Food Chemistry 130 (2012) 826831

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Food Chemistry
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Antibacterial activity of Thai edible plants against gastrointestinal pathogenic

bacteria and isolation of a new broad spectrum antibacterial polyisoprenylated
benzophenone, chamuangone
A. Sakunpak a, P. Panichayupakaranant a,b,
a
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand
b
Phytomedicine and Pharmaceutical Biotechnology Research Center, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Twenty-two edible plant extracts were subjected to evaluation of their antibacterial activity against some
Received 16 December 2010 gastrointestinal pathogenic bacteria, including Escherichia coli, Salmonella typhimurium, Salmonella typhi,
Received in revised form 11 May 2011 Shigella sonnei and Helicobacter pylori using the disc diffusion and broth microdilution methods. Sixteen of
Accepted 21 July 2011
the plant extracts exhibited antibacterial activity against one or more tested bacteria. Only Garcinia cowa
Available online 6 August 2011
leaf extracts exhibited antibacterial activity against all tested bacteria. Purication of the ethyl acetate
extract of G. cowa leaves using an antimicrobial assay-guided isolation afforded a new polyprenylated
Keywords:
benzophenone, chamuangone, that exhibited satisfactory antibacterial activity against Streptococcus
Garcinia cowa
Antibacterial
pyogenes (minimum inhibitory concentration (MIC) 7.8 lg/ml), Streptococcus viridans and H. pylori (MICs
Antimicrobial 15.6 lg/ml), and Staphylococcus aureus, Bacillus subtilis and Enterococcus sp. (MICs 31.2 lg/ml).
Chamuangone 2011 Elsevier Ltd. All rights reserved.
Benzophenone

1. Introduction Antimicrobial substances from natural sources like plants have

The gastrointestinal (GI) tract in humans is a long passage that
takes food from the mouth through to the anus where the wastes
are eliminated. The basic sequence is mouth, oesophagus, stomach,
duodenum, small intestine, large intestine, rectum, and anus. The
upper GI tract, including the oesophagus, stomach and small intes-
tine can harbour a wide variety of pathogens (Lamps, 2008). The GI
mucosa is the largest surface area in the body that is exposed to the
external environment. This large surface area allows for efcient
uptake of nutrients, but also exposes the body to potential external
toxins and pathogens (Sekirov, Russell, Antunes, & Finlay, 2010).
Some infections are involved in diseases that are ready dissemi-
nated. Bacterial infections of the GI tract leads either to symptoms
of vomiting and diarrhoea or to systemic disease. There are numer-
ous bacteria species that can infect the digestive system, such as
Salmonella spp., Shigella spp. Escherichia coli, Staphylococcus aureus,
Helicobacter pylori and Vibrio spp. In recent years, the emergence of
antibiotic resistant bacteria such as the nosocomal infective bacte-
rium methicillin-resistant S. aureus has been a serious problem and
investigations for new classes of antibiotics have become increas-
ingly important. Ideally antibiotic targets that microbes will nd
it impossible to develop resistance too are needed.
been investigated to achieve higher levels of food safety. For centu-
ries, indigenous plants have been used in herbal medicine for curing
various diseases, including enteritis. It has been reported that garlic,
onion, cinnamon, curry, mustard, basil, ginger and other spices exhi-
bit antimicrobial properties (Essawi & Srour, 2000; Otshudi, Foriers,
Vercruysse, Van Zeebroeck, & Lauwers, 1999; Srinivasan, Nathan,
Suresh, & Perumalsamy, 2001). The huge biodiversity in Thai plants
could be a great source for detecting new antibiotics (Temkitthawon
et al., 2008). In southern Thailand, people have a habit of eating rice
as a staple food together with several kinds of fresh vegetables
(Taungbudhitham, 1995). Some edible plants, specically located
in southern Thailand, are also used as traditional medicines, such
as Parkia speciosa and Azadirachta indica, that possess anti-diabetic
(Jamaluddin, Mohameda, & Lajis, 1994, 1995) and antimicrobial
properties (Alzoreky & Nakahara, 2003), respectively.
In this study, the anti-bacterial activity of extracts from twenty-
two edible plants commonly consumed in southern Thailand was
investigated. In addition, chamuangone, a new polyprenylated
benzophenone that possessed potent antibacterial activity was
puried from Garcinia cowa for the rst time.
2. Materials and methods

