Arch. Pharm. Res.

DOI 10.1007/s12272-013-0266-4

RESEARCH ARTICLE

Anti-Helicobacter pylori xanthones of Garcinia fusca

Jannarin Nontakham • Napaporn Charoenram •

Wanchalerm Upamai • Malai Taweechotipatr •

Sunit Suksamrarn

Received: 11 May 2013 / Accepted: 15 October 2013

Ó The Pharmaceutical Society of Korea 2013

Abstract A new geranylated xanthone derivative, fus- Introduction

caxanthone I (1), along with nine xanthones (2–9 and 11), a
biphenyl (10) and three biflavonoids (12–14) were isolated Oxygenated xanthones, secondary metabolites from higher
from the roots of Garcinia fusca Pierre. Compounds 8, 10 plants, in particular Garcinia species, revealed interesting
and 11–14 were reported from this plant species for the first biological profiles. These include, for example, antibacterial,
time. Their structures were elucidated by spectroscopic antifungal, antiviral, antioxidant, anti-inflammatory and cyto-
analyses, including 1D- and 2D-NMR and MS. The iso- toxic to cancer cells (Chin and Kinghorn 2008; Peres et al.
lated compounds were evaluated for antibacterial activity 2000). Garcinia fusca Pierre (Clusiaceae family) or Madan-paa
against Helicobacter pylori. Cowaxanthone (5) and fu- in Thai, is a native endemic tree grown in the northeastern part
kugiside (14) exhibited stronger inhibitory activity against of Thailand and many Asian countries. Its edible acidic fruit
H. pylori DMST reference strain at MICs 4.6 and 10.8 lM, and leaf are commonly used in food preparation. G. fusca is also
respectively, than that of the control metronidazole. Isoj- used as a spice in India and as flavoring in soup in Malaysia. The
acareubin (8) displayed the most potent activity against H. root, stem, leaf and fruit of this plant are ethno medically used
pylori HP40 clinical isolate with MIC 23.9 lM, which was for improvement of blood circulation, expectorant, treatment of
approximately two times greater than that of the standard coughs and indigestion, laxative, and the relief of fever (Poom-
drug amoxicillin. ipamorn and Kumkong 1997). Previous bioactive chemical
examination on G. fusca stem barks led to the identification of
Keywords Garcinia fusca  Xanthones  eight new xanthones, fuscaxanthones A–H and eight reported
Fuscaxanthone I  Antibacterial activity  xanthones, which played an important role in producing
Helicobacter pylori inhibitory effects on Epstein–Barr virus early antigen induction
(Ito et al. 2003). As part of the ongoing project to search for new
bioactive compounds from Garcinia species (Suksamrarn et al.
2003, 2006), a chemical investigation has been undertaken of
the roots of G. fusca. Reported herein are the isolation, structure
Electronic supplementary material The online version of this elucidation of a new oxygenated xanthones, fuscaxanthone I
article (doi:10.1007/s12272-013-0266-4) contains supplementary
material, which is available to authorized users. (1), along with 13 known compounds (Fig. 1) and anti-Heli-
cobacter pylori activity evaluation of these compounds.
J. Nontakham  N. Charoenram  W. Upamai 
S. Suksamrarn (&)
Department of Chemistry and Center of Excellence for
Innovation in Chemistry, Faculty of Science, Srinakharinwirot Materials and methods
University, Bangkok 10110, Thailand
e-mail: [email protected] General experimental procedures
M. Taweechotipatr 1
Department of Microbiology, Faculty of Medicine, H- and 13C-NMR, COSY, HMQC, HMBC and NOESY
Srinakharinwirot University, Bangkok 10110, Thailand spectra were run on a Bruker AVANCE 300 FT-NMR

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 J. Nontakham et al.

