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A new xanthone from the bark of

Calophyllum thorelii
a b
Le-Thu Thi Nguyen , Duc Minh Nguyen & Lien-Hoa Dieu Nguyen
a

a
Natural Product and Medicinal Chemistry Lab, Faculty of
Chemistry, Ho Chi Minh City University of Science, 227 Nguyen Van
Cu, Ho Chi Minh City, Vietnam
b
Faculty of Pharmacy, University of Medicine and Pharmacy in Ho
Chi Minh City, 41 Dinh Tien Hoang, Ho Chi Minh City, Vietnam
Version of record first published: 01 May 2012.

To cite this article: Le-Thu Thi Nguyen , Duc Minh Nguyen & Lien-Hoa Dieu Nguyen (2013): A new
xanthone from the bark of Calophyllum thorelii , Natural Product Research: Formerly Natural
Product Letters, 27:6, 563-567

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 Natural Product Research, 2013
Vol. 27, No. 6, 563–567, http://dx.doi.org/10.1080/14786419.2012.682992

A new xanthone from the bark of Calophyllum thorelii

Le-Thu Thi Nguyena, Duc Minh Nguyenb and Lien-Hoa Dieu Nguyena*
a
Natural Product and Medicinal Chemistry Lab, Faculty of Chemistry, Ho Chi Minh City
University of Science, 227 Nguyen Van Cu, Ho Chi Minh City, Vietnam; bFaculty of Pharmacy,
University of Medicine and Pharmacy in Ho Chi Minh City, 41 Dinh Tien Hoang, Ho Chi Minh
City, Vietnam
(Received 31 March 2011; final version received 4 March 2012)
Downloaded by [McGill University Library] at 23:34 11 April 2013

A new xanthone, calothorexanthone, together with five known compounds,

garbogiol, 1,4,8-trihydroxyxanthone, �-tocotrienol, 1,7-dihydroxyxanthone and
globuxanthone, was isolated from a petroleum ether extract of the bark of
Calophyllum thorelii. Their structures were established on the basis of spectro-
scopic methods, mainly one- and two-dimensional NMR. Antioxidant activity
of the isolated compounds was tested using DPPH free radical scavenging assay
and some exhibited remarkable effects with IC50 of 13.63–17.46 mg mL�1.
Keywords: Calophyllum thorelii; Guttiferae; calothorexanthone; xanthones; anti-
oxidant activity

1. Introduction
The genus Calophyllum (Guttiferae) is well known as a rich source of phenolic compounds
such as xanthones, coumarins and chromanones (Su et al., 2008). Xanthones have shown
to possess various biological activities, from antimicrobial, antitubercular, antitumour,
antileukaemic to antidepressant, antihepatotoxic and anti-inflammatory (Peres & Nagem,
1997; Peres, Nagem, & de Oliveira, 2000) as well as antioxidant properties (Hay et al.,
2004; Hiroyuki, Kuwayama, Yoshizawa, & Yoshiyasu, 1996). In the previous paper
(T.L.T. Nguyen, M.D. Nguyen, & D.L.H. Nguyen, 2010b), we have reported the isolation
of a new xanthone, thorexanthone (1), together with trans-cinnamic acid and two
polyisoprenylated benzophenones, guttiferone F and 30-epi-cambogin, from a petroleum
ether extract of the fruit of Calophyllum thorelii collected in central Vietnam. In addition,
five compounds, osajaxanthone, 1,4,8-trihydroxyxanthone, 1,3,5-trihydroxy-4-(3-methyl-
but-2-enyl)xanthone, 1,2,5-trihydroxyxanthone, 1,3,5,6-tetrahydroxyxanthone and proto-
catechuic acid, were obtained from an ethyl acetate extract of the bark (T.L.T. Nguyen,
M.D. Nguyen, & D.L.H. Nguyen, 2010a). In this article, we describe the isolation and
structure elucidation of a new and five known compounds from a petroleum ether extract
of the same bark. Antioxidant activity of the isolated compounds using DPPH free radical
scavenging assay was also reported.