Corresponding author at: Department of Pharmacognosy and Pharmaceutical 2.1. Plant materials
Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai,
Songkhla 90112, Thailand. Tel.: +66 74 428220, +66 74 288980; fax: +66 74 428220. Twenty-two plants (Table 1) were collected from the Hat-Yai Dis-
E-mail address: pharkphoom.p@psu.ac.th (P. Panichayupakaranant). trict, Songkhla Province, Thailand, in June 2008. Voucher specimens

0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.07.088
 A. Sakunpak, P. Panichayupakaranant / Food Chemistry 130 (2012) 826831 827

were deposited in the herbarium of the Faculty of Pharmaceutical
Sciences, Prince of Songkla University, Thailand. These plants were
dried at 50 C for 24 h in a hot air oven and were reduced to coarse
powders using a grinder, and the powder passed through a sieve
(No. 45).
2.2. Preparations of plant extracts
was 10 mg/disc. DMSO was used as a negative control, whilst stan-
dard noroxacin (30 lg/disc) and ampicillin (10 lg/disc) discs
were used as positive controls. Plates were then incubated under
suitable conditions for each bacterium.
All disc diffusion tests were performed in triplicate and antibac-
terial activity was expressed as the mean of their diameter of inhi-
bition zones (mm).

Dried plant powders were extracted twice with ethyl acetate
under reux for 1 h. The extracts were combined and concentrated
under reduced pressure to produce the ethyl acetate extracts. The
residues were subsequently extracted with methanol and concen-
trated under the same conditions to produce the methanol
extracts.
2.3. Microorganisms and media
S. typhimurium (DMST 562), S. sonnei (DMST 561), H. pylori
(DMST 20979) and S. pyogenase (DMST 17020) were obtained from
the Department of Medical Science Center, Thailand. S. typhi, E. coli
(ATCC 25922), S. aureus (ATCC 25923), S. viridians and Enterococci
sp. were obtained from the Department of Microbiology, Faculty
of Medicine, Prince of Songkla University. B. subtilis was obtained
from Faculty of Science, Prince of Songkla University. MullerHin-
ton agar (MHA), MullerHinton broth, BrainHeart infusion (BHI),
and agar were purchased from Becton Dickinson (Franklin Lakes,
NJ).
2.4. Antibacterial susceptibility testing
These experiments were performed by the disc diffusion meth-
od (NCCLS, 2008) with some modication. E. coli, S. typhimurium, S.
typhi, S. sonnei and S. aureus were incubated in MHA at 37 C for
24 h, B. subtilis and Enterococci sp. were incubated in BHI at 37 C
for 24 h, H. pylori and Streptococcus spp. were incubated in BHI in
37 C under microaerophilic conditions for 48 h, and volume ad-
justed with 0.85% sodium chloride to yield approximately
108 CFU/ml as compared to the turbidity of the McFarland No.
0.5 standard. The prepared inoculum was streaked onto the surface
of MHA or BHI with a cotton swab. A sterile paper disc (6 mm) was
impregnated with 10 ll of plant extract (1000 mg/ml) and the disc
was placed on the agar. The concentration of each plant extract
2.5. Determination of minimum inhibitory and bactericidal
concentration
The minimum inhibitory concentration (MIC) and minimal
bactericidal concentration (MBC) were determined by the broth
microdilution assay (NCCLS, 2008). Cultures of aerobic and micro-
aerophilic bacteria were 24 and 48 h broth cultures, respectively.
The MIC was dened as the lowest concentration of the com-
pound to inhibit the growth of microorganisms, and the MBC
was dened as the lowest concentration of the compound to kill
the microorganisms.
2.6. Bioassay guided isolation
The dried leaf powder of G. cowa (1 kg) was extracted with ethyl
acetate (3 L  3), under reux conditions for 1 h, to obtain (after sol-
vent evaporation) a greenish-brown extract (32 g). The ethyl acetate
extract was subjected to silica gel vacuum chromatography. The ex-
tract, which had been pre-adsorbed on silica gel, was applied to the
top of the silica gel column (13 cm in diameter and 6 cm in height),
and the column subsequently eluted with 500 ml of solvent with the
aid of a vacuum pump. Mixtures of hexane and ethyl acetate were
used for column elution, using a step-gradient elution starting from
100% hexane to 100% ethyl acetate. Based on TLC chromatograms of
each fraction (500 ml), ten pooled fractions (fractions 110) were
obtained. The fractions were then subjected to antibacterial assay.
The antibacterial fraction (fraction 2) was further puried by silica
gel column (5.5  50 cm) (1 g extract per 50 g silica gel) eluted with
a mixture of hexane and ethyl acetate (9:1, v/v, 250 ml each) to give
eight pooled fractions (fractions IVIII). The fractions were then sub-
jected to antibacterial assay. The antibacterial fraction (fraction III)
was further puried by a Sephadex LH-20 column (3.5  80 cm)
using methanol as eluent (10 ml each) to give six pooled fractions
(fractions AF). A yellowish oily liquid (GC1, 50 mg) was obtained