Fig. 1 Chemical structures of B O OH

25
compounds 1–14 23
H3CO 8 1
A

20
HO O 3 R
OH
24
18
2 R = OCH3, A = B =
16 14 OH
O OH
8 1 11
H3CO 8a 9a
9
15 4 R = OH, A = ,B=

HO 5
10a O 4a 3 OH
6 R = OH, A = B =
1
OH

7 R = OH, A = ,B=

O OH
O OH H3CO
H3CO
HO O OH
HO O O
5
3

O OH OCH3
1
O O OH
HO
6 3 H3CO
HO 5 O O
H3CO OCH3
OH
HO O OH
8 9 10

OH

O OH HO O
1
R2
OH O OH
HO O OH
OH R1O O 12 R1 = R2 = H
13 R1 = H, R2 =OH
11 14 R1 = O-β -D-Glu, R2 = H
OH O

spectrometer operating at 300 MHz (1H) and 75 MHz (13C). were performed on silica gel 60 (finer than 0.063 mm,
For the spectra taken in CDCl3, the residual nondeuterated Merck) and Sephadex LH-20 (Pharmacia). Precoated silica
solvent signals at d 7.24 and d 77.00 were used as references gel 60 F254 plates (Merck) were used for TLC and com-
for 1H- and 13C-NMR spectra, respectively. Mass spectra pounds were visualized by exposure under UV light and by
were recorded on a Thermo Finnigan LC-Q and a Bruker spraying with anisaldehyde–(95:5–5:95, v/v), aceto-
microTOF mass spectrometer. Melting points were deter- neH2SO4 followed by heating on a hot plate.
mined using a Griffin melting point apparatus and are
uncorrected. UV spectra were obtained on a UV-2401 PC Plant material
spectrophotometer (Shimadzu) in MeOH and IR spectra on a
FT-IR Spectrum BX (Perkin Elmer) in KBr. Optical rota- The roots of G. fusca were collected from Buayai Sub-
tions were measured on a JASCO-1020 digital polarimeter. district, Nampong District, Khon Kaen Province, Thailand,
Quick column chromatography (QCC) was carried out on in April 2008 and the plant species was identified by James
silica gel 60 F254 (Merck), column chromatography (CC) F. Maxwell. A voucher specimen (Jannarin Nontakham

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Anti-Helicobacter pylori xanthones

