Acylphloroglucinol and xanthones from Hypericum ellipticum

Article abstract of DOI:10.1016/j.phytochem.2011.01.032

An acylphloroglucinol, elliptophenone A, and two xanthones, elliptoxanthone A and elliptoxanthone B, were isolated from the aerial portions of Hypericum ellipticum together with three known xanthones, 1,3,7-trihydroxy-8-(3-methyl-2- butenyl)-9H-xanthen-9-

Full text of DOI:10.1016/j.phytochem.2011.01.032

Phytochemistry 72 (2011) 662–667
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Acylphloroglucinol and xanthones from Hypericum ellipticum
Kylie Manning a, Elyse Petrunak a, Michelle Lebo a, Antonio González-Sarrías b, Navindra P. Seeram b,
Geneive E. Henry a,
⇑
a Department of Chemistry, Susquehanna University, 514 University Avenue, Selinsgrove, PA 17870, USA
b Bioactive Botanical Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, 41 Lower College Road, University of Rhode Island,
Kingston, RI 02881, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An acylphloroglucinol, elliptophenone A, and two xanthones, elliptoxanthone A and elliptoxanthone B,
were isolated from the aerial portions of Hypericum ellipticum together with three known xanthones,
1,3,7-trihydroxy-8-(3-methyl-2-butenyl)-9H-xanthen-9-one, 1,6-dihydroxy-4-methoxy-9H-xanthen-9-
one, and 1,4,5-trihydroxy-9H-xanthen-9-one. Their structures were determined by spectroscopic analy-
ses. The acylphloroglucinol and xanthones were evaluated for cytotoxicity using three human colon can-
cer cell lines cell lines (HT-29, HCT-116 and Caco-2) and a normal human colon cell line (CCD-18Co).
Ó 2011 Elsevier Ltd. All rights reserved.
Received 29 September 2010
Received in revised form 8 January 2011
Available online 19 February 2011
Keywords:
Hypericum ellipticum
Clusiaceae
Acylphloroglucinol
Xanthone
Cytotoxicity
1. Introduction
and three known xanthones, 1,3,7-trihydroxy-8-(3-methyl-2-bute-
nyl)-9H-xanthen-9-one (4), 1,6-dihydroxy-4-methoxy-9H-xan-
The Hypericum genus (family Clusiaceae) contains over 400 spe-
cies distributed worldwide. Phytochemical studies of several mem-
bers of the Hypericum genus, including H. perforatum (St. John’s
wort), have yielded a number of structurally diverse secondary
metabolites from the naphthodianthrone, acylphloroglucinol,
flavonoid, and xanthone classes (Avato, 2005). These compounds
exhibit a wide range of biological properties including anticancer,
antimicrobial, anti-inflammatory, anti-HIV and antioxidant activi-
ties (Avato, 2005; Ciochina et al., 2006; El-Seedi et al., 2010).
As part of a study to characterize biologically active secondary
metabolites from Hypericum species growing in the state of
then-9-one (5) and 1,4,5-trihydroxy-9H-xanthen-9-one (6) from
an acetone extract of H. ellipticum. Herein, we describe the struc-
ture elucidation of compounds 1–3 and cytotoxicity evaluation of
compounds 1–2 and 4–6 against three human colon cancer cell
lines and one normal colon cell line.
2. Results and discussion
The acetone extract of the aerial portions of H. ellipticum was
subjected to column chromatography with silica gel followed by
reversed-phase HPLC purification to afford one previously unchar-
acterized acylphloroglucinol (1) and two xanthone derivatives (2
and 3) (Fig. 1). In addition, three known xanthones, 1,3,7-trihy-
Pennsylvania, USA, we recently reported the isolation of
a
benzophenone glucoside, 30-O-b- -300,400,600-triacetyl-glucopyrano-
D
syl-2,4,50,6-tetrahydroxybenzophenone from an acetone extract
of H. ellipticum, pale St. John’s wort (Petrunak et al., 2009). During
the isolation of the benzophenone glucoside, minor components
belonging to the xanthone and acylphloroglucinol classes were
detected by 1H NMR spectroscopy. However, owing to the paucity
of plant extracts, these compounds could not be isolated in suffi-
cient quantities for characterization. We now report the isolation
of a new acylphloroglucinol derivative, elliptophenone A (1), two
new xanthones, elliptoxanthone A (2) and elliptoxanthone B (3),
droxy-8-(3-methyl-2-butenyl)-9H-xanthen-9-one
(4)
(Morel
et al., 2002), 1,6-dihydroxy-4-methoxy-9H-xanthen-9-one (5)
(Seo et al., 2002) and 1,4,5-trihydroxy-9H-xanthen-9-one (6)
(Minami et al., 1995) were isolated and identified by NMR spectro-
scopic analyses. The structures of compounds 4–6 were confirmed
by comparison of the spectroscopic data with literature values.