*Corresponding author. Email: lienhoa-nguyen@vnn.vn

© 2013 Taylor & Francis

 564   L.-T.T. Nguyen et al.

O OH OH O OH
OH O OH 13
14

HO 1

O
8a 9a
H
O 7 11
O
12
O
OH O 4a 3 O 15
(1) 5
10a
OH (3)
(2)

O OH
OH
HO

O
O
OH
(5)
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(4)

Figure 1. Structures of 1–5.

2. Results and discussion

Column chromatography (CC) of the petroleum ether extract of the bark of C. thorelii
over silica gel, Diol silica, RP18 and Sephadex LH-20 furnished a new xanthone,
calothorexanthone (2), along with five known compounds, garbogiol (3) (Iinuma et al.,
1998), 1,4,8-trihydroxyxanthone (Dos Santos, Nagem, da Silva, & e Silva, 2000),
�-tocotrienol (4) (Merza et al., 2004), 1,7-dihydroxyxanthone (Yang, Xu, & Yang, 2001)
and globuxanthone (5) (Iinuma, Tosa, Tanaka, Asai, & Shimano, 1995) (Figure 1).
Calothorexanthone (2) was isolated as yellow needles, m.p. 154–155� C, [�]25 D �37.8
(c 0.27, EtOH). A positive test with alcoholic FeCl3 indicated its phenolic nature.
HRESIMS revealed the molecular formula to be C18H16O6 (m/z 329.1023 [M þ H]þ),
i.e. the molecule had 11 units of unsaturation. The UV (�max 241, 267, 273, 324 and
394 nm) and IR spectra [�max 3413 (O–H), 1652 (chelated C ¼ O) and 1610 (aromatic
ring) cm�1] were typical of polyhydroxylated xanthones (Terreaux, Maillard, Gupta, &
Hostettmann, 1995).
The 1H and 13C-NMR spectra contained resonances for two chelated hydroxyl groups
[�H 12.20 and 11.80 (1H each, s, 1-OH and 8-OH)] and the corresponding chelated
carbonyl carbon (�C 186.0, C-9), a free hydroxyl [�H 8.02 (1H, s, 7-OH)], two ortho-coupled
protons [�H 7.32 and 6.89 (1H each, d, J ¼ 9.0 Hz, H-6 and H-5); �C 124.5 and 106.9, C-6
and C-5], an isolated aromatic proton [�H 6.35 (1H, s, H-4); �C 90.4, C-4], a 2,3,3-
trimethyl-2,3-dihydrofuran ring [�H 4.59 (1H, q, J ¼ 6.5 Hz, H-12), 1.50 (3H, s, H3-14),
1.40 (3H, d, J ¼ 6.5 Hz, H3-15), and 1.26 (3H, s, H3-13); �C 92.1 (C-12), 44.0 (C-11), 25.4
(C-14), 20.8 (C-13), and 14.5 (C-15)] and nine substituted aromatic carbons, six of which
were oxygenated.
In the HMBC plot, the less shielded chelated hydroxyl proton (�H 12.20, 1-OH) showed
correlations with one oxygenated and two substituted aromatic carbons (�C 158.7, 117.9
and 103.3; C-1, C-2 and C-9a). The two tertiary methyl protons (H3-13 and H3-14)
correlated to C-2 (�C 117.9), revealing that the 2,3,3-trimethyl-2,3-dihydrofuran ring was
condensed with the xanthone A ring at C-2 and C-3 with the latter being oxygenated.
Cross-peaks between the isolated aromatic proton (�H 6.35) with C-2, C-9a and two
oxygenated aromatic carbons (�C 168.1 and 159.4, C-3 and C-4a) showed that the proton
was located on C-4. The chemical shifts of C-3 and C-4a could not be differentiated using
the HMBC spectrum and this was established by comparison of their shifts with those
 Natural Product Research   565

H
OH O O 13

HO 8a 9a
1 14

7 11
15

H 10a O 4a 3 O
5

H H
(2)

Figure 2. HMBC correlations for 2.

O OH 13 14

1
9a
11 H
12
O 4a 3 O 15
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Figure 3. NOE interactions in the dihydrofuran ring of 2.

Table 1. DPPH free radical antioxidant activity.

