, is alkaloids have been identified, such as the xanthine
second to cacao in terms of economic importance. T. alkaloids, caffeine, and theobromine.11,12
grandiflorum is a medium-sized tree, usually 6-10 m and As part of our continuing study of polyphenols from
up to 18 m tall. Its natural distribution is in the southern lesser-used tropical fruits, we have examined the antioxi-
half of the State of Pará, Brazil, and the adjacent Amazo- dant and cytotoxic activities of T. grandiflorum. An alcohol
nian Maranhão, where it grows at low densities in the rain extract of T. grandiflorum seeds was subjected to activity-
forest.1 guided fractionation using the DPPH method, and two new
“Cupuaçu” produces the largest pods among all Theo- polyphenols, theograndins I (1) and II (2), were identified.
broma species. In the past 20 years, “cupuaçu” has been The seeds of T. grandiflorum were extracted exhaus-
developed into a commercial crop in Amazonia.3 It is tively with MeOH and partitioned sequentially with hex-
frequently cultivated in Brazil as well as in the warm ane, EtOAc, and BuOH. The EtOAc and BuOH fractions
lowlands of other tropical American countries such as were subjected to activity-guided fractionation using the
Ecuador, Costa Rica, Venezuela, Colombia, Guyana, Suri- DPPH method, with an initial separation by Sephadex LH-
nam, and French Guiana. Although T. grandiflorum is 20 column chromatography (CC). Further separation of
morphologically distinct from T. cacao, the two species can active fractions over reversed-phase (RP-18) CC and Sepha-
be hybridized and the hybrids produce a high number of dex LH-20 CC yielded two new sulfated flavonoid glyco-
fruits. For that reason T. grandiflorum is considered to be sides, 1 and 2.
one of the closest relatives of T. cacao.4 Recent phytochemi-
cal studies have demonstrated that T. cacao seeds contain Compound 1, a yellow powder, gave a molecular ion at
potent polyphenolic antioxidants including (-)-epicatechin m/z 541.0297 corresponding to [M - H]- in the negative
and (+)-catechin.5,6 The effects of chocolate on human HRESIMS, which indicated a molecular formula of
health are being studied with considerable interest.7-9 Our C21H18O15S. Compound 1 displayed four main peaks in the
current research on the antioxidant constituents of “cu- negative ESIMS at m/z 541 [M - H]-, 461 [M - SO3 -
puaçu” is an outgrowth of this extensive research on H]-, 286 [M - SO3 - GlcA - H]-, and 255 [M - 286 - H]-
chocolate. and UV absorbance maxima at 271 and 335 nm, which
The “cupuaçu” fruit is appreciated for its acidic and suggested that 1 is a monosulfated flavonoid glycoside.13,14
strongly aromatic pulp that surrounds the seed. Because Acid hydrolysis of 1 confirmed the presence of glucuronic
of its strong flavor, the fruit pulp is not typically consumed acid by comparison with a commercial sugar standard.15
alone, but rather is used as an ingredient in various food The sulfate ion was verified after acid hydrolysis by
precipitating the aqueous layer with BaCl2.13 The 1H and
13C NMR data (Table 1) of 1 were similar to those of
* To whom correspondence should be addressed. Tel: (718) 960-1105.
Fax: (718) 960-8236. E-mail: [email protected]. isoscutellarein 8-O-β-D-glucuronopyranoside,16 except for
†
Lehman College and The Graduate Center, The City University of New the downfield chemical shifts observed for H-3′′ (+1.00
York.
‡
Columbia University College of Physicians and Surgeons. ppm) and C-3′′ (+8.3 ppm), showing that the sulfate group
§
The New York Botanical Garden. is linked to the C-3′′ hydroxyl of the glucuronic acid
10.1021/np034002j CCC: $25.00 © 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/23/2003
 1502 Journal of Natural Products, 2003, Vol. 66, No. 11 Notes

Table 1. NMR Data of Theograndins I (1) and II (2)