The molecular formula of compound 1 was deduced to be
C
24H28O4 based on HRESIMS data. The IR spectrum displayed
absorption bands at 3424 and 1653 cmꢀ1 indicating the presence
of hydroxyl groups and conjugated carbonyl carbons. The 1H NMR
spectrum (Table 1) of compound 1 displayed three aromatic sig-
nals at 7.45, 7.40 and 7.35 ppm, integrating for five protons.
HMBC correlations (Table 1) indicated that these protons
⇑
Corresponding author. Tel.: +1 570 372 4222; fax: +1 570 372 2752.
E-mail address: henry@susqu.edu (G.E. Henry).
0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.01.032

K. Manning et al. / Phytochemistry 72 (2011) 662–667
663
Fig. 1. Structures and important HMBC correlations for compounds 1, 2 and 3.
Table 1
NMR spectroscopic data of elliptophenone A (1) and dimethyl elliptophenone A (1a).
Position
1 [(CD3)2CO]
1a [CDCl3]
a
a
dC
dH
HMBCb
24
dC
dH
1
2
3
4
5
6
7
8
189.5
106.5
193.7
57.4
188.6
118.3
169.9
53.8
14,19
14, 15, 19, 20
14, 19, 24
24
9, 13
10,12
11
12
9,13
10
11
173.2
107.6
195.7
139.5
128.0
127.4
130.5
127.4
128.0
37.3
118.7
134.2
25.2
17.3
37.3
170.2
119.5
196.6
138.6
129.3
128.7
133.1
128.7
129.3
35.8
118.7
134.7
26.0
18.0
35.8
9
7.40 (1H, d, 7.3)
7.88 (1H, d, 7.0)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
3-OH
5-OH
3-OCH3
5-OCH3
7.35 (1H, dd, 7.3, 7.3)
7.45 (1H, dd, 7.3, 7.3)
7.35 (1H, dd, 7.3, 7.3)
7.40 (1H, d, 7.3)
2.65 (2H, br d, 7.4)
4.93 (1H, t, 7.4)
7.41 (1H, dd, 7.0, 7.0)
7.50 (1H, dd, 7.0, 7.0)
7.41 (1H, dd, 7.0, 7.0)
7.88 (1H, d, 7.0)
2.61 (2H, m)
4.93 (1H, t, 6.4)
15
14, 17, 18
14, 17, 18
15, 18
15, 17
20
19, 22, 23
19, 22, 23
20, 23
20, 22
1.62 (3H, s)
1.59 (3H, s)
2.65 (2H, br d, 7.4)
4.93 (1H, t, 7.4)
1.71 (3H, s)
1.60 (3H, s)
2.61 (2H, m)
4.93 (1H, t, 6.4)
118.7
134.2
25.2
118.7
134.7
26.0
1.62 (3H, s)
1.59 (3H, s)
1.90 (3H, s)
1.71 (3H, s)
1.60 (3H, s)
1.89 (3H, s)
17.3
18.0
6.6
9.8
18.49 (1H, br s)
9.87 (1H, br s)
59.6
61.5
3.56 (3H, s)
3.90 (3H, s)
a
Coupling contants (J, Hz) given in parentheses.
Data for compound 1. Proton correlating to carbon shift.
b
comprised a monosubstituted aromatic ring, A. The protons at
7.40 ppm showed HMBC correlations to the carbonyl carbon at
195.7 ppm in the 13C NMR spectrum (Table 1), confirming that
the carbonyl group was directly attached to ring A. The presence
of the modified phloroglucinol ring B was suggested by the 13C
NMR spectroscopic data (193.7, 189.5, 173.2, 107.6, 106.5 and
57.4 ppm) (Zhao et al., 2005). The following 1H NMR signals sug-
gested the presence of two 3-methyl-2-butenyl groups: 4.93 ppm
(2H, t), 2.65 ppm (4H, br d), 1.62 ppm (6H, s) and 1.59 ppm (6H,
s). These data were corroborated by characteristic 13C NMR reso-
nances at 134.2, 118.7, 37.3, 25.2 and 17.3 ppm, coupled with
HMBC correlations (Fig. 1). A singlet at 1.90 ppm in the 1H NMR
spectrum, integrating for three protons, confirmed the presence
of an additional methyl group. The points of attachment of the
methyl group and the two 3-methyl-2-butenyl groups to ring B
were determined based on HMBC correlations. The protons of
the prenyl moieties, H2-14/H2-19 (2.65 ppm) and H-15/H-20
(4.93 ppm), showed long-range correlations with the aliphatic
quaternary carbon at 57.4 ppm. Additionally, H2-14/H2-19
showed HMBC correlations to C-3 (193.7 ppm) and C-5
(173.2 ppm), establishing the placement of the two 3-methyl-2-
butenyl groups at position 4. The methyl protons at 1.90 ppm
showed long-range correlations to the quaternary carbons at C-
1 (189.5 ppm), C-5 (173.2 ppm), and C-6 (107.6 ppm), allowing
the placement of the methyl group at C-6. A proton singlet at
18.49 ppm, was attributed to the hydroxyl group at position 3,
with the proton in a hydrogen bonding relationship to the C-7
carbonyl carbon. Based on the foregoing data, compound 1 was
determined to be 2-benzoyl-3,5-dihydroxy-6-methyl-4,4-bis-(3-
methyl-2-butenyl)cyclohexa-2,5-dienone, named elliptophenone
A (Fig. 1). Although elliptophenone A can exist in more than
one tautomeric form, only a single tautomer was observed in ace-
tone and DMSO. The compound is prone to oxidative decomposi-
tion, so the hydroxyl groups were methylated in order to obtain
the compound in a more stable form. Methylation of compound
1 using diazomethane in ether afforded three di-O-methylated
derivatives, 1a, 1b and 1c (Fig. 2), with 1a being the major
form.

