Beruflich Dokumente
Kultur Dokumente
Abstract—Previous studies have revealed the inhibitory effects of an acidic polysaccharide purified from the root of Panax ginseng
against the adhesion of Helicobacter pylori to gastric epithelial cells and the ability of Porphyromonas gingivalis to agglutinate erythro-
cytes. In this study, this acidic polysaccharide from P. ginseng, PG-F2, was investigated further, in order to characterize its antiadhesive
effects against Actinobacillus actinomycetemcomitans, Propionibacterium acnes, and Staphylococcus aureus. The minimum inhibitory
concentrations (MIC) were found to be in a range of 0.25–0.5 mg/mL. However, results showed no inhibitory effects of PG-F2 against
Lactobacillus acidophilus, Escherichia coli, or Staphylococcus epidermidis. PG-F2 is a pectin-type polysaccharide with a mean MW
of 1.2 · 104 Da, and consists primarily of galacturonic and glucuronic acids along with rhamnose, arabinose, and galactose as minor
components. The complete hydrolysis of PG-F2 via chemical or carbohydrolase enzyme treatment resulted in the abrogation of its
antiadhesive activity, but limited hydrolysis via treatment with pectinase (EC. 3.2.1.15) yielded an oligosaccharide fraction, with activ-
ity comparable to the precursor PG-F2 (the MIC of ca. 0.01 mg/mL against H. pylori and P. gingivalis). Our results suggest that PG-F2
may exert a selective antiadhesive effect against pathogenic bacteria, while having no effects on beneficial and commensal bacteria.
2006 Elsevier Ltd. All rights reserved.
Keywords: Bacterial adhesion; Hemagglutination; Panax ginseng (Araliaceae); Antiadhesive activity; Helicobacter pylori; Porphyromonas gingivalis;
Actinobacillus actinomycetemcomitans; Propionibacterium acnes; Staphylococcus aureus
0008-6215/$ - see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.03.032
J.-H. Lee et al. / Carbohydrate Research 341 (2006) 1154–1163 1155
2. Results
In a previous study, the adherence of H. pylori to AGS The hydrolysis of PG-F2, accomplished by treatment
gastric cells was determined via scanning electron with NaNO2, a- and b-amylase, or cellulase, yielded
microscopy (SEM) by counting microcolonies of H. py- oligosaccharide fractions of lower molecular weight,
lori attached to the surfaces of gastric cells.25 Adminis- which were found to be non-inhibitory up to a concen-
tration of the acidic polysaccharide was found to tration of 2 mg/mL (data not shown). By contrast, a
attenuate the degree to which H. pylori attached to the limited enzymatic hydrolysis of PG-F2 by pectinase
gastric cells. This PG-F2-induced inhibition of the (EC. 3.2.1.15, poly-[1,4-a-D -galacturonide]glycano-
attachment of H. pylori to the AGS cells was re-exam- hydrolase) for 30 min at 37 C resulted in the pro-
ined using a urea phenol red assay. PG-F2 was not duction of oligosaccharide fragments, which were then
Figure 4. Elution profile of PG-OF1 by gel-filtration chromatography. A Bio-Gel P4 column (2.5 · 102 cm) was used at a flow rate of 0.3 mL/min.
The thin line represents absorbance at 490 nm from the phenol–sulfuric acid assay. PG-OF1 is indicated by the bar above the peak.
