International Journal of Antimicrobial Agents 23 (2004) 377–381

Isopanduratin A from Kaempferia pandurata as an active antibacterial

agent against cariogenic Streptococcus mutans
Jae-Kwan Hwang a,∗ , Jae-Youn Chung a , Nam-In Baek b , Jung-Hee Park a
a Department of Biotechnology & Bioproducts Research Center, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749, South Korea
b Graduate School of Biotechnology & Plant Metabolism Research Center, Kyunghee University, Yongin, South Korea

Received 24 March 2003; accepted 5 August 2003

Abstract

An antibacterial compound active against Streptococcus mutans was isolated from Kaempferia pandurata and identified as isopanduratin
A using 1 H NMR, 13 C NMR and EI-MS. The minimum inhibitory concentration (MIC) of isopanduratin A was 4 mg/l which was much
lower than that of some other natural anticariogenic agents such as sanguinarine (12 mg/l), green tea extract and carvacrol (125 mg/l), thymol
(250 mg/l) and isoeugenol and eucalyptol (500 mg/l). The bactericidal test showed that isopanduratin A completely inactivated S. mutans
at 20 mg/l in 1 min. Significant inhibitory activity of isopanduratin A was also observed against S. sobrinus, S. sanguinis and S. salivarius
with an MIC of 4 mg/l. Damage to the cell membrane and cell wall of S. mutans by isopanduratin A was shown using transmission electron
microscopy (TEM). These results suggest that isopanduratin A could be employed as a potential antibacterial agent for preventing dental
caries.
© 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Keywords: Antibacterial activity; Kaempferia pandurata; Isopanduratin A; Streptococcus mutans; Dental caries

1. Introduction Natural antibiotic agents from various medicinal plants

and food materials have been screened for antibacterial
Streptococcus mutans has been identified as a plaque activity. Spice extracts, cinnamon bark oil, papua-mace
forming bacterium, capable of producing dental caries in ex- extracts and clove bud oil have been reported to inhibit sig-
perimental animals and humans. The ability of S. mutans to nificantly the growth of oral bacteria [4]. Green tea extract
adhere firmly to tooth surfaces in the presence of sucrose is usually used after every meal as a means of preventing
and to release acids by fermenting various dietary sugars has dental caries in Japan, and several polyphenols in the tea ex-
been associated with its caries-inducing potential [1,2]. tract, catechin, epicatechin, gallocatechin, epigallocatechin,
Many attempts have been made to eliminate S. mu- epicatechin gallate, epigallocatechin gallate, etc. inhibit the
tans from the oral flora. Antibiotics such as chlorhexidine, growth of S. mutans [5]. Sanguinarine is an alkaloid iso-
penicillin, ampicillin, tetracycline, erythromycin and van- lated from the rhizome of Sanguinaria canadensis, with a
comycin are very effective in preventing dental caries in broad spectrum against various oral bacteria [6]. Sanguinar-
vivo and in vitro. However, their excessive use can result ine has been used as a natural agent in a wide range of oral
in alterations of the oral and intestinal flora and cause un- care products such as toothpaste and mouthwash due to its
desirable side effects such as the development of bacterial strong antibacterial activity [7]. However, since sanguinar-
tolerance, vomiting, diarrhoea and teeth stains [3]. These ine was reported to be associated with oral leukoplakia, its
problems necessitate further search for natural antimicro- industrial applications have been greatly reduced [8,9].
bial agents that are safe for humans and specific for oral Kaempferia pandurata Roxb. (Zingiberaceae), known
pathogens. as temu kunci in Indonesia or krachai in Thailand, has
been used traditionally for food and also medicinal pur-
poses for diarrhoea, and for its antimutagenic, antitumour,
∗ Corresponding author. Tel.: +82-2-2123-5881; fax: +82-2-362-7265. anti-inflammatory activities, etc. [10–12]. Flavonoids such
E-mail address: [email protected] (J.-K. Hwang). as boesenbergin A, boesenbergin B, panduratin A, cardomin,