001) was deposited at the Faculty of Science, Ram- furnish more amount of 1 (12 mg) and 1,3,5,6,-tetrahydr-
khamhaeng University, Thailand. oxyxanthone (11) (10 mg) as a pale yellow solid. A portion of
fraction K (500 mg from 9.1 g) was chromatographed over
Extraction and isolation silica gel (/ 2.5 9 40 cm) eluting with CH2Cl2–MeOH–H2O
(4:0.2:86) to give vokensiflavone (12) (15 mg) as a yellow
The air dried roots of G. fusca (1 kg) were ground into small solid. Another portion of fraction K (500 mg) was further
pieces and extracted with EtOAc (7 L) followed by with chromatographed over silica gel eluting with a gradient of
MeOH (7 L) by using Soxhlet extraction apparatus. Evapo- hexane–acetone (95:5–5:95, v/v) to yield 10 subfractions
ration of solvent under reduced pressure gave the EtOAc (K1–K10) and a yellow solid of morelloflavone or fukugetin
(60.1 g) and MeOH (45.8 g) extracts, respectively. The (13) (129 mg) was obtained after repeated silica CC (/
EtOAc soluble extract which showed anti-H. pylori HP40 2.0 9 35 cm) eluting with CH2Cl2–MeOH–H2O (8:82:0.2)
activity (MIC 62.5 lg/mL) was further subjected to isolation of subfraction K7 (160 mg). Fraction N (4.34 g) was chro-
and characterization of active compounds. Thus the EtOAc matographed over silica gel (/ 5.0 9 50 cm, CH2Cl2–
extract (40.0 g) was fractionated by QCC (/ 10 9 15 cm) MeOH–H2O, 4:86:0.2) to yield fukugiside (14) (108 mg) as
eluting with a gradient of n-hexane–acetone (95:5–5:95, v/v), yellow needles.
acetone–MeOH (95:5–0:100, v/v) and MeOH to afford 14
main fractions (A–N). Fractions C, D and E were combined Fuscaxanthone I (1)
(2.8 g) and chromatographed over silica gel (/ 5 9 50 cm)
eluted with a gradient of n-hexane–acetone (95:5–40:60 and Yellow amorphous solid; mp 103–105 °C; [a]26 D -9.5°
30:70–90:10, v/v) to yield 18 subfractions (CE1–CE18). (acetone, c 0.20); UV (MeOH) kmax (log e): 243 (4.6), 259
Subfraction CE9 (906 mg) was further chromatographed (4.5), 315 (4.4), 356 (3.9). IR mmax (KBr) cm-1: 3402,
over silica gel (/ 2.5 9 40 cm), eluting with a gradient of n- 3215, 2968, 2932, 1642, 1607, 1581, 1462, 1282, 1192,
hexane–acetone (99.5:0.5–98.5:1.5, v/v), to provide 8 sub- 993, 837; ESIMS: m/z (rel. int.): 511 ([M-H]-, 100);
fractions (CE9.1–CE9.8). Subfraction CE9.4 (168 mg) was HRTOFMS (APCI-): m/z 511.2338 [M-H]- (calcd for
then subjected to Sephadex LH-20 CC (in MeOH) to obtain C29H36O8-H, 511.2326); 1H-NMR (300 MHz, CDCl3 ? 2
b-mangostin (2) (65 mg) and fuscaxanthone A (3) (2 mg) drops CD3OD): d 13.55 (1H, s, 1-OH), 6.64 (1H, s, H-5),
as yellow solids. Cowanin 4 (94 mg) was precipitated out 6.15 (1H, s, H-4), 5.41 (1H, br t, J = 7.8 Hz, H-12), 5.12