J.-H. Lee et al. / Carbohydrate Research 341 (2006) 1154–1163 1157
purified by using Bio-Gel P-4 gel-filtration chromato- It is worth noting that PG-OF1, a partially hydro-
graphy (Fig. 4). One of the oligosaccharide fractions, lyzed oligosaccharide from PG-F2, also exhibited pro-
PG-OF1, exhibited activity comparable to that of the found antiadhesive properties against H. pylori and
precursor PG-F2 against H. pylori attachment, with an P. gingivalis, with an MIC of ca. 0.01 mg/mL, which is
MIC of 0.01 mg/mL. 10-fold lower than that of its precursor PG-F2 (Table
Carbohydrate analyses of PG-OF1 revealed the high- 2 and Fig. 6). The MICs of PG-OF1 against A. actino-
est uronic acid content among the fractions, and indi- mycetemcomitans and P. acnes were as high as those
cated a composition of galacturonic acid (72.7%) and of PG-F2, but it evidenced minimal inhibitory activ-
glucuronic acid (25.9%), with minor amounts of arabi- ity against S. aureus. Probably more importantly,
nose and rhamnose (Table 1). The molecular mass of PG-OF1 was also determined to exert no detectable
the predominant oligosaccharide in PG-OF1 was esti- activity against L. acidophilus, E. coli, or S. epidermidis,
mated by gel-filtration chromatography to be approxi- similarly to its precursor, PG-F2. The antiadhesive
mately 1 kDa and determined by MALDI-TOF mass effects of both PG-F2 and PG-OF1 against H. pylori
spectrometry to be about 815 Da (see Fig. 1 in the and P. gingivalis can likely be highly correlated with
Supplementary data). This suggests that PG-OF1 may their inherent high uronic acid content. In this con-
be equivalent to 4–5 sugar units, calculated from the text, other carbohydrates harboring negatively charged
average molecular weight of 180 Da per sugar unit. groups were examined in order to elucidate the struc-
ture–activity relationships for the carbohydrates.
2.4. Antiadhesive activity of PG-F2 or PG-OF1 against
oral, gastrointestinal, and skin bacteria 2.5. Antiadhesive activity of carbohydrates against oral,
gastrointestinal, and skin bacteria
PG-F2 was assessed with regard to its antiadhesive ef-
fects against A. actinomycetemcomitans, P. acnes and Among carbohydrates containing negatively charged
S. aureus, as well as H. pylori and P. gingivalis, by using groups, the antiadhesive effects evidenced by pectin were
a series of hemagglutination assays. Surprisingly, PG-F2 the most pronounced against P. gingivalis with an MIC
was found to inhibit the attachment of these gastric, of ca. 0.0001 mg/mL (Table 2). Pectin also exhibited sig-
oral, and skin pathogenic bacteria, in a range of 0.1– nificant antiadhesive properties against P. acnes and
0.5 mg/mL (Table 2 and Fig. 5). In contrast, when S. aureus with an MIC of 0.01 mg/mL. However, these
PG-F2 was examined regarding its possible effects effects disappeared at concentrations above 0.1 mg/
against the beneficial or commensal bacteria, L. acido- mL. Alginic acid has lower but broad inhibition against
philus, E. coli, and S. epidermidis, it showed little activ- bacteria at concentrations of 0.25–0.5 mg/mL. LMWH
ity against them, even at concentrations of 2.0 mg/mL and SOS were highly active against P. gingivalis, both
(Table 2). with MICs of 0.01 mg/mL (9 lM). Only xylitol was
Table 2. Inhibition of bacteria-induced hemagglutination by an acidic polysaccharide from Panax ginseng and by a range of carbohydrates
Inhibitorsb MW(Da) Minimum inhibitory concentrations (MIC) in mg/mLa
H. p P. g A. a P. a S. a S. e E. c L. a
PG-F2 12,000 0.1 0.1 0.25 0.5 0.5 — — —
PG-HMW 80,000 0.25 0.1 0.1 0.1 — — — —
PG-OF1 1000 0.01 0.01 0.5 0.25 — — — —
Pectinc 20,000 — 0.0001 — 0.01 0.01 — — —
Alginic acid 1,000,000 0.25 0.25 0.25 — 0.25 0.25
Heparin 3000 — 0.02 — 0.1 1.0 — — 0.1
SOS 1159 — 0.01 — 1.0 1.0 — — —
GlcAd 194 — — — 0.5 0.5 0.5 — 0.1
GalAd 212 — — — — — — — 0.01
GlcNAcd 212 — 0.01 — — — — — —
Xylitol 152 — — 0.01 — — — — —
Palatinose 342 — 0.1 — — — — 0.01 —
Trehalose 342 — 0.01 — — — — 0.1 0.01
a
All the values are the MIC in mg/mL, corresponding to the average in triplicates. ‘—’ represents no inhibition at high concentrations above
2.0 mg/mL. Aspartic and glutamic acids were used as non-carbohydrate acidic compounds and bacterial binding did not cause nonspecific reaction
(data not shown). H. p: Helicobacter pylori, P. g: Porphyromonas gingivalis, A. a: Actinobacillus actinomycetemcomitans, P. a: Propionicbacterium
acnes, S. a: Staphylococcus aureus, S. e: Staphylococcus epidermidis, E. c: Escherichia coli, L. a: Lactobacillus acidophillus.
b
PG-F2 and PG-OF1 represent a polysaccharide and its hydrolyzed oligosaccharide from P. ginseng, respectively.
c
Pectin showed the highest inhibition to P. gingivalis with an MIC of ca. 0.0001 mg/mL. However, pectin did not show any inhibitory activity above
0.1 mg/mL.
d
GlcA, GalA, and GlcNAc are D -glucuronic acid, D -galacturonic acid, and N-acetylglucosamine, respectively.
