Chinese Medicine, 2011, 2, 37-42
doi:10.4236/cm.2011.22007 Published Online June 2011 (htt p:// l/cm)
Copyright © 2011 SciRes. CM
Antimicrobial Activity of Traditional Chinese
Medicines on Common Oral Bacteria
Michelle K. Z. Yu en1, Ricky W. K. Wong1, Urban Hägg1, Lakshman Samaranayake2
1Orthodontics, Faculty of Dentistry, The University of Hong Kong, Hong Kong, China
2Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong, China
Received January 24, 2011; revised March 8, 2011; accepted March 15, 2011
Objective: To evaluate twenty Traditional Chinese Medicines (TCM) against four oral bacteria. Methods:
Twenty TCM were tested for sensitivity against Streptococcus mitis, Streptococcus sanguis, Streptococcus
mutans and Porphyromonas gingivalis. Aliquots of suspension of each bacterial species wer e i nocu lated on a
horse blood agar (HBA) plate, 6 mm diameter paper disks was soaked in different drug suspensions were
placed concentrically on a HBA plate. Disks soaked in 0.2% w/v chlorhexidine were used as positive con-
trols. These HBA pl ates were incubated for 48 hours anaerobically and the diameters of growth inhibition of
three different ar eas were mea sured u sing a cali brat ed co mputer s o ftware an d t he mean dia meter o b tain ed fo r
each bacteria. Broth microdilution assay was used to determine minimum inhibitory concentration (MIC)
and minimum bactericidal concentration (MBC). The experiment was repeated on three separate occasions.
Results: The TCMs that consistently against Porphyromonas gingivalis, included Folium artemisiae argyi,
Fructus crataegi, Rhizoma dryopteris crassirhizomae, Flos magnoliae, Rhizoma polygoni cuspidati, Radix
scrophulariae ningpoensis, Galla chinensis, Radix scutellariae baicalensis and Rhizoma coptidis; against
Streptococcus mutans included Fructus crataegi, Galla chinensis and Rhizoma copitidis; against Strepto-
coccus mitis and Streptococcus sanguis included Galla chinensis and Rhizoma copitidis. Conclusion: Rhi-
zoma copitidis and Galla chinensis had inhibitory effects on Streptococcus mitis, Streptococcus sanguis,
Streptococcus mutans and Porphyromonas gingivalis in vitro.
Keywords: Chinese Medicine, Antimicrobial Activity, Streptococcus mutans, Streptococcus sanguis,
Streptococcus mitis, Porphyromonas gingivalis, Oral Biofilm
1. Introduction
Dental caries is a common human disease that affects a
vast majority of people. It is a chronic endogenous infec-
tion caused by the normal oral commensal flora [1]. Oral
biofilm develop on all natural or artificial shedding and
non-shedding surfaces of the oral cavity. Microorganisms
in oral biofilm are the major aetiological agents of dental
caries. Other than caries, oral biofilm can cause many oral
infections including periodontal disease and candidiasis.
Plaque formation involves initial colonization and multi-
plication by pioneer species, followed by secondary colo-
nization by other species and finally becoming a climax
community [2]. The pioneer speciesof oral biofilm are
Streptococcus oralis, Streptococcus mitis and Streptococ-
cus sanguis. There are also specific bacteria that are closely
related to specific dental diseases, for example, Str e pto-
coccus mutans and Porphyromonas gingivalis are associ-
ated with dental caries and pe riodontal disease, respectively
Traditional Chinese Medicines (TCM) has been used
in China to treat various infectious diseases for more
than four thousand years. Different from the western-
medicine, TCM works as a formula of herbs that is tai-
lored to individual patient under their specific condition.
They are designed in the form of remedy that uses one
or two main ingredients that target the illness, with many
other ingredients also added to adjust the formula to suit
patient’s condition [3]. Recently the mechanism of one
of these formulae has been investigated at molecular,
cellular and organism levels [4]. TCM possess a variety
of biological properties that has the potential to be de-
Copyright © 2011 SciRes. CM
veloped as effective drugs. Certain TCM have been
shown to have antibacterial properties and so far none
has shown to have any known resistance. Currently, a
number of TCMs has already been used in oral health-
care products such as toothpaste according to their ef-
fects. Yet few studies have been performed to screen
these TCM and evaluate their effectiveness against oral
bacteria forming oral biofilm.