as yellow amorphous solid from fraction G (339 mg) and (1H, br t, J = 7.4 Hz, H-21), 4.25 (2H, s, H-14), 3.81 (3H, s,
the filtrate was further subjected to silica gel CC (/ 2.0 9 7-OCH3), 3.41 (2H, br t, J = 7.8 Hz, H-11), 3.31 (2H, br t,
35 cm) eluting with n-hexane–acetone (100:1–80:20 and J = 7.2 Hz, H-16), 2.06 (2H, m, H-20), 1.86 (2H, m,
70:30–60:40, v/v) to yield 16 subfractions (G1–G16). Sub- H-17), 1.75 (3H, s, H-15), 1.66, 1.61 (2 9 3H, each s, H-25
fractions G8 and G9 were identified to be cowaxanthone (5) and 23), 1.55 (2H, m, H-19), 1.26 (3H, s, H-24); 13C-NMR
(49 mg) and a-mangostin (6) (107 mg) as yellow solid. (75 MHz, CDCl3 ? 2 drops CD3OD): d 181.6 (C-9), 161.7
Fraction H (2.2 g) was subjected to silica gel CC (/ 5.0 9 (C-3), 160.1 (C-1), 155.6 (C-6), 155.4 (C-10a), 154.7
50 cm) eluting with CH2Cl2 and CH2Cl2–MeOH (99:1– (C-4a), 142.8 (C-7), 138.2 (C-8), 133.8 (C-13), 131.4 (C-
88:12 and 85:15–60:40, v/v) to give 16 subfractions (H1– 22), 126.2 (C-12), 124.5 (C-21), 111.0 (C-8a), 108.7 (C-2),
H16), and two successive CC over silica gel of subfraction H9 102.8 (C-9a), 101.8 (C-5), 93.0 (C-4), 72.8 (C-18), 61.8 (C-
(83 mg) eluting with n-hexane–acetone (/ 1.5 9 20 cm, 14), 61.4 (OCH3), 42.0 (C-19), 41.7 (C-17), 26.2 (C-24),
85:15, v/v) afforded cowanol (7) (67 mg) and isojacareubin 25.6 (C-25), 22.7 (C-20), 22.4 (C-15), 21.4 (C-16), 21.2
(8) (10 mg) as orange solids. Subfraction H12 (620 mg) was (C-11), 17.5 (C-23).
subjected to a Sephadex LH-20 column (MeOH) to afford
fuscaxanthone G (9) (13 mg) as an orange solid and more Preparation of fuscaxanthone A (3)
amount of compound 7 (406 mg). Fraction I (4.2 g) was
chromatographed over silica gel (/ 5.0 9 50 cm) using A solution of cowanin (4) (24 mg, 0.05 mmol) in CH2Cl2
CH2Cl2–EtOAc (100:5, 80:20 and 5:95 v/v), to give 15 sub- (1 mL) was added to 2,3-dichloro-5,6-dicyanobenzoqui-
fractions (I1–I15). Subfraction I4 was further purified by a none (DDQ) (15 mg, 0.06 mmol) and the reaction mixture
Sephadex LH-20 column (MeOH) to give fuscaxanthone I (1) was stirred at room temperature for 18 h. Water was then
(7 mg) as a yellow solid. Fraction J (639 mg) was purified by added and the mixture was extracted with EtOAc. The
silica gel CC (/ 2.5 9 40 cm) eluting with CH2Cl2–EtOAc combined organic phase was washed with H2O, dried over
(100:1–88:12 and 85:15–50:50, v/v) to give 15 subfractions anhydrous Na2SO4 and the solvent was removed under
(J1–J15). Subfraction J2 was proved to be nigrolineabiphenyl vacuum. The reaction mixture was purified by column
B (10) (14 mg) as a brown solid. Subfraction J11 (113 mg) chromatography using n-hexane–acetone (95:1) to yield
was subjected to a Sephadex LH-20 column (MeOH) to compound 3 (13 mg, 54 %) as yellow amorphous solid.