1158 J.-H. Lee et al. / Carbohydrate Research 341 (2006) 1154–1163
Figure 5. Micrograph images of a hemagglutination inhibition assay by pathogenic bacteria (magnification ·100). Hemagglutination was inhibited
by PG-F2 from P. ginseng, which is active against H. pylori (H. p), P. gingivalis (P. g), A. actinomycetemcomitans (A. a), P. acnes (P. a), and S. aureus
(S. a). The minimum inhibitory concentrations were in the range of 0.1–0.5 mg/mL. A positive control (without inhibitor) is shown along with each
pathogenic bacterium.
Figure 6. Micrograph images of a hemagglutination inhibition assay by pathogenic bacteria (magnification ·100). Hemagglutination was inhibited
by the oligosaccharide PG-OF1, which is active against H. pylori (H. p), P. gingivalis (P. g), A. actinomycetemcomitans (A. a), P. acnes (P. a), and
S. aureus (S. a), with minimum inhibitory concentrations between 0.01 and 0.5 mg/mL. A positive control (without inhibitor) is shown along with
each pathogenic bacterium.
the present study, the PG-F2 polysaccharide further Bacillus anthracis, and Y. pestis.35 In addition, citrus pec-
demonstrated its profound antiadhesive effects against tin has proved effective with regard to the inhibition of
A. actinomycetemcomitans, P. acnes, and S. aureus, in adhesion to the specific cell-surface receptors.33 Our find-
a concentration range of 0.25–0.5 mg/mL. Given its ings also indicated that pectin exerts a profound anti-
apparent molecular weight of 12 kDa, the effective con- adhesive effect against P. gingivalis, P. acnes, and S.
centrations of PG-F2 occurred in a range of 20–40 lM. aureus. These data are, therefore, suggestive that anti-
These active concentrations are comparable to those of adhesive activity might be strongly associated with high
sialyl-3 0 -lactose, which has been considered to be the uronic acid content, implying the potential role of carbo-
most active oligosaccharide.27 In fact, sialyl-3 0 -lactose hydrates harboring negatively charged groups in host–
exhibited no significant inhibitory effects at a concentra- bacterial adhesion. In this context, it was not surprising
tion of 0.1 mg/mL (ca. 150 lM), which is lower than to find that pectin, alginic acid, LMWH, and SOS were
that of PG-F2 with regard to the inhibition of H. pylori- all effective in the prevention of H. pylori or P. gingi-
mediated adhesion to AGS cells.25 Whereas the inhibi- valis-mediated hemagglutination.
tory effect of sialyl-3 0 -lactose depended on H. pylori As is shown in Table 2, although pectin exhibited a
strains,27 PG-F2 had similar activities against strains high degree of antiadhesive activity against P. gingivalis
43504 and 26695, in addition to 49503 previously with an MIC of 0.0001 mg/mL, it did not exhibit activity
reported.23 at concentrations above 0.1 mg/mL. Alginic acid, which
Notably, PG-F2 did not inhibit the adhesion capabil- is composed of guluronic acid and mannuronic acid,
ities of beneficial or commensal bacteria, including showed inhibition against bacteria at 0.25–0.5 mg/mL.
L. acidophilus, E. coli, and S. epidermidis. Although cer- Alginic acid was found to cause red cell hemolysis at
tain deleterious or pathogenic strains of E. coli, such as concentrations above 1.0 mg/mL, and its inhibitory
strain O157, are known to cause human disease,28 many effect may be partly due to hemolysis. These results of
other strains live naturally and harmlessly within the pectin and alginic acid, although reproducible, have
human digestive system. L. acidophilus has been shown yet to be clearly elucidated. Glucuronic and galacturonic
to provide health benefits.29 Whereas S. epidermidis acids were not shown to have any inhibitory activity
can be regarded as a pathogen primarily associated with against the bacteria used in this study. Thus, we cannot
nosocomial infections contracted from blood-contacting rule out the possibility that specific carbohydrate com-
devices within hospitals,30 it also exerts beneficial effects ponents of PG-F2, other than uronic acids, may also
as a normal constituent of the flora of healthy human play a role in the antiadhesion in host–bacterial interac-
skin.21 Collectively, our findings indicate that, whereas tions, thereby implying that host–bacterial adhesion
PG-F2 exhibits profound antiadhesive activity against may rely on multiple interactions.