0924-8579/$ – see front matter © 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
doi:10.1016/j.ijantimicag.2003.08.011
378 J.-K. Hwang et al. / International Journal of Antimicrobial Agents 23 (2004) 377–381

cardomonin, pinostrobin, pinocembrin, alpinetin and 5- by elution with n-hexane and ethyl acetate solution (5:1, v/v)
hydroxy-7-methoxyflavanone are recognised as the bioac- and 10 ml volumes of eluant were collected in tubes. The
tive components of K. pandurata [13,14]. However, few collected tubes were divided into four fractions (Fr. I–IV)
studies have been conducted on the antibacterial activity of following silica TLC (thin layer chromatography, Merck, 60
K. pandurata. F254 ) and each fraction was assayed for antibacterial activity.
This research was aimed to isolate the antibacterial
compound from K. pandurata against S. mutans and to in- 2.4. Instrumentation
vestigate its effectiveness compared with other well-known
commercial antibacterial agents. 1H NMR and 13 C NMR spectra were measured using
JNM-LA500 (CD3 OD, 500 MHz, JEOL, Japan). Mass spec-
tra (EI-MS) were carried out using VG PlatformII (FISONS,
2. Materials and methods UK) at an ionization voltage of 40 eV.

2.1. Plant materials 2.5. Determination of minimum inhibitory concentration

(MIC) and minimum bactericidal concentration (MBC)
Kaempferia pandurata Roxb. (Zingiberaceae) was col-
lected from the Biofarmaka Research Center of Bogor Agri- Isopanduratin A and other antibacterial agents, dissolved
cultural University (Indonesia) and identified by Dr. Latifah in 1% DMSO, was added to the first tube containing 1 ml
K. The plant material was shade dried and ground to pow- of BHI broth and serially diluted by the two-fold method
der. A reference specimen has been deposited at 4 ◦ C in the [15,16] to obtain concentrations of 1000-1 mg/l. A bacterial
Bioproducts Research Center, Yonsei University, Seoul, Ko- suspension (0.1 ml) containing 2 × 105 colony forming units
rea. All reagents were purchased from Sigma (USA), unless (CFU)/ml was added to each tube and incubated for 24 h
otherwise stated. at 37 ◦ C. The MIC was determined by judging visually the
bacterial growth in the series of test tubes. The MBC was
2.2. Bacterial strains and culture conditions the concentration in which S. mutans was unable to remain
viable. The control included an inoculated growth medium
The following seven oral microorganisms were used in without the test compound and some commonly available
this study: S. mutans ATCC 25175, S. sanguinis ATCC antibacterial agents used for caries control were employed
35105, S. sobrinus ATCC 27351 and Actinomyces visco- as positive controls. All experiments were performed in du-
sus ATCC 15987 were purchased from the American Type plicate and the average values were reported as MIC and
Culture Collection (Rockville, MD, USA). Lactobacillus MBC.
acidophilus KCCM 32820, L. casei KCCM 35465 and S.
salivarius KCCM 40412 were obtained from the Korean 2.6. Measurement of bactericidal activity
Culture Center of Microorganisms (Seoul, Korea). S. mu-
tans, S. sanguinis, S. sobrinus and S. salivarius were cultured The viable cell count method was employed to examine
in Brain Heart Infusion (BHI, Difco, USA) at 37 ◦ C for 24 h the bactericidal activity of isopanduratin A. S. mutans was
aerobically. Yeast malt extract and LBS broth were used for cultured in BHI broth, washed twice with 50 mM potassium
the culture of A. viscosus and L. acidophilus at 37 ◦ C for 24 h phosphate buffer (pH 7.0) and diluted to give a final concen-
anaerobically. L. casei was cultured in beef extract (1%), tration of 2 × 105 CFU/ml. A mixture of 4 ml isopanduratin
1N NaOH (2.5%), trypticase (3%), yeast extract (0.5%), A or sanguinarine and 1 ml cell suspension was incubated
K2 HPO4 (0.5%), cysteine–HCl (0.05%), 0.025% resazurin at 37 ◦ C for 48 h, and the viability of the incubated mixture
solution (0.4%), haemin solution (0.1%) and menadione was determined by the pour plating method [17].
solution (0.002%) at 37 ◦ C for 24 h anaerobically.
2.7. Morphology
2.3. Isolation of an antibacterial compound
The control and isopanduratin A-treated cells were fixed
One hundred grams dried rhizome of Kaempferia pan- with 2% glutaraldehyde and 1% osmium tetraoxide at room
durata were ground and extracted twice with 75% aqueous temperature. After eliminating the remaining glutaraldehyde
methanol (400 ml, v/v) for 24 h at room temperature and the and osmium tetraoxide, a dehydration process was con-
extract was concentrated, frozen and lyophilized (11 g). The ducted with 30, 50, 70, 80, 95, and 100% ethyl alcohol. Each
MeOH extract was further fractionated successively with step was performed for 10 min at room temperature. The
ethyl acetate, n-butanol and water. Each fraction was evapo- fixed cells were embedded with araldite and small blocks
rated and dried under reduced pressure (ethyl acetate fraction of bacteria in the araldite were cut with an ultramicrotome
5.6 g, butanol fraction 1.0 g, water fraction 4.4 g). The ethyl (MT-X, RMC, USA). Ultra thin sections were cut and ob-
acetate fraction was further separated using silica gel col- served with a transmission electron microscope (JEOL, JEM
umn chromatography (Merck Kieselgel 60, 70–230 mesh) 1010, Japan).
 J.-K. Hwang et al. / International Journal of Antimicrobial Agents 23 (2004) 377–381 379