664
K. Manning et al. / Phytochemistry 72 (2011) 662–667
Fig. 2. Structures and important HMBC correlations for compounds 1a, 1b and 1c.
The structures of 1a–1c were confirmed by extensive NMR
Table 2
spectroscopic analyses. The 1H and 13C NMR data of 1a (Table 1)
are similar to those of compound 1, except that the hydroxyl pro-
ton signals in compound 1 were replaced by methoxyl proton sig-
nals at 3.90 and 3.56 ppm. The placement of these protons at C-5
and C-3, respectively, was based on HMBC correlations between
the protons at 3.90 ppm and C-5 and between the protons at
3.56 ppm and C-3 (Fig. 2). The NMR data of compound 1a are con-
sistent with those for dimethyl grandone (de Oliveira et al., 1996),
which contains a 3-methyl-2-butenyl in place of the C-24 methyl
group. The structures of 1b and 1c were distinguished by examin-
ing the long-range correlations between the protons in the groups
attached directly to ring B and the carbon atoms in the ring (Fig. 2).
For compound 1b, the methylene protons at C-15/C-20 showed
correlations to the C-1 carbonyl carbon and C-5, whereas the
methyl protons at C-14 showed correlations to C-3 and C-5. For
compound 1c, the methyl protons at C-7 were correlated to both
the C-1 carbonyl carbon and C-3, whereas the methylene protons
C-15/C-20 were correlated to the C-1 carbonyl carbon and C-5.
The UV spectra of compounds 1b and 1c displayed absorption
bands at 341 and 345 nm, respectively. These absorptions bands
are indicative of an extended conjugated system containing a die-
none moiety (Porto et al., 2000), distinguishing 1b and 1c from 1a
which contains a cross-conjugated dienone moiety.
NMR spectroscopic data for elliptoxanthone
(CD3)2CO.
A (2) and elliptoxanthone B (3) in
2
3
Position
dC
dH (J in Hz)a
Position
dC
dH (J in Hz)a
1
2
3
4
4a
5
6
7
8
154.0
108.3
122.7
136.7
143.7
116.4
123.9
151.6
128.2
118.9
184.8
109.4
151.5
25.5
1 2 3
2
120.6
133.0
76.1
8.03 (1H, d, 10.2)
5.93 (1H, d, 10.2)
6.57 (1H, d, 8.8)
7.23 (1H, d, 8.8)
3
4a
5
6
6a
7a
8
9
138.3
153.5
102.7
153.5
143.5
136.7
122.3
108.7
153.9
109.2
183.5
107.9
120.1
26.3
7.35 (1H, d, 8.8)
7.43 (1H, d, 8.8)
6.87 (1H, s)
8a
9
7.20 (1H, d, 8.8)
6.55 (1H, d, 8.8)
10
11
9a
10a
11
12
13
14
15
1-OH
4-OH
6-OH
7-OH
11a
12
12a
12b
13
4.16 (2H, d, 7.0)
5.29 (1H, t, 7.0)
123.3
130.8
25.2
1.63 (3H, s)
1.82 (3H, s)
12.40 (1H, s)
9.50 (1H, s)b
1.46 (3H, s)
1.46 (3H, s)
9.50 (1H, s)
9.50 (1H, s)
12.38 (1H, s)
17.5
14
26.3
5-OH
8-OH
11-OH
9.80 (1H, s)b
a
Compound 1 is structurally related to the hops bitter acid, lupu-
lone, which like grandone contains a modified phloroglucinol ring
with three C-prenyl groups attached. Incubation studies have
established that lupulone is biosynthesized from an acylphloroglu-
cinol and three units of dimethylallyl pyrophosphate (DMAPP), cat-
alyzed by prenyltransferases (Zuurbier et al., 1995, 1998).
Compound 1 may be biosynthesized by a similar process with
the C-24 methyl group being introduced by S-adenosylmethionine
(SAM), catalyzed by a methyltransferase enzyme.