pathogenic bacteria, it exerts no or minimal inhibitory Asaccharolytic P. gingivalis was shown to be the most
effects against commensal bacteria. This suggests, then, sensitive to the carbohydrates used in this study, which
that PG-F2 is selectively antiadhesive against human involves binding to a variety of receptors containing col-
pathogenic bacteria. lagen, fibronectin, laminin, vitronectin and cytokera-
PG-F2 may be a pectin-type polysaccharide composed tin.7,36 The adhesion of S. aureus to the skin, as is seen
primarily of galacturonic and glucuronic acids (93%). in cases of atopic dermatitis, induces the formation of
This uronic acid content appears to be significantly high- a biofilm, which binds to fibronectin, fibrinogen, laminin,
er than previously reported,25 and this can probably be and sphinganine.15,16,21,22 Multiple agents specifically
attributed to improved techniques for the separation inhibiting each type of receptor adhesions, or a single
and calculation of neutral and acidic polysaccharides. agent exhibiting a broad spectrum antiadhesion activity,
Acidic polysaccharides obtained from plant sources have could be considered as candidates for the development of
been shown to exhibit a variety of biological activities, an antiadhesive therapeutic modality.14,17 In this connec-
including immunostimulatory, antioxidant, antitumor, tion, PG-OF1, a partially hydrolyzed oligosaccharide
and antiviral properties.31–33 An acidic polysaccharide from PG-F2, may mimic the effects of the core carbohy-
with immunomodulating activity, which was obtained drate moiety in host–pathogen interactions, and should
from P. ginseng leaves, was found to consist of a highly prove useful in the development of new carbohydrate-
branched glycan structure, composed of arabinose, based antiadhesive agents with broad-spectrum activity.
galactose, rhamnose and galacturonic acid with a b-
(1!3)-linked galactan backbone.34 Glycosaminoglycan,
N-acetylneuraminic acid and glucuronic acid were all 4. Experimental
previously implicated in P. gingivalis-mediated cell adhe-
sion.13,14 Negatively charged polysaccharides, such as 4.1. Materials
dextran sulfate, also inhibit the adhesion of respiratory
pathogens, including P. aeruginosa, Burkholderia ceno- The ginseng root extract concentrates were kindly sup-
depacia, Burkholderia pseudomallei, L. pneumophila, plied by S&D Co. (Cheon-ahn, Korea). H. pylori (ATCC
J.-H. Lee et al. / Carbohydrate Research 341 (2006) 1154–1163 1161
43504 and 26695), P. gingivalis (ATCC 33277), A. actino- and grown in LB broth without antibiotics for 6 h at
mycetemcomitans (ATCC 29522), P. acnes (ATCC 6919), 37 C. All bacterial cell stocks were maintained in a
and L. acidophilus (ATCC 4356) cells were acquired from liquid nitrogen tank until required. L. acidophilus was
the Korean Culture Center of Microorganisms (KCCM) directly used from the stocks obtained from KCCM,
(Seoul, Korea). The E. coli strains, BL21(DE3) and without further growth.
BL21(DE3)pLysS, were purchased from Novagen (Mad-
ison, WI, USA). S. aureus, S. epidermidis, and human 4.3. Extraction and isolation of polysaccharide PG-F2
erythrocytes were obtained from Korea University
Hospital (Ansan, Korea). Pectinase (EC. 3.2.1.15, poly- The extract (20 g) from the roots of P. ginseng was pre-
[(1!4)-a-D -galacturonide]glycanohydrolase), a- and b- pared as described previously,23,37 which was dissolved
amylases (EC 3.2.1.1. and EC 3.2.1.2, respectively), in hot distilled water (200 mL). After centrifugation to
trypsin (EC 3.4.21.4), D -glucuronic acid, D -galacturonic remove insoluble materials, the supernatant was subse-
acid, trehalulose (a-D -glucosylpyranosyl-(1!1)-D -fructo- quently precipitated using abs EtOH (final, 70% concen-
furanose), palatinose (a-D -glucopyranosyl-(1!6)-D - tration) at 4 C. The precipitate was then dissolved in
fructofuranose), NaNO2, low-molecular-weight heparin PBS containing 10 mM MgCl2 and 1 mM CaCl2, and
(LMWH), citrus pectin, alginic acid sodium salt, ribonu- incubated for 3 h with RNase and DNase at 37 C.