Table 1
Plant materials used in this study.

Family Botanical name Voucher number Part used

Amaranthaceae Amaranthus gracilis Desf. SKP 007 01 19 01 Leaf
Anacardiaceae Anacardium occidentale Linn. SKP 009 01 15 01 Leaf
Araliaceae Polyscias fruticosa (L.) Hams SKP 017 16 06 01 Leaf
Lecythidaceae Careya sphaerica Roxb. SKP 022 03 19 01 Leaf
Boraginaceae Gnetum gnemon Linn. var. tenerum Markgr. SKP 028 07 07 01 Leaf
Compositae Emilia sonchifolia (L.) Dc. SKP 051 05 19 01 Leaf
Euphorbiaceae Glochidion wallichianum Mull.Arg. SKP 071 07 23 01 Leaf
Guttiferae Garcinia cowa Roxb. ex DC. SKP 083 07 03 01 Leaf
Malvaceae Abelmoschus esculentus (L.) Moench. SKP 109 01 05 01 Pod
Athyriaceae Diplazium esculentum (Retz) SW. SKP 110.1 01 05 01 Leaf
Mimosoideae Archidendron jiringa (Jack) I.C. Nielsen SKP 117 01 10 01 Seed
Leucaena leucocephala (Lam.) de Wit SKP 115 12 12 01 Leaf
Parkia speciosa Hassk. SKP 071 16 19 01 Seed
Parkia timoriana Merr. SKP 115 16 20 01 Seed
Moraceae Artocarpus heterophyllus Lam. SKP 117 01 08 01 Seed
Peperomiaceae Peperomia pellucida (L.) Humb., Bonpl. & Korth SKP 143.1 16 16 01 Leaf
Rubiaceae Morinda citrifolia Linn. SKP 165 13 03 01 Leaf
Rutaceae Micromelum minutum (G.Forst.) Wight & Arn. SKP 116 13 13 01 Leaf
Solanaceae Solanum stramonifolium Jacq. SKP 180 19 19 01 Fruit
Euphorbiaceae Antidesma ghaesembilla Gaertn. SKP 183.1 01 07 01 Fruit
Zingiberaceae Globba malaccensis Ridl. SKP 206 07 13 01 Rhizome
Etlingera elatior (Jack) R.M.Sm. SKP 206 14 05 01 Flower
828 A. Sakunpak, P. Panichayupakaranant / Food Chemistry 130 (2012) 826831