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 J. Nontakham et al.

Bacterial strains and inoculums preparation 2003), cowanin (4) (Na Pattalung et al. 1994), cowax-
anthone (5) (Na Pattalung et al. 1994), a-mangostin (6)
The clinical isolate of H. pylori HP40 was obtained from (Suksamrarn et al. 2006), cowanol (7) (Na Pattalung et al.
King Chulalongkorn Memorial Hospital kindly provided 1994), isojacareubin (8) (Rath et al. 1996), fuscaxanthone
by Dr. Thanitta Chatsuwan, Chulalongkorn University, G (9) (Ito et al. 2003) and 1,3,5,6-tetrahydroxyxanthone
Thailand. H. pylori DMST 20165 was obtained from the (11) (Frahm and Chaudhuri 1979), a biphenyl, nigrolinea-
Department of Medical Science, Ministry of Public Health, biphenyl B (10) (Rukachaisirikul et al. 2005) and three
Thailand and H. pylori ATCC 43504 was used as the ref- biflavonoids with dextrorotatory optical rotation : (?)-
erence strain. Bacterial strains were grown on Brain Heart vokensiflavone (12) ([a]25 D ?142.0°, MeOH, c 0.10) (lit
Infusion (BHI) agar (Becton–Dickinson, USA) supple- [a]20
D ?1.6°, Chen et al. 1975), (?)-morelloflavone or
mented with 10 % horse serum (GIBCO Invitrogen, Eng- fukugetin (13) ([a]25 29
D ?161.6°, MeOH, c 0.20) (lit [a]D 0°,
29
land) and incubated at 37 °C for 3 days under micro- MeOH, Konoshima et al. 1969; [a]D 170°, MeOH,
aerobic conditions (5 % O2, 10 % CO2, 85 % N2) gener- Konoshima et al. 1969; [a]20 D ?17°, Chen et al. 1975; [a]D
25
20
ated by using a micro-aerobic GasPak (Mitsubishi, Japan) ?188°, MeOH, c 0.1, Li et al. 2002; [a]D ?10°, DMSO,
in an anaerobic box (Mitsubishi, Japan). To prepare inoc- c 0.25, Elfita et al. 2009) and (?)-morelloflavone glucoside
ula, test bacteria were prepared in BHI broth by adjusting or (?)-fukugiside (14) ([a]28 D ?150.0°, MeOH, c 0.28) (lit
to 0.5 McFarland standard that had 1.5 9 108 colony [a]25
D ?165°, MeOH, c 1.07, Elfita et al. 2009; Konoshima
forming units (CFU)/mL. and Tkeshiro 1970) (Fig. 1). Compounds 8 and 10–14 were
not found previously in this plant (Ito et al. 2003). To
Antibacterial assay obtain a greater amount of compound 3 for sufficient
antibacterial testing, treatment of cowanin (4) with DDQ at
A broth micro dilution method (NCCLS 1997; Wangchuk room temperature gave 3 in 51 % yield after chromato-
et al. 2011) carried out in a 96-well sterilized micro plate graphic purification.
was used to determine the minimum inhibitory concentra- The new compound 1 was isolated as a yellow amorphous
tion (MIC) of the pure compounds. MICs were determined solid, mp 103–105 °C and the molecular formula C29H36O8
as the lowest concentration that produces complete growth was deduced from its HRTOFMS at m/z 511.2338 [M-H]-.
inhibition of the tested bacteria. Test samples were dis- The IR spectrum showed intense absorptions for the
solved in dimethyl sulfoxide (DMSO, Merck). Serial hydroxyls (3402 and 3215 cm-1) and conjugated carbonyl
twofold dilutions of the test samples were mixed with BHI (1642 cm-1) (Panthong et al. 2009). The UV spectrum of 1
broth in 96-well microtiter plates (Corning, USA) in an exhibited absorptions of a xanthone nucleus at kmax 243, 259,
initial concentration of 250 lg/mL. The suspension of each 315 and 356 nm (Ito et al. 2003). The 1H-NMR spectrum of 1
tested bacteria (1 9 106 CFU/mL) was added in each well displayed signals for a 1,3,6,7-tetraoxygenated xanthone,
in equal volume of test sample (50 lL). The microtiter which included one hydrogen-bonded hydroxyl group (dH
plates were incubated as described above. H. pylori HP40 13.55, s), two aromatic singlets (dH 6.64, H-5 and 6.15, H-4)
isolated from clinical specimen and reference H. pylori and a methoxyl (dH 3.81, 3H), in addition to two modified
ATCC 43504 and H. pylori DMST 20165 were used as test prenyl and geranyl units. The oxymethylene (dC 61.8) and
strains. MICs were recorded by reading the lowest con- carbinol carbon (dC 72.8) resonances were identified from its
13
centration that inhibited visible growth. The test was per- C-NMR and DEPT spectra. Analysis of its 1H, 13C, DEPT,
formed in triplicates. Amoxicillin, clarithromycin and COSY and HMQC spectra and comparison with related
metronidazole (Government Pharmaceutical Organization, compounds (Panthong et al. 2009; Ito et al. 2003) led to the
Thailand) were used as positive control drug. establishment of a 4-hydroxy-3-methylbut-2-enyl moiety
and a hydroxylated geranyl unit. The 1H- and 13C-NMR data
of compound 1 were similar to those of the known xanthone,
Results and discussion cowanol (7) (Na Pattalung et al. 1994), except for a double
bond of the geranyl chain in 1 was hydroxylated at C-18. The
From the ethyl acetate extract obtained from the roots of G. HMBC correlations between the methylene resonance at d
fusca, a new geranylated xanthone, fuscaxanthone I (1), 3.31 (H-16) and C-7 at d 142.8 and C-8 at d 138.2, in addition
was isolated and characterized. In addition, by detailed to the NOESY interactions with methoxyl signal at d 3.81
analysis of the 2D-NMR data and comparison the 1H- and and methyl group (H-24) at d 1.26, allowed the placement of
13
C-NMR and MS including optical rotation values with the hydroxygeranylated unit at C-8 carbon. The HMBC
those of previous reports, the 13 known compounds were connections between this latter methyl signal to carbon res-
determined as nine oxygenated xanthones: b-mangostin (2) onances at d 41.7 (C-17), d 72.8 (C-18) and d 42.0 (C-19),
(Suksamrarn et al. 2006), fuscaxanthone A (3) (Ito et al. and also NOESY cross peak with H-20 (d 2.06) were also

123
Anti-Helicobacter pylori xanthones

Table 1 MIC values (lM) of compounds 1–14 against H. pylori

ATCC 43504, H. pylori DMST20165 and H. pylori HP40
Compound MIC
ATCC43504 DMST20165 HP40