3. Results and discussion Table 1

MIC and MBC of isopanduratin A and other antibacterial compounds
against S. mutans
3.1. Isolation and identification of an active antibacterial
compound Compounds MIC (mg/l) MBC (mg/l)

Isopanduratin A 4 8
Since the antibacterial activity against S. mutans was cle- Chlorhexidine 1 2
arly observed in the ethyl acetate fraction, further sep- Vancomycin 1 2
aration was performed on this fraction using silica gel Hexamedine 6 12
Sanguinarine 12 24
column chromatography, and the purification procedures of Green tea extract 125 250
the active compound were monitored by MIC. Compound Carvacrol 125 250
II-b, prepared as described in the methodology, had a Thymol 250 500
molecular weight = 406 from the EI-MS spectrum (m/z, Isoeugenol 500 500
406, M+ ). Careful comparison of several spectral data of Eucalyptol 500 500
compound II-b including 13 C NMR, 1 H NMR and EI-MS
with those in the literature suggested the chemical structure
to be isopanduratin A (Fig. 1) or (2-methoxy-4,6-dihydroxy- agents. The activity was almost comparable with chlorhex-
phenyl)-(3 -methyl-2 -(3 -methylbut-2 -enyl)-6 -phenylcy- idine, vancomycin, and hexamedicine. However, antibiotics
clohex-3 -enyl) methanone [13,14]. The chemical structure are very limited in extended applications in oral care as they
was identified as an isomer of panduratin A [18]. may produce detrimental side effects such as discolouration
of teeth, reduction of the immune defence, disruption of nor-
mal ecology of plaque, diarrhoea, vomiting, etc. [20,21].
3.2. Antibacterial activity of isopanduratin A
Fig. 2 compares the bactericidal activities of isopanduratin
A and sanguinarine at the same concentration (20 mg/l). It is
Antibacterial activity of isopanduratin A was investigated
noteworthy that 20 mg/l of isopanduratin A showed consid-
in terms of minimum inhibitory concentration (MIC) and
erable killing of S. mutans in one minute. This fast bacteri-
minimum bactericidal concentration (MBC) compared with
cidal effect is of practical importance, since isopanduratin A
some commercially available agents. As shown in Table 1,
in toothpaste or mouthwash should be effective within a few
the MIC and MBC of isopanduratin A for S. mutans were
minutes. Similar bactericidal activity has also been reported
4 and 8 mg/l, respectively. Its MIC is much lower than
for xanthorrhizol from Curcuma xanthorrhiza [22]. Fig. 2
those of other natural antibacterial agents, such as green
also shows that the bactericidal activity of isopanduratin A
tea extract and carvacrol (125 mg/l), thymol (250 mg/l) and
against S. mutans was much more efficient than that of san-
isoeugenol and eucalyptol (500 mg/l). It is also interesting
guinarine. Accordingly, isopanduratin A might have more
to note in Table 1 that isopanduratin A (4 mg/l) had a sig-
potential application in oral care products in place of san-
nificantly lower MIC than sanguinarine (12 mg/l), which is
guinarine. The strong bactericidal activity of isopanduratin
a natural compound isolated from Sanguinaria canadensis
A at a concentration as low as 5 mg/l and dose-dependence
widely used for industrial toothpaste and mouthwash prod-
is shown in Fig. 3.
ucts [6,19]. This indicates that isopanduratin A has much
stronger antibacterial activity than other natural commercial
0