1 2
δH (int., mult., 1H-1H COSY HMBC δH (int., mult., 1H-1HCOSY HMBC
C/H δC J in Hz) (1H No.) (13C No.) δC J in Hz) (1H No.) (13C No.)
2 166.8 s 167.2 s
3 105.2 d 6.62 (1H, s) 1′, 10 103.8 d 6.62 (1H, s) 1′, 10
4 183.9 s 183.7 s
5 159.3 s 158.9 s
6 100.5 d 6.26 (1H, s) 8, 10 100.7 d 6.30 (1H, s) 8, 10
7 159.1 s 158.9 s
8 126.9 s 127.3 s
9 151.2 s 151.2 s
10 103.2 s 104.8 s
1′ 122.8 s 123.5 s
2′ 130.3 d 8.10 (1H, d, 8.8) 3′ 2, 4′, 6′ 114.5 d 7.71 (1H, d, 2.3) 2, 4′, 6′
3′ 117.2 d 6.96 (1H, d, 8.8) 2′ 1′, 5′ 146.9 s
4′ 162.9 s 150.7 s
5′ 117.2 d 6.96 (1H, d, 8.8) 6′ 1′, 3′ 116.8 d 6.92 (1H, d, 8.0) 6′ 1′, 3′
6′ 130.3 d 8.10 (1H, d, 8.8) 5′ 2, 2′, 4′ 120.9 d 7.43 (1H, dd, 8.0, 2.3) 5′ 2, 2′, 4′
1′′ 107.8 d 4.86 (1H, d, 8.0) 2′′ 3′′, 5′′, 8 108.3 d 4.80 (1H, d, 7.6) 2′′ 3′′, 5′′, 8
2′′ 74.1 d 3.89 (1H, m) 1′′, 3′′ 4′′ 74.1 d 3.90 (1H, m) 1′′, 3′′ 4′′
3′′ 84.4 d 4.38 (1H, t, 9.2) 2′′, 4′′ 1′′, 5′′ 84.2 d 4.39 (1H, t, 9.0) 2′′, 4′′ 1′′, 5′′
4′′ 72.1 d 3.89 (1H, m) 3′′, 5′′ 2′′, 6′′ 71.8 d 3.90 (1H, m) 3′′, 5′′ 2′′, 6′′
5′′ 78.9 d 3.80 (1H, d, 9.6) 4′′ 1′′, 3′′ 79.4 d 3.82 (1H, d, 9.6) 4′′ 1′′, 3′′
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6′′ 174.8 s 176.0 s

Publication Date (Web): October 23, 2003 | doi: 10.1021/np034002j

moiety.16,17 Furthermore, negative ESIMS displayed a

fragment at m/z 255 [M - 286 - H]-, indicative of the loss
of the isoscutellarein aglycon. The location of the glycosyl
linkage was determined to be at C-8 since the anomeric
proton signal at δ 4.86 showed a HMBC correlation with
an oxygenated quaternary carbon at δ 126.9, assignable
to C-8 on the basis of the correlation with H-6 (δ 6.26). In
the 1H NMR spectrum, the coupling constant of H-1′′ (J )
8.0 Hz) confirmed a β-glycosyl linkage.16 The “D” configu-
ration of glucuronic acid was determined after acid hy-
drolysis of 1 by isolating the sugar using preparative HPLC
and measuring the purified sugar’s optical rotation. From
these results, theograndin I (1) was determined to be
isoscutellarein 8-O-β-D-glucuronopyranoside 3′′-O-sulfate.
Compound 2 was obtained as a yellow powder, and
HRESIMS of 2 in the negative mode gave m/z 557.0231
[M - H]-, indicating a molecular formula of C21H18O16S.
The molecular formula of 2 differs from 1 by one additional
oxygen atom. The 1H NMR spectrum of 2 was similar to
that of 1, except that the spectrum of 2 displayed an AMX
spin system comprised of signals at δ 7.71 (1H, d, J ) 2.3
Hz), 7.43 (1H, dd, J ) 8.0, 2.3 Hz), and 6.92 (1H, d, J )
8.0 Hz). The 1H and 13C NMR data (Table 1) indicated that
the flavonoid skeleton for 2 is hypolaetin.16 In a manner
similar to 1, acid hydrolysis of 2 confirmed the presence of
a sulfate group and D-glucuronic acid. Comparison of the
1H and 13C data of the glucuronic acid moiety of 2 with