from the pooled antibacterial fraction (fraction D) after being puri-
ed using a semi-preparative RP-C18 HPLC column and acetonitrile
100% as eluent, with a ow rate 3.0 ml/min.
2.7. Identication of GC1
GC1. Yellow oil; UV kmax (MeOH) nm: 280; IR vmax (KBr) cm 1:
3350 (br) (OH), 1730 and 1670 (C@O), 16001530 (C@C, Ar),
14601380 (C@C), 1240 (CO); EIMS m/z: 502 [M]+, 298
[M 3  C5H8]+, 105 [C7H5O]+; HRFAB-MS m/z: 503.3166 [M+H]+
(calc. 502.3083 for C33H42O4), 13C and 1H NMR: Table 5.
3. Results and discussion
Amongst the forty-four extracts of twenty-two edible plants
that were investigated for antibacterial activity against Gram-neg-
ative pathogenic bacteria, sixteen plant extracts, including the
ethyl acetate extracts of G. cowa, G. gnemon, A. ghaesembilla, S. stra-
monifolium, P. speciosa and P. timoriana, and the methanol extract
of C. sphaerica, L. leucocephala, G. cowa, A. gracilis, A. occidentale,
A. ghaesembilla, S. stramonifolium, M. minutum P. timoriana and G.
wallichianum were capable of inhibiting the growth of one or more
tested bacteria at the concentration of 10 mg/disc (Table 2). Only
the ethyl acetate and methanol extracts from G. cowa exhibited
inhibitory effect against all tested bacteria. Ten edible plants used,
including A. esculentus, P. pellucida, A. heterophyllus, E. elatior, A.
jiringa, G. malaccensis, D. esculentum, E. sonchifolia, M. citrifolia,
and P. fruticosa did not produce any inhibitory activity against
any tested bacteria.
The extracts that possessed inhibitory effect were determined
for their MIC and MBC values by the broth microdilution method.
The MIC and MBC values of the plant extracts are shown in Tables
3 and 4. The methanol extract of A. occidentale exhibited the high-
est antibacterial activity against E. coli and S. sonnei with MICs of
1.25, 0.31 mg/ml, and MBCs of 1.25, 2.5 mg/ml, respectively. The
methanol extract of A. gracilis showed the strongest bactericidal
activity against S. typhimurium and S. typhi with MICs and MBCs
of 1.25 and 2.5 mg/ml, respectively. In addition, the ethyl acetate
extract of G. cowa exhibited the strongest antibacterial activity
against H. pylori with MIC and MBC values of 1.25 and 5 mg/ml,
respectively. There has been only one report of antibacterial
activity from fruit rinds of G. cowa against some foodborne patho-
gens and spoilage bacteria, such as Bacillus cereus, Bacillus coagu-
lans, B. subtilis, S. aureus and E. coli (Negi, Jayaprakasha, & Jena,
2008). However, the antibacterial active compound from G. cowa
fruit rinds has not yet been identied. In this study, only G. cowa

Table 2
Diameter of inhibition zones of the plant extracts and standard drugs.

Edible plants Diameters of inhibitions zones (mm)

Ethyl acetate extracts Methanol extracts
Ec Sm St Ss Hp Ec Sm St Ss Hp
A. ghaesembilla 22.6 11.8 15.6 17.6 14.2 17.5 n.i. 9.6 13.8 13.9
A. gracilis n.i. n.i. n.i. n.i. n.i. 20.5 13.3 15.5 23.3 11.0
A. occidentale n.i. n.i. n.i. n.i. n.i. 24.0 12.3 13.9 22.7 n.i.
C. sphaerica n.i. n.i. n.i. n.i. n.i. n.i. n.i. 9.6 n.i. 16.9
G. cowa 25.0 11.4 11.5 13.8 16.9 21.3 9.4 9.9 11.5 22.8
G. gnemon 10.3 n.i. n.i. 12.1 n.i. n.i. n.i. n.i. n.i. n.i.
G. wallichianum n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 11.1
L. leucocephala n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 13.2 17.8
M. minutum n.i. n.i. n.i. n.i. n.i. 20.4 11.9 13.1 22.9 12.6
P. speciosa 12.0 n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. 14.4
P. timoriana n.i. n.i. n.i. 11.7 n.i. n.i. n.i. n.i. n.i. n.i.
S. stramonifolium 10.3 n.i. n.i. n.i. n.i. 11.5 n.i. n.i. n.i. n.i.
Noroxacin 25.0
Ampicillin 28.2 22.4 12.7 26.6