The twenty TCMs are selected according to their anti-
bacterial properties that enable them to treat infection
and disease at different part of body. The aim of this
study was to evaluate in vitro twenty TCMs that ar e cur-
rently used to treat infectious diseases; for their antim-
icrobial activity against oral biofilm bacteria and caries
and periodontal disease. The hypothesis is to investigate
the TCMs investigated in the study have effect against
the four common oral bacteria.
2. Methods
2.1. Organism and Culture Condition
Frozen isolates of type cultures of Streptococcus mitis
(ATCC 15914), Streptococcus sanguis (ATCC 10556),
Streptococcus mutans (ATCC 35668) and Porphyromo-
nas gingivalis (ATCC 33277) were thawed and their
identity reconfirmed using standard methodology. They
were then inoculated onto horse blood agar (HBA) and
incubated anaerobically at 37˚C for 3 days. For sensitiv-
ity studies the bacterial cultures were suspended in
phosphate buffered saline at a concentration of 1 × 106
cells/mL (0.5 MacFarland Standard Units).
2.2. Identification and Preparation of TCM
The twenty TCM were purchased from a local Chinese
Medicine store and were identified morphologically,
histologically and chemically using standard Chinese
herbal identification procedures [5].
Aqueous extracts of TCM were prepared using stan-
dard protocol [5]. Briefly, 4mL distilled water was added
to 10 g of TCM powder. The mixture was boiled with
constant stirring for 4 hours with occasional adding of
distilled water to prevent drying. Distilled water was
added at the end to make up the volume of the mixture to
4 mL. The mixture was cooled, centrifuged and filtered.
This pro duc ed 2.5 g/mL of one TCM extract.
The twenty TCM chosen for the study were Rhizoma
coptidis, Radix arnebia, Herba artemisiae, Flos magno-
liae, Radix bupleuri, Galla chinensis, Folium artemisiae
argyi, Radix scrophulariae ningpoensis, Radix scutel-
lariae baicalensis, Rhizoma polygoni cuspidati, Folium
isatidis, Fructus crataegi, Herba patriniae cum radice,
Rhizoma dryopteris crassirhizomae, Spica prunellae
vulgaris, Radix sophorae, Fructus gardeniae jasminoidis,
Anemarrhena aspodeloidea Bunge, Cortex fraxini and
Tarxacum mongolicum. Chlorhexidine gluconate, a
common oral antiseptic, at a concentration of 0.2% w/v
was used as a positive control for all experiments.
2.3. Agar Diffusion Assay
The standard agar diffusion assay for sensitivity testing
was performed according to a standard protocol [1]. 20
μL aliquots of suspension of each bacterial species were
inoculated on horse blood agar (HBA) plates using glass
rods, then 6 mm diameter paper disks soaked in 10 μL of
each of the 2.5 g/mL TCM extract were placed concentr-
ically on the HBA plate. Positive controls were disks
soaked in 10 μL of 0.2% w/v chlorhexidine placed in the
HBA plates. These HBA plates were incubated anaerob-
ically for 48 hours at 37˚C. After that, measurements of
any growth inhibition zone were evaluated using a cali-
brated computer software (Image J 1.40 for Windows,
NIH Image, Maryland, USA). The diameters of growth
inhibition of three different directions were measured
and the mean diameter of growth inhibition was calcu-
lated for each organism. The experiment was repeated on
thre e separate occasions. In cases wh ere no clear bacteria
growth inhibition could be seen but the bacteria surface
appearance changed, partial inhibitory effect was rec-
2.4. Broth Microdilution Susceptibility Test
The TCMs, Rhizoma coptidis and Galla chinensis, which
showed potent antimicrobial activity against the four
tested bacteria in the agar diffusion assay-screening test,
were selected for minimum inhibitory concentration
(MIC) determination using the standard broth microdilu-
tion assay [6]. Inocula of 24 hour s bacteria cultures were
standardized to a turbidity equivalent of 0.5 McFarland
standard at 520 nm with a spectrophotometer. The sus-
pensions were further diluted in Rosewell Park Memorial
Institute (RPMI) 1640 medium (Life technologies, New
York, USA) to yield an inoculum concentration of ap-
proximately 1 × 104 CFU mL–1. MIC assay was per-
formed in 96-well round-bottomed microtiter plates
(Iwaki, Tokyo, Japan) and each of the bacteria was ex-
posed to a double dilution of each of TCM agents. The
plates were covered with a lid and incubated for 72 hours
at 37˚C in an anaerobic chamber to evaluate the growth
kinetics. The MIC of each TCM drug was defined as the
lowest concentration that prevents visible tur bidity of the
broth. Same procedure had also been carried out on a
Copyright © 2011 SciRes. CM
horseblood agar plate to evaluate the minimal bacter icid-
al concentration (MBC) which classified as the lowest
concentration of drug that kills at least 99.9% of the
CFUs contained in the original inoculums. Experiments
were repeated on three different occasions with duplicate
determinations on each occasion.