Fuscaxanthone I (1) 30.5 15.2 122.0

b-Mangostin (2) 18.3 18.3 147.3
Fuscaxanthone A (3) 131.2 16.3 131.2
Cowanin (4) 130.6 16.3 130.6
Cowaxanthone (5) 152.3 4.6 152.3
Fig. 2 Selected HMBC and NOESY correlations for compound 1 a-Mangostin (6) 19.0 19.0 76.1
Cowanol (7) 126.4 15.7 126.4
observed (Fig. 2). By the same analogy, the methylene Isojacareubin (8) 23.9 23.9 23.9
protons at d 3.41 (H-11) exhibited HMBC cross peak with Fuscaxanthone G (9) 32.6 16.3 130.6
C-2 at d 108.7 and NOESY interaction with hydroxyl Nigrolineabiphenyl B (10) 226.3 56.5 226.3
methylene protons at d 4.25 (H-14). Furthermore, the HMBC 1,3,5,6-Tetrahydroxyxanthone 240.3 29.9 240.3
correlations between H-14 and the methyl singlet at d 1.75 (11)
(H-15) and the methine triplet at d 5.41 (H-12) clearly Vokensiflavone (12) 115.7 14.4 115.7
showed the location of the 4-hydroxy-3-methylbut-2-enyl Morelloflavone (13) 112.3 14.0 112.3
moiety at C-2. The structure of fuscaxanthone I (1) was Fukugiside (14) 87.0 10.8 174.0
therefore established to be 1,3,6-trihydroxy-8-(3-hydroxy- Amoxicillina 0.3 0.6 42.7
3,7-dimethyloct-6-enyl)-2-(4-hydroxy-3-methylbut-2-enyl)- Metronidazolea – 11.1 –
7-methoxyxanthone. However, the absolute configuration at Clarithromycina 41.8 – 0.6
C-18 could not be resolved. a
Positive control
Helicobacter pylori have been shown to have an etio-
logical role in chronic gastritis, gastric ulcer and gastric
cancer (Chey and Wong 2007). Effective medication shows DMST with MICs of 4.6 and 10.8 lM, respectively, than
that more than one type of antibiotics are needed due to that of the control metronidazole (MIC 11.1 lM). Isoj-
higher chance of resistance of the bacteria against antibi- acareubin (8) demonstrated the most activity against H.
otics (Wang and Huang 2005). The use of medicinal plant pylori HP40 at MIC 23.9 lM and was approximately two
extracts and/or their metabolites is therefore an alternative times more potent than that of amoxicillin (MIC 42.7 lM),
for prevention and treatment of diseases caused by H. but much lower activity compared with clarithromycin
pylori infection. Xanthones from the medicinal Garcinia (MIC 0.4 lM). It should be noted that the 1,3,5,6-oxy-
plants are well known for their antibacterial properties genated function comprising substitutions at C-4 and at the
(Peres et al. 2000), including anti-H. pylori activity of 3-hydroxyl positions in xanthone nucleus played important
mangostins and their synthetic derivatives (Hasegawa et al. roles for strong inhibitory activity against the H. pylori
1996). In our work, the in vitro antibacterial effect of all ATCC 43504 and HP40 strains as observed in compound 8
isolated compounds from G. fusca was evaluated against when compared with no substitution in 11.
metronidazole-resistant H. pylori ATCC43504, reference
Acknowledgments This work was supported by the Center of
H. pylori DMST20165, reference strains, and metronida- Excellence for Innovation in Chemistry (PERCH-CIC), Office of the
zole-resistant clinical isolate H. pylori HP40. The results Higher Education Commission and Srinakharinwirot University
(Table 1) revealed that b-mangostin (2) (MIC 18.3 lM) (Grant number 231/2554). Authors thank Mr. Maurice Broughton,
and a-mangostin (6) (MIC 19.0 lM) exhibited similar Chulabhorn Research Institute, Thailand, for proof reading the article.
effect against H. pylori ATCC43504, though none of the
compounds assayed was as effective as the control amox-
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