Ec, E. coli; Sm, S. typhimurium; St, S. typhi; Ss, S. sonnei; Hp, H. pylori; n.i., no inhibition zone; , not performed.
Diameter of a sterile disc is 6 mm. Concentration of each plant extract was 10 mg/disc. Data are mean (n = 3).

Table 3
Minimum inhibitory concentration of the plant extracts and standard drugs.

Edible plants Minimum inhibitory concentrations (mg/ml)

Ethyl acetate extracts Methanol extracts
Ec Sm St Ss Hp Ec Sm St Ss Hp
A. ghaesembilla 5 10 5 2.5 5 5 n.d. 10 2.5 10
A. gracilis n.d. n.d. n.d. n.d. n.d. n.d. 1.25 1.25 0.62 5
A. occidentale n.d. n.d. n.d. n.d. n.d. 1.25 2.5 1.25 0.31 n.d.
C. sphaerica n.d. n.d. n.d. n.d. n.d. 1.25 n.d. 2.5 n.d. 5
G. cowa 5 10 5 5 1.25 5 1.25 2.5 5 5
G. gnemon 10 n.d. n.d. 10 n.d. n.d. n.d. n.d. n.d. n.d.
G. wallichianum n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 5
L. leucocephala n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 1.25 5
M. minutum n.d. n.d. n.d. n.d. n.d. 2.5 2.5 5 0.62 5
P. speciosa 10 n.d. n.d. 20 n.d. n.d. n.d. n.d. n.d. n.d.
P. timoriana n.d. n.d. n.d. n.d. 20 n.d. n.d. n.d. n.d. 20
S. stramonifolium 20 n.d. n.d. n.d. n.d. 20 n.d. n.d. n.d. n.d.
Noroxacina 0.06
Ampicillina 0.25 0.25 4 0.25

Ec, E. coli; Sm, S. typhimurium; St, S. typhi; Ss, S. sonnei; Hp, H. pylori; n.d., not determined. n = 3.
a
MIC (lg/ml).
 A. Sakunpak, P. Panichayupakaranant / Food Chemistry 130 (2012) 826831 829

Table 4
Minimum bactericidal concentrations of the plant extracts and standard drugs.

Edible plants Minimum bactericidal concentrations (mg/ml)

Ethyl acetate extracts Methanol extracts
Ec Sm St Ss Hp Ec Sm St Ss Hp
A. ghaesembilla 5 10 10 10 5 10 n.d. 20 10 10
A. gracilis n.d n.d. n.d. n.d. n.d. n.d. 2.5 2.5 1.25 10
A. occidentale n.d. n.d. n.d. n.d. n.d. 1.25 2.5 10 2.5 n.d.
C. sphaerica n.d. n.d. n.d. n.d. n.d. 2.5 n.d. 20 n.d. 5
G. cowa 10 20 20 20 5 10 20 20 10 10
G. gnemon >20 n.d. n.d. >20 n.d n.d. n.d. n.d. n.d. n.d.
G. wallichianum n.d n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 20
L. leucocephala n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 5 5
M. minutum n.d n.d. n.d. n.d. n.d. 10 20 20 10 10
P. speciosa >20 n.d. n.d. >20 n.d n.d. n.d. n.d. n.d. n.d.
P. timoriana n.d. n.d. n.d. n.d. >20 n.d. n.d. n.d. n.d. 20
S. stramonifolium 20 n.d. n.d. n.d. n.d. 20 n.d. n.d. n.d. n.d.
Noroxacina 0.12
Ampicillina 0.25 0.5 8 0.25

Ec, E. coli; Sm, S. typhimurium; St, S. typhi; Ss, S. sonnei; Hp, H. pylori; n.d.: not determined. n = 3.
a
MBC (lg/ml).