those of 1 and hypolaetin 8-O-β-D-glucuronopyranoside16

demonstrated that the sulfate group of 2 is connected to
C-3′′, and the HMBC experiment confirmed the D-glucu-
ronic acid to be at the C-8 position. Therefore, theograndin
II (2) was elucidated as hypolaetin 8-O-β-D-glucuronopy-
ranoside 3′′-O-sulfate.
In addition to the two new sulfated flavonoid glycosides,
four known flavonoids, (+)-catechin (3), (-)-epicatechin (4),
quercetin (9), and kaempferol (10), were identified from T.
grandiflorum seeds by comparing them with authentic
compounds by RP-18 TLC and ESIMS. Using MS and NMR
data from the literature, five known flavonoids, isoscutel-
larein 8-O-β-D-glucuronide (5),16 hypolaetin 8-O-β-D-glucu-
ronide (6),16 quercetin 3-O-β-D-glucuronide (7),18 quercetin
3-O-β-D-glucuronide 6′′-methyl ester (8),18 and isoscutella-
rein 8-O-β-D-glucuronide 6′′-methyl ester (11),19 were iden-
tified from T. grandiflorum seeds.
 Notes Journal of Natural Products, 2003, Vol. 66, No. 11 1503

Theograndin I (1) showed low antioxidant activity (IC50 eter (Perkin-Elmer, Boston, MA). 1H NMR and 13C NMR
) 341.1 µM) in the DPPH free-radical assay and displayed spectra were recorded using a JEOL GX-400 MHz, operating
weak cytotoxicity in the HCT-116 and SW-480 human colon at 400 and 100 MHz, respectively. 2D NMR experiments were
cancer cell lines with IC50 values of 205 and 164 µM, run on a Varian Inova 400 MHz. All compounds were mea-
respectively. Theograndin II (2) displayed higher antioxi- sured in CD3OD. ESIMS was performed with a ThermoFinni-
dant activity (IC50 ) 120.2 µM) in the DPPH free-radical gan LCQ instrument (San Jose, CA) equipped with Xcalibur
assay and cytotoxicity in the HCT-116 and SW-480 human software. Samples were dissolved in MeOH and introduced by
colon cancer cell lines with IC50 values of 143 and 125 µM, direct injection. The capillary voltage was 10 V, the spray
voltage was 4.5 kV, and the tube lens offset was 0 V. The
respectively. The nine known compounds, 3-11, were also
capillary temperature was 230 °C. HRESIMS was performed
screened for their antioxidant capacities in the DPPH
on a 70-SE-4F mass spectrometer (Micromass). Samples were
assay, and all displayed high antioxidant activity, with IC50
dissolved in MeOH. HPLC analyses were carried out on a
values of 49.0, 49.0, 68.1, 58.2, 44.4, 41.6, 39.7, 89.7, and Waters 2690 separations module equipped with a Waters 996
69.2 µM, respectively. Therefore, the order of antioxidant photodiode array detector and Waters Millenium32 software
potency, as defined by IC50 values in the DPPH assay, was using a Phenomenex Aqua C18 column (4.6 × 250 mm, 5 µm)
quercetin (9) > quercetin 3-O-β-D-glucuronide (7) ≈ quer- and a solvent system of 5:95 to 50:50 MeCN/H2O linear
cetin 3-O-β-D-glucuronide 6′′-methyl ester (8) > (+)-cat- gradient, a flow rate of 1 mL/min, column at room tempera-
echin (3) ) (-)-epicatechin (4) > hypolaetin 8-O-β-D- ture, 20 min run time for analysis of subfractions, and an
glucuronide (6) > isoscutellarein 8-O-β-D-glucuronide (5) isocratic solvent system of MeCN/H2O (70:30), flow rate of 1
≈ isoscutellarein 8-O-β-D-glucuronide 6′′-methyl ester (11) mL/min, column at room temperature, 10 min run time for
> kaempferol (10) > theograndin II (2) > theograndin I sugar identification. Preparative HPLC was carried out using
(1). These results are in agreement with previous reports a Waters 600 controller with a Waters 486 tunable absorbance
of structure-activity relationship of antioxidant polyphe- detector and Waters Empower software with a Phenomenex
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nols. For example, increasing the number of hydroxyl Nucleosil 10 C18 column (21.1 × 250 mm, 10 µm) and an
Publication Date (Web): October 23, 2003 | doi: 10.1021/np034002j