-1

-2
Log survival ratio

-3

-4

-5

-6

-7
0 2 4 6 8 10 12
Treatment time (min)

Fig. 2. Comparison of bactericidal activity between isopanduratin A and

sanguinarine: (䉬) control (0.05% DMSO); (䊏) 20 mg/l sanguinarine; (䉱)
Fig. 1. The chemical structure of isopanduratin A. 20 mg/l isopanduratin A.
380 J.-K. Hwang et al. / International Journal of Antimicrobial Agents 23 (2004) 377–381

-1

-2
Log survival ratio

-3

-4

-5

-6

-7
0 2 4 6 8 10 12
Treatment time (min)

Fig. 3. Bactericidal activity of isopanduratin A against of S. mutans: (䉬)

control (0.05% DMSO); (䊉) 5 mg/l; (䊏) 10 mg/l; (䉱) 20 mg/l.

As shown in Table 2, isopanduratin A also exhibited pref-

erential antimicrobial spectrum against other oral bacteria
such as S. sobrinus, S. sanguinis and S. salivarius with MIC
values of 4 mg/l. In contrast, no significant activity was ob-
served against L. acidophilus, L. casei and A. viscosus.

3.3. Morphology
Fig. 4. Transmission electron microscopy of isopanduratin A-treated S.
mutans (magnification: 40,000×): (a) control (0.05% DMSO); (b) 10 mg/l
Transmission electron microscopy (TEM) was carried isopanduratin A.
out to see any morphological changes in S. mutans brought
about by isopanduratin A treatment. Compared with the
intact cell in (Fig. 4a), significant morphological changes caused by the growth of Streptococcus spp. Further studies
were observed in the cells treated with 10 mg/l isopan- are necessary for elucidating the antibacterial mechanism of
duratin A (Fig. 4b). After isopanduratin A treatment, the isopanduratin A at the molecular level.
cell surface was remarkably disintegrated and the cytoplasm
membrane was detached from the cell wall. A similar re-
sult was also reported for kuwanon G from Morus alba Acknowledgements
[23].
In this study, isopanduratin A exhibited specific activity This work was supported by the Korea Science and Engi-
against the oral bacteria S. mutans, S. sobrinus, S. sanguinis neering Foundation (KOSEF) through the Bioproducts Re-
and S. salivarius. In particular, isopanduratin A showed high search Center at Yonsei University.
activity against S. mutans at 20 mg/l in one minute. These
data suggest that isopanduratin A could be employed as
a natural anticariogenic agent for preventing dental caries References

[1] Hamada S, Koga T, Ooshima T. Virulence factors of Streptococcus

Table 2 mutans and dental caries prevention. J Dent Res 1984;63:407–11.
MIC and MBC of isopanduratin A against oral microorganisms [2] Oh S, Lee JH, Kim GT, Shin JG, Baek YJ. Anti-cariogenic activity
of a bacteriocin produced by Lactococcus sp. HY449. Food Sci
Microorganisms MIC (mg/l) MBC (mg/l) Biotech 2003;12:9–12.
[3] Chen CP, Lin CC, Namba T. Screening of Taiwanese crude drugs for
Streptococcus mutans ATCC 25175 4 8
antibacterial activity against Streptococcus mutans. J Ethonopharma-
Streptococcus salivarius KCCM 40412 4 6
col 1989;27:285–95.
Streptococcus sanguis ATCC 35105 4 8
[4] Saeki Y, Ito Y, Okuda K. Antimicrobial action of natural substances
Streptococcus sobrinus ATCC 27351 4 8
on oral bacteria. Bull Tokyo Dent Coll 1989;30:129–35.
Lactobacillus acidophilus KCCM 32820 500 750
[5] Otake S, Makimura M, Kuroki T. Anticaries effects of polypheno-
Lactobacillus casei KCCM 35465 500 750
lic compounds from Japanese green tea. Caries Res 1991;25:438–
Anctinomyces viscosus ATCC 15987 500 750
43.
 J.-K. Hwang et al. / International Journal of Antimicrobial Agents 23 (2004) 377–381 381