Compound 2 was assigned the molecular formula C18H16O5 on
the basis of HRESIMS data. The UV spectrum with absorption max-
ima at 409, 335, 269 and 239 nm indicated a xanthone skeleton
(Nguyen and Harrison, 1998). The IR spectrum exhibited absorp-
tion bands at 3447 and 1653 cmꢀ1 indicative of one or more hydro-
xyl groups and a conjugated carbonyl, respectively. The conjugated
carbonyl was confirmed by a 13C NMR signal at 184.8 ppm. A sharp
singlet at 12.40 ppm in the 1H NMR spectrum (Table 2) indicated
that the carbonyl group was hydrogen bonded to a phenolic proton
(OH-1). This relationship was confirmed by HMBC correlations be-
tween OH-1 and C-1, C-2 and C-9a (Fig. 1). The 1H NMR spectrum
also displayed signals corresponding to two additional phenolic
protons (9.80 and 9.50 ppm), two pairs of ortho coupled aromatic
protons (7.23/6.57 ppm and 7.43/7.35 ppm; J = 8.8 Hz), and a 3-
methyl-2-butenyl group (5.29 ppm, 1H, t; 4.16 ppm, 2H, d;
1.82 ppm, 3H, s; 1.63 ppm, 3H, s). Both the protons at 7.23 and
6.57 ppm showed HMBC correlations to C-1 and C-4, establishing
the placement of these two protons at C-2 and C-3, with a phenolic
group at position 4. The placement of the phenolic group at posi-
Coupling contants (J, Hz) given in parentheses.
Observed in DMSO-d6.
b
tion 4 was further supported by HMBC correlation between the
C-4 phenolic hydrogen and C-4a. The points of attachment of the
3-methyl-2-butenyl group and the remaining hydroxyl substituent
to ring B were established based on HMBC correlations. The place-
ment of the 3-methyl-2-butenyl group at C-8 was based on corre-
lations between the methylene protons (4.16 ppm) and C-8 and C-
8a. The methylene protons appear downfield of typical values ow-
ing to the deshielding effect of the carbonyl group (Duan et al.,
2010). The location of the hydroxyl group at position 7 was deter-
mined by correlations between the hydroxyl proton and C-8. The
foregoing data required that the remaining set of ortho coupled
protons (7.43 and 7.35 ppm) be placed at positions 5 and 6 on ring
B, confirmed by HMBC correlations between both protons and C-
10a and between H-5 and C-8a. Based on the spectroscopic data,
compound 2 was identified as 1,4,7-trihydroxy-8-(3-methyl-2-
butenyl)-9H-xanthen-9-one, named elliptoxanthone A.
The molecular formula of compound 3 was determined to be
C18H14O6 on the basis of HRESIMS data. The UV spectrum with
absorption maxima at 410, 321 and 291 nm indicated a xanthone
nucleus (Nguyen and Harrison, 1998). The IR spectrum was similar
to that of compound 2 with absorption bands indicative of hydro-
xyl (3406 cmꢀ1) and conjugated carbonyl (1653 cmꢀ1) groups. The
1H NMR spectroscopic data (Table 2) showed signals at 12.38 ppm
(s), 9.50 (br, s), 7.20 ppm (d) and 6.55 ppm (d), indicating a

K. Manning et al. / Phytochemistry 72 (2011) 662–667
665
dihydroxylated pattern in ring A, identical to that of compound 2.
Two proton doublets (J = 10.2 Hz) at 8.03 ppm (H-1) and 5.93 ppm
(H-2) coupled with a singlet at 1.46 ppm (H3-13, H3-14), integrat-
ing for six protons, indicated the presence of a gem-dimethylpyran
ring. The downfield position of H-1 indicated that it was located
near to the carbonyl carbon (Ishiguro et al., 1993). A third phenolic
group (9.50 ppm) was positioned at C-5. A sharp singlet at
6.87 ppm was attributed to a hydrogen at position 6. These place-
ments were based on HMBC correlations between the proton sig-
nal at 6.87 ppm and C-4a, C-5, C-6a and C-12a. The spectroscopic
data led to the identification of compound 3 as 5,8,11-trihy-
droxy-3,3-dimethyl-3H,12H-pyrano[3,2-a]xanthen-12-one, named
elliptoxanthone B. The 1H and 13C resonances for ring B and the
pyran ring were in close agreement with those reported for toxy-
loxanthone (Ishiguro et al., 1993). Toxyloxanthone is an isomer
of elliptoxanthone containing a hydroxyl group at position 9 in-
stead of position 8.