groups in a flavonoid often increases antioxidant activity.20 isocratic solvent system of MeCN/H2O (70:30), a flow rate of 5
The O-dihydroxy structure in the B ring of a flavonoid mL/min, column at room temperature, and 30 min run time.
confers higher stability to the radical form and participates TLC analyses were performed on RP-18 F254 plates (Merck,
in electron delocalization,21 and thus the dihydroxylation Darmstadt, Germany), with compounds visualized by spraying
in the 3′, 4′ positions of the B ring plays an important role with 1.0 g of vanillin in 10 mL of H2SO4 (concentrated) and
90 mL of EtOH. Sephadex LH-20 (25-100 µm; Pharmacia Fine
in antioxidant activity, as in both (+)-catechin (3) and (-)-
Chemicals, Piscataway, NJ) and C18 reversed-phase silica gel
epicatechin (4). The glycosylation of flavonoids may reduce
(40 µm; J. T. Baker, Phillipsburg, NJ) were used for column
their antioxidant activity when compared to that of the
chromatography.
corresponding aglycons, such as quercetin (9) and its
Plant Material. Theobroma grandiflorum fruits were col-
glycoside quercetin 3-O-β-D-glucuronide (7).22 The unsat- lected in Cayenne, French Guiana (May 1999), and identified
uration in the C ring as in quercetin (9) and kaempferol by Dr. Scott Mori, Nathaniel Lord Britton Curator of Botany
(10) allows electron delocalization across the molecule for at The New York Botanical Garden.
stabilization of aryloxy radicals due to existing conjuga- Extraction and Isolation. The dried-powder seeds (962.3
tion.21 The C-3 and C-5 hydroxyl groups with a 4-oxo g) of T. grandiflorum were extracted with MeOH three times
function in the A and C rings allow for maximum radical- at room temperature. The MeOH was removed in vacuo, and
scavenging potential, such as in quercetin (9).21 the resulting brown extract (60.8 g) was suspended in H2O
The antioxidant activities of sulfate flavonoid glycosides and partitioned sequentially with hexane, EtOAc, and BuOH.
The hexane (FrH, 12.8 g), EtOAc (FrEA, 5.5 g), and BuOH (FrB,
1 and 2 are significantly less than those of their corre-
9.8 g) extracts were tested in the DPPH assay, and their IC50
sponding flavonoid glycosides 5 and 6. The decreased values were calculated as 1204.8, 40.9, and 137.0 µg/mL,
antioxidant activity may be due to the inter- and intramo- respectively.
lecular hydrogen bond formed between the oxygen of the Fraction FrEA was subjected to column chromatography over
sulfated group and the hydrogen of the hydroxyl group Sephadex LH-20 (100.0 g) and eluted with an isocratic system
connected to a flavonoid skeleton. This hydrogen bond may using MeOH, and 25 fractions (50 mL) were collected. Frac-
prevent the ionization of hydrogen from the hydroxyl group tions were combined based upon HPLC analysis to 11 sub-
and reduce the available hydrogen donors and therefore fractions (FrEA1-11). Fraction FrB was separated and combined
decrease the antioxidant activity of sulfated flavonoid in a similar fashion, and nine subfractions (FrB1-9) were
glycosides. The low water solubility of many flavonoids obtained.
limits their therapeutic application.15 Sulfated flavonoid Fraction FrB7 (1.2 g) was separated by RP-18 (50.0 g) CC
eluting with a gradient system of 1:9 to 1:0 MeOH/H2O to yield
glycosides are highly soluble in water, although the anti-
six subfractions (FrB7a-f), and two of these subfractions, FrB7b
oxidant activities of sulfated flavonoid glycosides are less (100.2 mg) and FrB7e (163.5 mg), were purified by Sephadex
than their corresponding flavonoids and flavonoid glyco- LH-20 (10.0 g) CC eluting with an isocratic system of MeOH
sides. The sulfated flavonoid glycosides might improve to obtain theograndin I (1) (5.0 mg) and isoscutellarein 8-O-
bioavailability because they are both easily dissolved in β-D-glucuronide 6′′-methyl ester (11) (7.0 mg), respectively.
water and hydrolyzed to their corresponding flavonoid Fraction FrB9 (1.6 g) was separated by RP-18 (50.0 g) CC
glycosides and flavonoids; however, in vivo and clinical eluting with a gradient system of 1:9 to 1:0 MeOH/H2O to yield
studies are needed to understand the significance of the seven subfractions (FrB9a-h), and subfraction FrB9d (230.0 mg)
sulfated flavonoid glycosides to human health. was purified by Sephadex LH-20 (10.0 g) CC eluting with an
isocratic system of MeOH to obtain theograndin II (2) (7.2 mg).
Experimental Section The fractions FrEA3 (0.9 g), FrEA4 (0.6 g), FrEA8 (2.1 g), and
FrB1 (0.5 g) were separated by RP-18 (50.0 g) CC and Sephadex
General Experimental Procedures. Melting points were LH-20 (10.0 g) CC, using a gradient system of 1:9 to 1:0 MeOH/
determined on a Mel-Temp II melting point apparatus (Labo- H2O for RP-18 CC and an isocratic system of MeOH for
ratory Devices Inc., Holliston, MA) and are uncorrected. Sephadex LH-20 CC to yield (+)-catechin (3) (8.6 mg) and (-)-
Optical rotations were measured on an Autopol III automatic epicatechin (4) (6.2 mg) from FrEA3, to afford isoscutellarein
polarimeter (Rudolph Research Analytical, Flanders, NJ). UV 8-O-β-D-glucuronide (5) (3.4 mg) from FrEA4, to obtain hypo-
spectra were measured on a Lambda 2 UV/vis spectrophotom- laetin 8-O-β-D-glucuronide (6) (277.6 mg), quercetin 3-O-β-D-
 1504 Journal of Natural Products, 2003, Vol. 66, No. 11 Notes