Table 5
23 13
C NMR (125 MHz) and 1H NMR (500 MHz) data of GC1.
22
21 d d
Position C (CDCl3) H (CDCl3)

20 1 64.8 3.4 s
2 191.4
19 3 115.6
4 194.6
18 27 5 64.6
6 42.6 1.44ax t (13)
17 28
26 2.05eq dd (3.2, 13)
24
25 7 40.9 1.55 m
15 8
7 8 48.5
16 1 9 206.5
14
2 6 10 198.0
9
11 137.1
11 5 12 128.5 7.45 d (7.3)
13 10 3 13 127.8 7.35 t (7.8)
12 4 29 14 132.4 7.50 d (7.3)
15 127.8 7.35 t (7.8)
16 128.5 7.45 d (7.3)
30 17 17.7 0.95 s
31 32 18 38.5 1.36a dt (3.7, 12.7)
1.64b overlapped
33 19 21.9 1.95a m
2.34b overlapped
Fig. 1. Structure of chamuangone. 20 123.8 5.07 m
21 132.1
22 17.8 1.69 overlapped
23 25.7 1.64 overlapped
extracts possessed antibacterial activity against all tested Gram-
24 28.1 1.60a overlapped
negative bacteria. The ethyl acetate extract of G. cowa was there- 2.10b overlapped
fore selected and subjected to isolation of the antibacterial active 25 121.9 4.87 m
compound. 26 133.4
On the basis of antibacterial assay-guided purication, GC1 was 27 18.1 1.60 overlapped
28 25.7 1.50 overlapped
obtained as an antibacterial active compound from G. cowa leaves. 29 30.4 2.35a overlapped
GC1 was identied as chamuangone, a new polyprenylated benzo- 2.52b dd (9.5, 13.9)
phenone (Fig. 1) by the following spectroscopic data. The proton 30 119.6 5.17 m
noise decoupled 13C NMR spectrum (Table 5) showed that the mol- 31 134.8
32 17.9 1.63 overlapped
ecule contained 33 carbons, and the results of the high resolution
33 26.0 1.73 overlapped
mass spectrum (HRESIMS) gave a molecular formula of C33H42O4
(m/z: 503.3166 [M+H]+; calc. 502.3083 for C33H42O4). Eleven qua- Coupling constants (J in Hz) are given in parentheses.
ternary, ten methine, ve methylene, and seven methyl carbon sig-
nals were observed. A fragment peak at m/z 105 in the EIMS
spectrum, and the carbon signals at d 127.8 (2C), 128.5 (2C), as well as two strong IR absorption bands at 1670 and 1730 cm 1.
132.4 (1C) and 137.1 (1C), as well as proton resonances at d 7.35 Three isopentenyl groups were identied by the carbon signals at
(2H), 7.45 (2H) and 7.50 (1H) in the NMR experiments corroborated d 21.9 (C-19), 123.8 (C-20), 132.1 (C-21), 17.8 (C-22), 25.7 (C-23),
the existence of a monosubstituted phenyl group. The presence of 28.1 (C-24), 121.9 (C-25), 133.4 (C-26), 18.1 (C-27), 25.7 (C-28),
two carbonyl groups in this compound were established by the 30.4 (C-29), 119.6 (C-30), 134.8 (C-31), 17.9 (C-32), and fragment
appearance of carbon resonances at d 191.4 (C-4) and 206.5 (C-9) peaks at m/z 69 in the EIMS. A bicyclo [3,3,1] nonane-2,4,9-trione
830 A. Sakunpak, P. Panichayupakaranant / Food Chemistry 130 (2012) 826831