Compounds 1–2, 4–6, and the positive control, etoposide, were
examined for cytotoxicity against three human colon cancer cell
lines, namely, HT-29, HCT-116 and Caco-2 as well as a normal co-
lon cell line (CCD-18Co). Compound 3 was not tested owing to the
limited supply of the sample. All five compounds showed moder-
ate activity against the four cell lines. Overall, the acylphloroglu-
cinol, 1, and prenylxanthones, 2 and 4, showed weaker activity
ization on an Agilent G6520A Q-TOF high resolution mass spec-
trometer (NIH: 1S10RR022649-01 and CUNY Instrumentation
Fund).
Preparative HPLC was performed using a Waters Delta 600 sys-
tem equipped with a Waters 2487 dual wavelength absorbance
detector. Column chromatography (CC) was performed using Fish-
er Scientific silica gel (230–400 mesh), and analytical TLC was per-
formed using Sigma–Aldrich polyester backed plates precoated
with silica gel UV254. All solvents were HPLC grade and were ob-
tained from Fisher Scientific and Aldrich Chemical Co.
3.2. Plant material
The aerial parts of H. ellipticum were collected in Clearfield
County, Pennsylvania, USA, in August 2009. The plant material
was authenticated by Joseph and Bonnie Isaac and a voucher spec-
imen (20714) has been deposited at the Carnegie Museum Herbar-
ium in Pittsburgh, Pennsylvania, USA.
3.3. Extraction and isolation
Aerial parts of H. ellipticum were oven dried at 38 °C and ground
to a fine powder using a coffee grinder. The plant material (459 g)
was extracted sequentially at room temperature using hexanes
(3 ꢁ 1 L), acetone (3 ꢁ 1 L) and MeOH (3 ꢁ 1 L). Each 1 L aliquot
of solvent added was allowed to percolate for 24 h. The combined
extracts for each solvent (e.g. hexanes) were concentrated in vacuo.
The acetone extract (14.6 g) was fractionated using silica gel CC to
(IC50: 84–104
lg/mL) than the two non-prenylated xanthone
derivatives, 5 and 6, (IC50: 71–95
lg/mL). The HCT-116 and Caco-
2 tumor cells lines were more sensitive to all of the compounds
compared to CCD-18Co normal colon cells. However, there was
no significant difference between the IC50 values for the HT-29 co-
lon tumor cells and the CCD-18Co normal colon cells. Etoposide
afford fractions A1–A11, eluted with
a solvent gradient of
(CH3)2CO-hexanes (5:95 to 100:0, v/v). Fraction A3 (509 mg),
eluted with (CH3)2CO-hexanes (1:3, v/v) was further fractionated
with EtOAc-hexanes (1:4 to 1:3, v/v) to afford a brown solid
(145 mg) which was purified by HPLC (Atlantis dC18 column,
showed greater selectivity for the three tumor cells lines (IC50
9.5–16.2 g/mL) relative to the normal cells (IC50: 43.9 g/mL).
However, unlike compounds 1, 2, 4, 5 and 6, it showed greatest
activity with the HT-29 cells (IC50: 9.5 g/mL).
:
l
l
l
5
lm, 19 ꢁ 150 mm, MeOH–H2O (4:1, v/v) isocratic elution, flow
Several prenylated and non-prenylated xanthones have exhib-
ited moderate to strong activity against HT-29 and HCT-116 tumor
cell lines (El-Seedi et al., 2010), but anticancer studies of these
compounds against Caco-2 cells is limited. However, studies have
been conducted with Caco-2 cells and xanthones when the Caco-
2 monolayer is utilized as an epithelial cell culture model to assess
absorption (Bumrungpert et al., 2009).
rate 10 mL/min) to afford compound 1 (28.0 mg). Fraction A4
(1.05 g), eluted with (CH3)2CO-hexanes (25:75 to 40:60, v/v) was
further fractionated by silica gel CC using EtOAc-hexanes (30:70
to 35:65, v/v) to afford a yellow-orange solid (310 mg). The solid
was re-applied to a silica gel column using EtOAc-hexanes (1:4,
v/v) to afford a yellow solid (87.0 mg), containing predominantly
compound 1. The sample was treated with excess diazomethane
in ether, prepared from diazald according to standard protocol
(Chen, 1988), and purified by HPLC (Phenomenex Luna C18 col-
2.1. Concluding remarks
umn, 5
lm, 10 ꢁ 250 mm, MeOH–H2O (85:15, v/v), flow rate
2.0 mL/min) to afford compounds 1a (11.9 mg), 1b (5.9 mg) and
1c (6.4 mg). Subfractions B1–B7 resulted from further fractionation
of A6 (655 mg) using tert-butyl methyl ether-hexanes (40:60 to
65:35, v/v). Fractions B2 (34.7 mg), B3 (67.3 mg) and B5
(108.8 mg) were purified by HPLC (Atlantis dC18 column, flow rate
10 mL/min). Fraction B2 was eluted with MeOH–H2O (70:30, v/v)
to afford compound 5 (3.4 mg), fraction B3 was eluted with
MeOH–H2O (63:37 to 65:35, v/v) to give compound 4 (5.1 mg),
and fraction B5 was eluted with MeOH–H2O (60:40, v/v) to yield
compounds 2 (21.1 mg), 3 (2.4 mg) and 6 (4.2 mg).