glucuronide (7) (5.8 mg), quercetin 3-O-β-D-glucuronide 6′′- plicate wells were assayed for each condition, and mean as
methyl ester (8) (3.6 mg), and quercetin (9) (12.2 mg) from well as standard deviations were determined. The IC50 values
FrEA8, and to give kaempferol (10) (4.2 mg) from FrB1. were determined by linear regression analysis.
Theograndin I (Isoscutellarein 8-O-β-D-glucuronopy-
ranoside 3′′-O-sulfate) (1): yellow powder, mp 164.2-165.8 Acknowledgment. The authors wish to acknowledge Dr.
°C; [R]D20 +62.35° (c 0.00085, MeOH); UV (MeOH) λmax (log ) Michael Blumenstein (Hunter College, City University of New
271 (4.1), 335 (4.2) nm; 1H and 13C NMR, see Table 1; negative York) for his considerable assistance with NMR measure-
ESIMS m/z 541 [M - H]-, 461 [M - SO3 - H]-, 286 [M - SO3 ments. Dr. Richard Franck (Hunter College, City University
- GlcA - H]-, 255 [M - 286 - H]-; negative HRESIMS m/z of New York) is thanked for use of equipment employed in this
541.0297 [M - H]- (calcd for C21H17O15S, 541.0288). study. HRESIMS were obtained in the Mass Spectrometry
Theograndin II (Hypolaetin 8-O-β-D-glucuronopyra- Laboratory, School of Chemical Sciences, University of Illinois
noside 3′′-O-sulfate) (2): yellow powder, mp 157.0-158.6 °C; at Urbana-Champaign, and their 70-SE-4F spectrometer was
[R]D20 +110° (c 0.0008, MeOH); UV (MeOH) λmax (log ) 270
purchased in part with funds from the National Institute of
(4.0), 355 (3.9) nm; 1H and 13C NMR, see Table 1; negative
General Medical Sciences, NIH (GM 27029). Dr. Bei Jiang
ESIMS m/z 557 [M - H]-, 477 [M - SO3 - H]-, 301 [M - SO3
- GlcA - H]-, 255 [M - 302 - H]-; negative HRESIMS m/z (Columbia University) and Mr. Kurt Reynertson (Lehman
557.0231 [M - H]- (calcd for C21H17O16S, 557.0237). College, City University of New York) are thanked for their
Acid Hydrolysis. Compounds 1 (0.5 mg) and 2 (0.3 mg) critical reading of the manuscript. This research was supported
were each refluxed separately in a mixture of MeOH (1 mL) by the funds from the NIH-National Institute of General
and 2% HCl (1 mL) for 2 h.15 After evaporation of MeOH, the Medical Sciences SCORE award S06GM08225 and the Profes-
aglycons were extracted with EtOAc; the water layer was sional Staff Congress of The City University of New York (PSC-
neutralized with a saturated solution of NaHCO3. The water CUNY) award 669662.
layers of 1 and 2 were purified by preparative HPLC to yield
0.12 and 0.09 mg of sugar, respectively. The sugar constituent References and Notes
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plate reader (Bio-Tek Instruments Inc., Winooski, VT). Octu- NP034002J