18 Table 6
H3C Minimum inhibitory concentration and minimum bactericidal concentration of
17 chamuangone.
15 8 Gram Microorganisms MICs (lg/ml) MBCs (lg/ml)
O
1
O7
Positive B. subtilis 31.2 >1000
14 16 2
9 6 Enterococcus sp. 31.2 62.5
H S. aureus 31.2 125
13 11 5 S. pyogenes 7.8 31.2
10 3 H S. viridans 15.6 125
12 4
Negative E. coli 500 >1000
H. pylori 15.6 62.5
H O OH S. sonnei 250 >1000
S. typhimurium 250 >1000

constant between H-6ax and H-7 conrmed this observation

(J = 13 Hz). In addition, the C-7 carbon signal showed an upeld shift
when its substituent is equatorial. Thus, GC1 was clearly identied
H3C
CH3 as chamuangone, a new polyisoprenylated benzophenones with a
23 bicyclo [3.3.1]-nonane-2,4,9-trione system (Fig. 1).
22
21 The antibacterial activity of chamuangone was evaluated against
S. pyogenes, S. viridans, H. pylori, B. subtilis, S. aureus, Enterococcus sp.,
20 S. sonnei, S. typhimurium, and E. coli using the broth microdilution
19
method. The results revealed that chamuangone exhibited inhibi-
H CH3 tory effect against all tested bacteria (Table 6). In addition, chamuan-
18 H 27 gone possessed satisfactory bactericidal activity against S. pyogenes,
H3C
17 28 S. viridans, H. pylori, Enterococcus sp., and S. aureus with MIC values of
26 CH3
24 7.8, 15.6, 15.6, 31.2, and 31.2 lg/ml, and MBC values of 31.2, 62.5,
15 8 25
O 125, 62.5, and 125 lg/ml, respectively. However, chamuangone
1 O7
14 16
H did not show bactericidal activity against B. subtilis, E. coli, S. sonnei
2 6
9 and S. typhimurium (MBC > 1000 lg/ml).
11 5 H Our ndings indicate that G. cowa is an edible plant that may
13 10 3 protect against some GI infections. In addition, chamuangone
12 4 29 may be used as an indicative marker for the quality control of G.
H
cowa leaf extracts as well as a lead compound for the development
H O OH
H 30 of anti-infectious drugs.
32
31 CH3
33 Acknowledgments
H3C

The authors wish to thank Thailand Research Fund through the

Fig. 2. Important 13
C1H long-range correlations observed by heteronuclear COSY Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0207/2550) for
of GC1. nancial support in this research.

system was established by the carbon signals at d 64.8 (C-1), 115.6
(C-3), 64.6 (C-5), 42.6 (C-6), 40.9 (C-7) and 48.5 (C-8), two carbonyl
groups at d 191.4 (C-2) and 206.5 (C-9), and an oxygen-bearing car-
bon at d 194.6 (C-4). The bicyclo and three isopentenyl groups were
also identied by 1H1H COSY and HMBC experiments. The position
of the isopentenyl group at C-18 was explained by the 1H1H COSY
spectrum that showed a correlation between H-18 and H-19. The
substituent of the isopentenyl group at positions C-5 and C-7 was
conrmed by HMBC spectrum that showed correlations between
C-5 and H-29, and C-7 and H-24, respectively (Fig. 2). The basic
bicyclic ring system requires the benzoyl group at C-3, and other
correlations are shown in Fig. 2.
The relative stereochemistry of the side-chains of chamuangone
was established by comparing the 13C NMR spectral data of refer-
ence compounds, nemorosone and clusianone (Cuesta-Rubio,
Velez-Castro, Frontana-Uribe, & Cardenas, 2001; Piccinelli et al.,
2005). The isopentenyl group at C-5 was assigned to an equatorial
orientation. The 13C chemical shift of the methyl carbon at C-17ax
(d 17.7) indicated an equatorial position for the isopentenyl group
at C-7. The upeld chemical shift of the C-17ax signal resulted from
a steric compression of gauche interaction between this carbon and
the C-24 of the equatorial isopentenyl group on C-7. The coupling
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