clease A from bovine pancreas (RNase), deoxyribonucle- The enzyme reactions were stopped by heating at
ase I from bovine pancreas (DNase), bovine serum 100 C for 5 min. After centrifugation, cold ethanol
albumin (BSA), methyl-b-cyclodextrin (CD), and di- was added to the supernatant (final, 70% concentration).
methyl sulfoxide (DMSO) were obtained from Sigma– The precipitate was then dialyzed against 20 mM Tris–
Aldrich (St. Louis, USA). Sucrose octasulfate (SOS) HCl (pH 8.0), and applied to a Q-Sepharose fast-flow
was purchased from Toronto Research Chemicals (North column (2.5 · 10 cm) equilibrated with the same buffer.
York, ON, Canada). Carbazole, phenol, and H2SO4 were After extensive washing, the polysaccharide fractions
purchased from Junsei Chemicals (Tokyo, Japan). The were eluted with an increasing linear gradient of NaCl
GC internal standards, myo-inositol and mannonic acid (0–1 M). The fractions were detected at 226 nm and ana-
lactone, were also obtained from Sigma–Aldrich (St. lyzed by the phenol–sulfuric acid and carbazole assays
Louis, USA). at 490 nm and 525 nm, respectively. The polysaccharide
fractions eluted at 0.2–0.3 M NaCl that exhibited anti-
4.2. Bacterial cell growth adhesive activity were then pooled, dialyzed against
distilled water, and lyophilized. It was dissolved in a
The H. pylori cells were grown in Brucella broth (Difco) minimum volume of 20 mM Tris–HCl buffer and
containing 10% (v/v) fetal bovine serum (FBS), 0.2% (w/v) applied to a Sephacryl S200 gel-filtration column
2,6-di-O-methyl-b-cyclodextrin (CD) and antibiotics (1.5 · 68 cm), which generated a major peak of polysac-
(cefsulodin, vancomycin, trimethoprim, and amphoteri- charide (PG-F2) and a minor peak of high-molecular-
cin B). The cells were incubated in a 10% CO2 atmo- weight polysaccharide (PG-HMW).
sphere at 37 C for 43 h. All stocks were maintained
with 20% (v/v) glycerol in a liquid nitrogen tank until 4.4. Partial hydrolysis of polysaccharide by enzyme
use. The H. pylori cells were quickly thawed and grown treatment
in Brucella broth containing 10% (v/v) FBS, CD, and
antibiotics at 37 C in a shaker for 43 h under a micro- In order to gain deeper insight into the structural and
aerobic environment afforded by BBL Campypak-Plus chemical characteristics of the functional moiety of the
envelopes (Cockeyville, USA). The cultured bacteria highly antiadhesive PG-F2, we attempted its partial
were then identified via urease and catalase reactions, hydrolysis using enzymatic and chemical methods. Par-
and the identifications were confirmed via polymerase tial HNO2 cleavage was conducted as previously de-
chain reactions based on VagA, Cag1, IceA1, IceA2, scribed38 with some slight modifications. PG-F2 was
BabA, and UreA primers (data not shown).25 P. gingiva- dissolved in a 2% HOAc solution and NaNO2 was
lis and A. actinomycetemcomitans were grown for 2 days added. After incubation at room temperature for 3 h,
in trypticase soy broth (Difco) supplemented with 0.6% it was neutralized with 1 N NaOH and purified by gel-
yeast extract, hemin (10 mL/L) and vitamin K3 (0.2 mL/L) filtration chromatography over a Superdex peptide col-
(for P. gingivalis) at 37 C under conditions of 80% N2, umn (bed volume, 24 mL). The oligosaccharide frag-
10% H2 and 10% CO2. P. acnes were grown for 2 days in ments were also acquired via the partial hydrolysis of
brain–heart infusion (Difco) at 37 C in an atmosphere PG-F2 with enzyme treatment using either pectinase
of 80% N2, 10% H2 and 10% CO2. S. aureus and S. epi- (final activity, 0.5 units/mg polysaccharide) in 20 mM
dermidis were grown aerobically in brain–heart infusion NaOAc (pH 5.0) for 30 min,23 or a- and b-amylase for
at 37 C. E. coli was aerobically cultured on Luria– 0.5–1 h. The hydrolyzed oligosaccharides with signifi-
Bertani (LB) agar plates. The colony was inoculated cant antiadhesive activities were pooled and subjected
1162 J.-H. Lee et al. / Carbohydrate Research 341 (2006) 1154–1163
incubation for 15 min. The percentage of attached 14. Ofek, I.; Sharon, N. Curr. Top. Microbiol. Immunol. 1990,