An acylphloroglucinol and five xanthones were isolated from
the aerial portions of H. ellipticum. The acylphloroglucinol and four
of the xanthone derivatives were evaluated for cytotoxicity against
three human colon tumor cell lines (HCT-116, HT-29 and Caco-2)
and a normal colon cell line (CCD-18Co). All tested compounds
demonstrated selective but moderate cytotoxicity for the HCT-
116 and Caco-2 tumor cells when compared to normal colon cells
(CCD-18Co) indicating a possible cytotoxic selectivity towards co-
lon cancer cells.
3. Experimental
3.4. Cytotoxicity assays
3.1. General experimental procedures
Cell lines included three human colon cancer cells: HT-29 (hu-
man colon adenocarcinoma), HCT-116 (human colon carcinoma)
and Caco-2 (human epithelial colorectal adenocarcinoma). In addi-
tion, normal human colon cells were included: CCD-18Co (human
colon fibroblasts). All cell lines were obtained from the American
Type Culture Collection (ATCC, Rockville, MD, USA) and maintained
at the University of Rhode Island. Caco-2 and CCD-18Co cells were
grown in EMEM medium supplemented with 10% v/v fetal bovine
UV data were obtained using a Varian Cary 4000 UV–Vis spec-
trophotometer. FTIR data were measured using a Nicolet Magna
IR 560 spectrometer. NMR spectra were recorded on a JEOL ECP
400 MHz spectrometer (1H, 400 MHz; 13C, 100 MHz) using CDCl3,
(CD3)2CO and DMSO-d6 as solvents and TMS as internal standard.
Gradient HMQC and HMBC data were obtained using standard
pulse programs. HRMS data were acquired using electrospray ion-
serum, 1% v/v nonessential amino acids, 1% v/v L-glutamine and 1%

666
K. Manning et al. / Phytochemistry 72 (2011) 662–667
v/v antibiotic solution (Sigma). HT-29 and HCT-116 cells were
grown in McCoy’s 5a medium supplemented with 10% v/v fetal bo-
vine serum, 1% v/v nonessential amino acids, 2% v/v Hepes and 1%
v/v antibiotic solution. For each cell line, passage numbers between
22 and 35 were used. Cells were maintained at 37 °C in an incuba-
tor under a 5% CO2/95% air atmosphere at constant humidity and
maintained in the linear phase of growth.
Cytotoxicity studies were carried out using a MTS [3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfenyl)-
2H-tetrazolium, inner salt] assay as described previously (Cory
et al., 1991). Briefly, test samples and a positive control, etoposide
3.8. 4-Benzoyl-3,5-dimethoxy-2-methyl-6,6-bis(3-methyl-2-
butenyl)cyclohexa-2,4-dienone
(1c): pale yellow oil; UV (MeOH) kmax (log
e
;
) 249 (4.30), 345
1H NMR (CDCl3)
(3.59) nm; IR (NaCl) mmax 1653, 1559, 1457 cmꢀ1
d: 7.95 (2H, d, J = 7.0 Hz, H-10/14), 7.56 (1H, dd, J = 7.0, 7.0 Hz, H-
12), 7.48 (2H, t, J = 7.0, 7.0 Hz, H-11/13), 4.93 (2H, t, J = 7.0 Hz, H-
16/21), 3.55 (3H, s, 5-OCH3), 3.48 (3H, s, 3-OCH3), 2.61 (4H, m,
H2-15/20), 1.79 (3H, s, H3-7), 1.68 (3H, s, H3-18/23), 1.58 (3H, s,
H3-19/24); 13C NMR (CDCl3) d: 202.0 (C-1), 195.4 (C-8), 169.5 (C-
3), 165.7 (C-5), 138.8 (C-9), 134.5 (C-17, C-22), 133.5 (C-12),
129.2 (C-10, C-14), 128.9 (C-11, C-13), 118.8 (C-16, C-21), 115.6
(C-2), 112.2 (C-4), 61.1 (5-OCH3), 59.6 (3-OCH3), 58.8 (C-6), 39.2
(C-15, C-20), 26.0 (C-18, C-23), 17.9 (C-19, C-24), 8.7 (C-7); HRE-
SIMS: m/z 409.2358 [M+H]+ (calcd for C26H33O4: 409.2379).