Compound IC50 (mg mL�1)

Calothorexanthone (2) 17.46 � 0.58

Garboginol (3) 15.07 � 0.63
1,4,8-Trihydroxyxanthone 13.63 � 0.57
�-Tocotrienol (4) 23.83 � 0.31
Globuxanthone (5) 38.19 � 0.52
Ascorbic acid 10.68 � 1.04

Note: The data presented are means of three experiments � SD.

reported for mangoxanthone (Nilar, Nguyen, Venkatraman, Sim, & Harrison, 2005). The
xanthone B ring therefore carried the remaining two hydroxyl groups. The second chelated
hydroxyl proton (�H 11.80, 8-OH) correlated to one substituted aromatic carbon (�C 108.2)
and two oxygenated aromatic carbons (�C 147.9 and 141.4), revealing that C-7 and C-8
were hydroxylated. The two ortho-coupled protons were therefore attached to C-5 and
C-6. Other HMBC correlations (Figure 2) supported the structure of the subunit. The
relative configuration of 2 was determined using NOESY experiment. The NOE
correlation between the oxymethine H�-12 (�H 4.59) and the methyl H3(�)-14 (�H 1.50)
indicated that the proton and the methyl group were in the same side. C�-13 and C�-15
methyls (�H 1.26 and 1.40, respectively) therefore had the same orientation, and this
was confirmed by an NOE correlation between the corresponding protons (Figure 3).
The compound thus had structure 2, which is named calothorexanthone. The absolute
configuration of 2 remained undetermined.
Antioxidant capacity of the isolated compounds was tested using DPPH free radical
scavenging assay (Hatano, Kagawa, Yasuhara, & Okuda, 1988). The scavenging activities
of the isolated compounds and ascorbic acid as a positive control are shown in Table 1.
All test compounds showed remarkable antioxidant activities except for 1,7-dihydroxyx-
anthone, which exhibited a low scavenging effect of 14.2% at the concentration
of 200 mg mL�1 and its IC50 was therefore not measured. 1,4,8-Trihydroxyxanthone
showed the strongest activity and the antioxidant capacity followed the order of
1,4,8-trihydroxyxanthone 4 garboginol (3) 4 calothorexanthone (2) 4 �-tocotrienol (4) 4
globuxanthone (5).
 566   L.-T.T. Nguyen et al.

3. Experimental
3.1. General experimental procedures
Optical rotations were measured on an A. Krüss Optronic polarimeter spectrophotometer.
Melting points were determined on a Wagner & Munz Polytherm A hot stage microscope
and were uncorrected. UV spectra were obtained with an Agilent 8453 spectrophotometer
and IR spectra were recorded in KBr using a Bruker Tensor 37 spectrophotometer.
HRESIMS was performed on a Bruker micrOTOF-QII (80 eV). NMR spectra were
measured using a Bruker AV 500 [500 MHz (1H) and 125 MHz (13C)] with TMS as an
internal standard and acetone-d6 as the solvent. Multiplicities were determined using the
DEPT pulse sequence. CC was run on silica gel (Merck, 40–63 mm), Diol (Merck,
Lichroprep, 40–63 mm), or RP18 (Merck, 40–63 mm) bonded phases. For gel permeation
chromatography (GPC), Sephadex LH-20 (GE Healthcare) with CHCl3–MeOH 1 : 1 as
eluent was used. TLC was carried out on precoated glass TLC plates normal phase
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(Merck, 250 mm) and RP18 (Merck, 200 mm). TLC plates were visualised using UV light,
staining with I2 or spraying with ethanolic FeCl3 for detection of phenolic compounds.
Petroleum ether refers to the fraction of b.p. 50–90� C.

3.2. Plant material

The bark of C. thorelii Pierre was collected in Binh Dinh Province, central Vietnam, and
was identified by Mr Nguyen Thien Tich, Department of Botany, Ho Chi Minh City
University of Science (HCMUS). Herbarium samples (CVB-Song Kon-2006) are
deposited in the Natural Product and Medicinal Chemistry Lab, Faculty of Chemistry,
HCMUS.