H. pylori cells was calculated as follows: attached % = 151, 91–113.
15. Ofek, I.; Hasty, D. L.; Sharon, N. FEMS Immunol. Med.
100 [(ODexperimental ODnegative)/(ODpositive ODnegative) ·
Microbiol. 2003, 38, 181–191.
100]. The negative controls contained only epithelial 16. Mack, D.; Bartscht, K.; Dobinsky, S.; Horstkotte, M. A.;
monolayers without bacteria. The positive controls con- Kiel, K.; Knobolch, J. K. M.; Schafer, P. Staphylococcal
tained bacteria and monolayers without added inhibitor, Factors Involved in Adhesion and Biofilm Formation of
and these were used to establish 100% attachment. Each Biomaterials. In Handbook of Bacterial Adhesion—Princi-
ples, Methods, and Applications; An, Y. H., Friedman, R.
experiment was conducted in triplicates on different
J., Eds.; Human Press: Totowa, 2000; pp 307–322.
days. 17. Mahdavi, J.; Sonden, B.; Hurtig, M.; Olfat, F.; Forsberg,
L.; Roche, N.; Angstrom, J.; Larsson, T.; Teneberg, S.;
Karlsson, K. A.; Altraja, S.; Wadstrom, T.; Kersulyte, D.;
Acknowledgements Berg, D. E.; Dubois, A.; Petersson, C.; Magnusson, K.-E.;
Norberg, T.; Lindh, F.; Lundskog, B. B.; Arnqvist, A.;
Hammarstrom, L.; Boren, T. Science 2002, 297, 573–578.
We wish to thank Mr. K. M. Yeo at S&D Co. for sup-
18. Ilver, D.; Arnqvist, A.; Ogren, J.; Frick, I.-M.; Kersulyte,
plying the P. ginseng extract samples. This work was D.; Incecik, E. T.; Berg, D. E.; Covacci, A.; Engstrand, L.;
supported by a grant from the BioGreen 21 Program Boren, T. Science 1998, 279, 373–377.
(2005-0301034369). J.L. was supported by the BK21 19. Agnani, G.; Tricot-Doleux, S.; Houalet, S.; Bonnaure-
Program of the Ministry of Education, Korea. Mallet, M. Infect. Immun. 2003, 71, 991–996.
20. Wadstrom, T.; Ljungh, A. J. Med. Microbiol. 1999, 48,
223–233.
21. Alugupalli, K. R.; Kalfas, S. Acta. Pathol. Microbiol.
Supplementary data Immunol. Scand. 1995, 103, 154–160.
22. Masako, K.; Hidyuki, I.; Shigeyuki, O.; Zenro, I. J.
Supplementary data associated with this article can be Dermatol. Sci. 2005, 38, 197–205.
found, in the online version, at doi:10.1016/ 23. Belogortseva, N. I.; Yoon, J. Y.; Kim, K. H. Planta Med.
2000, 66, 217–220.
j.carres.2006.03.032.
24. Woo, J. S.; Ha, B. H.; Kim, T. G.; Lim, Y.; Kim, K. H.
Biotechnol. Lett. 2001, 23, 507–511.
25. Lee, J.-H.; Park, E. K.; Uhm, C. S.; Chung, M.-S.; Kim,
References K. H. Planta Med. 2004, 70, 615–619.
26. Lee, J.-H.; Lee, J. S.; Chung, M.-S.; Kim, K. H. Planta
1. Tannock, G. W. The Normal Microflora: an Introduction. Med. 2004, 70, 566–569.
In Medical Importance of the Normal Microflora; Tan- 27. Simon, P. M.; Goode, P. L.; Mobasseri, A.; Zope, D.
nock, G. W., Ed.; Kluwer Academic: Dordrecht, 1999; pp Infect. Immun. 1997, 65, 750–757.