4 lg/mL (Sigma), were solubilized in DMSO by sonication. All sam-
ples were diluted with media to the desired treatment concentra-
tion and the final DMSO concentration per well did not exceed
0.5%. Control wells were also included on all plates. Following a
72 h drug-incubation period at 37 °C with serially diluted test com-
pounds, MTS, in combination with the electron coupling agent,
phenazine methosulfate, was added to the wells and cells were
incubated at 37 °C in a humidified incubator for 3 h. Absorbance
at 490 nm (OD490) was monitored with a spectrophotometer
(SpectraMax M2, Molecular Devices Corp., operated by SoftmaxPro
v.4.6 software, Sunnyvale, CA, USA) to obtain the number of surviv-
ing cells relative to control populations. The results are expressed
as the median cytotoxic concentrations (IC50 values) and were cal-
culated from six-point dose response curves using 4-fold serial
dilutions. Each point on the curve was tested in. Data are expressed
as means SE for three replications on each cell line.
3.9. 1,4,7-Trihydroxy-8-(3-methyl-2-butenyl)-9H-xanthen-9-one
(2): yellow solid; UV (MeOH) kmax (log e) 239 (3.35), 269 (3.34),
335 (1.72), 409 (1.81) nm; IR (KBr) mmax 3447, 1653, 1613, 1593,
1494 cmꢀ1; For 1H NMR and 13C NMR spectroscopic data, see Table
2; HRESIMS m/z 313.1069 [M+H]+ (calcd for C18H17O5: 313.1076).
3.10. 5,8,11-Trihydroxy-3,3-dimethyl-3H,12H-pyrano[3,2-a]xanthen-
12-one
(3): yellow solid; UV (MeOH) kmax (log e) 291 (3.61), 321 (3.59),
410 (3.22) nm; IR (KBr) mmax 3406, 1653, 1647, 1619, 1576, 1506,
1472, 1456 cmꢀ1; For 1H NMR and 13C NMR spectroscopic data,
see Table 2; HRESIMS m/z 327.0867 [M+H]+ (calcd for C18H15O6:
327.0869).
3.5. 2-Benzoyl-3,5-dihydroxy-6-methyl-4,4-bis(3-methyl-2-butenyl)-
cyclohexa-2,5-dienone
(1): white amorphous solid; UV (MeOH) kmax (log e) 249
Acknowledgements
(3.89) nm; IR (KBr) mmax 3424, 1653, 1588, 1560, 1508, 1493,
1448 cmꢀ1; For 1H NMR, 13C NMR and HMBC spectroscopic data,
see Table 1; HRESIMS: m/z 379.1919, [MꢀH]ꢀ (calcd for
This work was supported by a grant from Research Corporation
for Science Advancement (award #7252). We are grateful to Joe
Isaac for the collection of plant material. We thank Dr. Cliff Jones
at the CUNY Mass Spectrometry Facility at Hunter College for the
HRMS analyses.
C
24H27O4: 379.1909).
3.6. 2-Benzoyl-3,5-dimethoxy-6-methyl-4,4-bis(3-methyl-2-
butenyl)cyclohexa-2,5-dienone
References
(1a): pale yellow oil; UV (MeOH) kmax (log e) 250 (4.38) nm; IR
Avato, P., 2005. A survey on the Hypericum genus: secondary metabolites and
bioactivity. Stud. Nat. Prod. Chem. 30, 603–634.
Bumrungpert, A., Kalpravidh, R.W., Suksamrarn, S., Chaivisuthangkura, A.,
(NaCl) mmax 1674, 1656, 1607, 1458 cmꢀ1; For 1H NMR and 13C
NMR spectroscopic data, see Table 1; HRESIMS: m/z 409.2375,
[M+H]+ (calcd for C26H33O4: 409.2379).
Chitchumroonchokchai,
C.,
Failla,
M.L.,
2009.
Bioaccessibility,
biotransformation, and transport of alpha-mangostin from Garcinia
mangostana (Mangosteen) using simulated digestion and Caco-2 human
intestinal cells. Mol. Nutr. Food Res. 53 (Suppl. 1), S54–S61.
Chen, C.L., 1988. Characterization of lignin by oxidative degradation: use of gas
chromatography–mass spectrometry technique. Methods Enzymol. 161, 110–
136.
Ciochina, R., Grossman, R.B., Demirkiran, O., 2006. Polycyclic polyprenylated
acylphloroglucinols. Chem. Rev. 106, 3963–3986.
3.7. 2-Benzoyl-3,5-dimethoxy-4-methyl-6,6-bis(3-methyl-2-
butenyl)cyclohexa-2,4-dienone
Cory, A.H., Owen, T.C., Barltrop, J.A., Cory, J.G., 1991. Use of an aqueous soluble
tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun.
3, 207–212.
DeOliveira, C.M.A., Porto, A.M., Bittrich, V., Vencato, I., Marsaioli, A.J., 1996. Floral
resins of Clusia spp.: chemical composition and biological function. Tetrahedron
Lett. 37, 6427–6430.
Duan, Y.-H., Dai, Y., Wang, G.-H., Zhang, X., Chen, H.-F., Chen, J.-B., Yao, X.-S., Zhang,
X.-K., 2010. Bioactive xanthones from the stems of Cratoxylum formosum ssp.
pruniflorum. J. Nat. Prod. 73, 1283–1287.