3.3. Extraction and isolation

The air-dried and ground material (4.5 kg) was extracted by continuous percolation with
petroleum ether. Concentration of the solution gave a crude petroleum ether extract (96 g).
CC of the extract over silica gel (acetone–petroleum ether step gradient) yielded six
fractions.
Fraction 4 (5.16 g) was subjected to CC (silica gel, EtOAc–hexane) to afford six
fractions (4.1–4.6). Fraction 4.1 was purified using GPC to give calothorexanthone (2)
(14 mg). Fraction 4.4 was fractionated by CC (RP18, MeOH–H2O and then GPC) to yield
garbogiol (3) (25 mg). Fraction 4.5 was subjected to CC (RP18, MeOH–H2O) to produce
1,4,8-trihydroxyxanthone (10 mg). Fraction 2 (21.8 g) was repeatedly subjected to CC
(silica gel, acetone–hexane; EtOAc–hexane and then CH2Cl2–hexane) to give �-tocotrienol
(4) (40 mg). Fraction 3 (1.37 g) was separated using CC (silica gel, CH2Cl2–hexane) to yield
nine fractions (3.1–3.9). Fraction 3.6 was purified by CC (RP18, MeOH–H2O) to afford
1,7-dihydroxyxanthone (11.2 mg). Fraction 3.9 was subjected to CC (RP18, acetone–H2O)
to furnish globuxanthone (5) (15.5 mg).
Calothorexanthone (2): yellow needles, m.p. 154–155� C; [�]25 D �37.8 (c 0.27, EtOH).
UV �max (EtOH) nm (log "): 241 (4.37), 267 (4.38), 273 (4.40), 324 (4.25) and 394 (3.50);
IR �max cm�1: 3413, 1652, 1610, 1470, 1374, 1277, 1153, 1093, 811; 1H NMR (500 MHz,
acetone-d6): � 12.20 (1H, s, 1-OH), 11.80 (1H, s, 8-OH), 8.02 (1H, s, 7-OH), 7.32 (1H, d,
J ¼ 9.0 Hz, H-6), 6.89 (1H, d, J ¼ 9.0 Hz, H-5), 6.35 (1H, s, H-4), 4.59 q (1H, q, J ¼ 6.5 Hz,
H-12), 1.50 (3H, s, H3-14), 1.40 (3H, d, J ¼ 6.5 Hz, H3-15), and 1.26 (3H, s, H3-13).
13
C NMR (125 MHz, acetone-d6): � 186.0 (C-9), 168.1 (C-3), 159.4 (C-4a), 158.7 (C-1),
149.7 (C-10a), 147.9 (C-8), 141.4 (C-7), 117.9 (C-2), 124.5 (C-6), 108.2 (C-8a), 106.9 (C-5),
103.3 (C-9a), 92.1 (C-12), 90.4 (C-4), 44.0 (C-11), 25.4 (C-14), 20.8 (C-13) and 14.5 (C-15).
HMBC: H-4 ! C-2, C-3, C-4a, C-9a; H-5 ! C-7, C-8a, C-9, C-10a; H-6 ! C-7, C-8,
 Natural Product Research   567

C-10a; H-12 ! C-14; H-13 ! C-2, C-11, C-12, C-14; H-14 ! C-2, C-11, C-12, C-13;
H-15 ! C-11, C-12; 1-OH ! C-1, C-2, C-9a; 7-OH ! C-6, C-7, C-8 and 8-OH ! C-7,
C-8, C-8a. HRESIMS m/z: 329.1023 [M þ H]þ (Calcd for C18H17O6, 329.1025).

3.4. DPPH radical scavenging assay

The scavenging activity of all the isolated compounds on DPPH free radical was
determined spectrophotometrically using the method of Hatano et al. (1988). Calculation
of IC50 values was performed by testing the samples in five different concentrations.
One millilitre of the sample in MeOH at a series of concentrations of 5, 10, 20, 30 and
50 mg mL�1, respectively, in each reaction was mixed with 4 mL of 75 mM DPPH in MeOH.
The mixture was shaken vigorously, left to stand for 1 h in dark and at room temperature,
and the reduction of the DPPH free radical was measured by reading the absorbance of the
mixture at 517 nm using an Agilent 8453 spectrophotometer. L-Ascorbic acid was used as
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the positive control. All the tests were carried out in triplicate and averaged.

Supplementary material
Figure S1, showing the HMBC spectra, is available online.

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