1–23. 28. Paton, A. W.; Voss, E.; Manning, P. A.; Paton, J. C.
2. Casadevall, A.; Pirofski, L.-A. Infect. Immun. 2000, 68, Infect. Immun. 1997, 65, 3799–3805.
6511–6518. 29. Patti, J. M.; Allen, B. L.; McGavin, M. J.; Hook, M.
3. Buck, G. E. Clin. Microbiol. Rev. 1990, 3, 1–12. Annu. Rev. Microbiol. 1994, 48, 585–617.
4. Goodwin, C. S. Lancet 1988, 2, 1467–1469. 30. Vuong, C.; Otto, M. Microbes Infect. 2002, 4, 481–489.
5. Talley, N. J.; Zinsmeister, A. R.; Weaver, A.; DiMagno, 31. Gao, Q.-P.; Kiyohara, H.; Cyong, J.-C.; Yamada, H.
E. P.; Carpenter, H. A.; Pérez-Pérez, G. I.; Blaser, M. J. Plant Med. 1989, 55, 9–12.
J. Natl. Cancer Inst. 1991, 83, 1734–1739. 32. Shin, K.-S.; Kiyohara, H.; Matsumoto, T.; Yamada, H.
6. Hussell, T.; Issacson, P. G.; Crabtree, J. E.; Spencer, J. Carbohydr. Res. 1997, 300, 239–249.
Lancet 1993, 342, 571–574. 33. Nangia-Marker, P.; Conklin, J.; Hogan, V.; Raz, A.
7. Haffajee, A. D.; Socransky, S. S. Periodontology 2000, 5, Trends Mol. Med. 2002, 8, 187–194.
78–111. 34. Tomoda, M.; Hirabayashi, K.; Shimizu, N.; Gonda, R.;
8. Meyer, D. H.; Sreenivasan, P. K.; Fives-Taylor, P. M. Ohara, N. Biol. Pharm. Bull. 1994, 17, 1287–1291.
Infect. Immun. 1991, 59, 2719–2726. 35. Thomas, R.; Brooks, T. J. Med. Microbiol. 2004, 53, 833–
9. Holland, K. T.; Aldana, O.; Bojar, R. A.; Cunliffe, W. J.; 840.
Eady, E. A.; Holland, D. B.; Ingham, E.; McGeown, C.; 36. Zambon, J. J. J. Clin. Periodontol. 1985, 12, 1–20.
Till, A.; Walters, C. Dermatology 1998, 196, 67–68. 37. Steinmetz, I.; Rohde, M.; Brenneke, B. Infect. Immun.
10. Projan, S. J.; Novick, R. P. The Molecular Basis of 1995, 63, 3959–3965.
Pathogenicity. In The Staphylococci in Human Disease; 38. Turnbull, J. E.; Hopwood, J. J.; Gallangher, J. T. Proc.
Crossley, K. B., Archer, G. L., Eds.; Churchill Living- Natl. Acad. Sci. U.S.A. 1999, 96, 2698–2703.
stone: New York, 1997; pp 55–82. 39. Chaplin, M. F. Monosaccharides. In Carbohydrate Anal-
11. Worst, D. J.; Otto, B. R.; de Graaff, J. Infect. Immun. ysis; Chaplin, M. F., Kennedy, J. F., Eds.; IRL Press:
1995, 63, 4161–4165. Oxford, 1986; pp 1–36.
12. Nakayama, K.; Ratnayake, D. B.; Tsukuba, T.; Kado- 40. Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.
waki, T.; Yamamoto, K.; Fujimura, S. Mol. Microbiol. 41. Merkle, R. K.; Poppe, I. Methods Enzymol. 1994, 230,
1998, 27, 51–61. 1–14.
13. Shi, Y.; Ratnayake, D. B.; Okamoto, K.; Abe, N.; 42. Jones, T. M.; Albersheim, P. Plant Physiol. 1972, 49, 926–
Yamamoto, K.; Nakayama, K. J. Biol. Chem. 1999, 274, 936.
17955–17960. 43. Molin, G. Am. J. Clin. Nutr. 2001, 73, 380S–385S.