[6] Joann LD, Sigmund SS. Comparative in vitro activity of sanguinar- [14] Pandji CW, Grimm C, Wray V. Insecticidal constituents from four
ine against oral microbial isolates. Antimicrob Agents Chemother species of the Zingiberaceae. Phytochemistry 1993;34:415–9.
1985;27:663–5. [15] Bert F, Woolfrey MD, Joan M. Comparison of minimum inhibitory
[7] Eley BM. Antibacterial agents in the control of supragingival plaque-a concentration values determined by three antimicrobic dilution meth-
review. Br Dent J 1999;186:286–96. ods for Pseudomonas aeruginosa. Am J Clin Pathol 1981;75:39–44.
[8] Mascarenhas AK, Allen CM, Loudon J. The association between [16] Chand SI, Lusunzi DA, Veal LR. Rapid screening of the antimicrobial
Viadent use and oral leukoplakia. Epidemiology 2001;12:741– activity of extracts and natural products. J Antibiot 1994;47:1295–
3. 304.
[9] Munro IC, Delzell ES, Nestmann ER. Viadent usage and oral leuko- [17] Jones RN, Pfaller MA, Jacobs MR. Cefditoren in vitro activity and
plakia: a spurious association. Regul Toxicol Phamacol 1999;30:182– spectrum: a review of internation studies using reference methods.
96. Diagn Microbiol Infect Dis 2001;41:1–14.
[10] Trakoontivakorn G, Nakahara K, Shinmoto H. Structural analysis of [18] Jaipetch T, Kanghae S, Pancheroen O. Constituents of Boesenbergia
a novel antimutagenic compound, 4-hydroxypanduratin A, and an- pandurata (syn. Kaempferia pandurata): Isolation, crystal structure
timutagenic activity of flavonoids in a Thai spice, fingerroot (Boe- and synthesis of (±)-boesenbergin A. Aust J Chem 1982;35:351–61.
senbergia pandurata Schult.) against mutagenic heterocyclic amines. [19] Southard GL, Boulware RT, Walbonr DR. Sanguinarine, a new an-
J Agric Food Chem 2001;49:3046–50. tiplaque agent: retention and plaque specificity. J Am Dent Assoc
[11] Murakami A, Kondo A, Nakamura Y. Anti-tumor promoting prop- 1984;108:338–43.
erties of edible plants from Thailand, and identification of an active [20] Marsh PD. Microbiological aspects of the chemical control of plaque
constituent, cardamonin, of Boesenbergia pandurata. Biosci Biotech and gingivitis. J Dent Res 1992;71:1431–8.
Biochem 1993;57:1971–3. [21] Daneshmand N, Jorgensen MG, Nowzari H. Initial effect of con-
[12] Panthong A, Tassaneeyakul W, Kanjanapothi D. Anti-inflammatory trolled release chlorhexidine on subgingival microorganisms. J Peri-
activity of 5,7-dimethoxyflavone. Planta Med 1989;55:133–6. odontal Res 2002;37:375–9.
[13] Pancharoen O, Picker K, Reutrkul V. Constituents of Zingiberaceae. [22] Hwang JK, Shim JS, Baek NI, Pyun YR. Xanthorrhizol: a potential
Diastereomers of [7-hydroxy-5-methoxy-2-methyl-2-(4 -methylpent- antibacterial agent from Curcuma xanthorrhiza against Streptococcus
3 -enyl)-2H-chromen-8-yl][3 -methyl-2 -(3 -methylbut-2 -enyl)- mutans. Planta Med 2000;66:196–7.
6 -phenylcyclo-hex-3 -enyl]methanone (panduratin B), a constituent [23] Park KM, You JS, Lee HY, Baek NI, Hwang JK, Kuwanon G. An
of the red rhizomes of a variety of Boesenbergia pandurata. Aust J antibacterial agent from the root bark of Morus alba against oral
Chem 1987;40:455–9. pathogens. J Ethnopharmacol 2003;84:181–5.