El-Seedi, H.R., El-Barbary, M.A., El-Ghorab, D.M.J., Bohlin, L., Borg-Karlson, A.-K.,
Göransson, U., Verpoorte, R., 2010. Recent insights into the biosynthesis and
biological activities of natural xanthones. Curr. Med. Chem. 17, 854–901.
Ishiguro, K., Fukumoto, H., Nakajima, M., Isoi, K., 1993. Xanthones in cell suspension
cultures of Hypericum paturum. Phytochemistry 33, 839–840.
Minami, H., Takahashi, E., Fukuyama, Y., Kodama, M., Yoshizawa, T., Nakagawa, K.,
1995. Novel xanthones with superoxide scavenging activity from Garcinia
subelliptica. Chem. Pharm. Bull. 43, 347–349.
(1b): pale yellow oil; UV (MeOH) kmax (log
e
;
) 247 (4.53), 341
(4.08) nm; IR (NaCl)
m
max 1653, 1558, 1457 cmꢀ1
1H NMR (CDCl3)
d: 7.85 (2H, d, J = 7.0 Hz, H-9/13), 7.50 (1H, dd, J = 7.0, 7.0 Hz, H-
11), 7.39 (2H, dd, J = 7.0, 7.0 Hz, H-10/12), 4.91 (2H, t, J = 7.2 Hz,
H-16/21), 3.84 (3H, s, 5-OCH3), 3.68 (3H, s, 3-OCH3), 2.59 (2H, dd,
J = 14.0, 7.2 Hz, H-15/20), 2.43 (2H, dd, J = 14.0, 7.2 Hz, H-15/20),
1.95 (3H, s, H3-14), 1.68 (3H, s, H3-18/23), 1.53 (3H, s, H3-19/24);
13C NMR (CDCl3) d: 199.9 (C-1), 196.7 (C-7), 171.8 (C-3), 167.9
(C-5), 138.8 (C-8), 134.5 (C-17, C-22), 133.0 (C-11), 129.4 (C-9, C-
13), 128.5 (C-10, C-12), 118.8 (C-16, C-21), 115.6 (C-4), 114.5 (C-
2), 61.9 (5-OCH3), 60.7 (3-OCH3), 58.7 (C-6), 37.7 (C-15, C-20),
26.0 (C-18, C-23), 18.2 (C-19, C-24), 10.7 (C-14); HRESIMS: m/z
409.2368 [M+H]+ (calcd for C26H33O4: 409.2379).

K. Manning et al. / Phytochemistry 72 (2011) 662–667
667
Morel, C., Hay, A.-E., Litaudon, M., Sévenet, T., Séraphin, D., Bruneton, J., Richomme,
P., 2002. Thirteen new xanthone derivatives from Calophyllum caledonicum.
Molecules 7, 38–50.
Kinghorn, 2002. Cytotoxic prenylated xanthones and the unusual compounds
anthraquinobenzophenones from Cratoxylum sumatranum. J. Nat. Prod. 65, 299–
305.
Nguyen, L.H.D., Harrison, L.J., 1998. Triterpenoid and xanthone constituents of
Cratoxylum cochinchinense. Phytochemistry 50, 471–476.
Petrunak, E., Kester, A.C., Liu, Y., Bowen-Forbes, C.S., Nair, M.G., Henry, G.E., 2009.
New benzophenone O-glucoside from Hypericum ellipticum. Nat. Prod.
Commun. 4, 507–510.
Porto, A.L.M., Machado, S.M.F., de Oliveira, C.M.A., Bittrich, V., Amaral, M.d.C.E.,
Marsaioli, A.J., 2000. Polyisoprenylated benzophenones from Clusia floral resins.
Phytochemistry 55, 755–768.
Seo, E.-K., Kim, N.-C., Wani, M.C., Wall, M.E., Navarro, H.A., Burgess, J.P., Kawanishi,
K., Kardono, L.B.S., Riswan, S., Rose, W.C., Fairchild, C.R., Farnsworth, N.R.,
Zhao, F., Watanabe, Y., Nozawa, H., Daikonnya, A., Kondo, K., Kitanaka, S., 2005.
Prenylflavonoids and phloroglucinol derivatives from hops (Humulus lupulus). J.
Nat. Prod. 68, 43–49.
Zuurbier, K.W.M., Fung, S.-Y., Scheffer, J.J.C., Verpoorte, R., 1995. Formation of
aromatic intermediates in the biosynthesis of bitter acids in Humulus lupulus.
Phytochemistry 35, 77–82.
Zuurbier, K.W.M., Fung, S.-Y., Scheffer, J.J.C., Verpoorte, R., 1998. In vitro prenylation
of aromatic intermediates in the biosynthesis of bitter acids in Humulus lupulus.
Phytochemistry 49, 2315–2322.