2.5. Statistic Analysis
Data were analysed with a statistical analysis computer
software (SPSS 15.0 for Windows©, C hica go, USA). Data
were performed with one-way ANOVA to compare the
effects of different TCMs. Differences of pair-wise
comparison of each TCM with the positive control were
considered significant when the p value was less than
0.05. All TCMs that were showed effective against indi-
vidual bacteria in this experiment proved to be signifi-
cant except when Folium artemisiae argyi was tested
against P . gingivalis (Table 1).
3. Results
3.1. Initial Biofilm forming Bacteria (Strepto-
coccus mitis, Streptococcus sanguis)
The mean of inhibition zones of chlorhexidine measured
were 7.4 mm against Streptococcus mitis compared to
Galla chinensis and Rhizoma copitidis, which were of
5.8 mm and 6.2 mm respectively (Table 1, Figures 1
and 2). The average mean of inhibition zone measured in
diameter of chlorhexidine was 9.1 mm against Strepto-
coccus sanguis, where as Galla chinensis and Rhizoma
copitidis were measured as 10.6 mm and 7.6 mm. Rhi-
zoma copitidis demonstrated a comparable effect to
chlorhexidine against Streptococcus mitis, while Galla
chinensis showed sign of stronger effect than the chlor-
hexidine (9.1 mm) against Streptococcus sanguis.
3.2. Caries Causing Bacteria (Streptococcus
The mean of inhibition zones of chlorhexidine measured
were 12.1 mm against Streptococcus mutans. Three out
of twenty TCM extracts tested demonstrated consistent
antimicrobial activities with zones of growth inhibition
ranging from 5 mm to 12 mm aga inst Streptococcus mu-
tans. The average mean of inhibition zones measured in
diameter of Fructus crataegi, Galla chinensis and Rhi-
zoma copitidis to Streptococcus mutans, are 5.4 mm, 9.6
mm and 11.4 mm respectively (Table 1, Figure 3).
When compared to the positive control, chlorhexidine,
which is a highly potent antibacterial agent, Rhizoma
copitidis demonstrated a similar effect to chlorhexidine
(12.1 m m).
Table 1. Table showing antimicrobial property of TCMs.
S. mutans S. mitis S.
sanguis P.
Cortex fraxini - - - P
Flos magnoliae - - -
Folium artemisiae
argyi - - - 7.2
Folium isatidis - - - P
Fructus crataegi
- P
Fructus gardeniae
jasminoidis - - - -
Galla chinensis
Herba artemisiae - - - P
Herba patriniae
cum radice
- - - -
Herba taraxaci
mongolici cum
radice - - P P
Radix arnebia - - - P
Radix bupleuri - - - -
Radix scrophulariae
- - P
Radix scutellariae
baicalensis - - P 25.3
Radix sophorae - - P -
Rhizoma anemarrhenae - - - -
Rhizoma coptidis 11.4
0)* 6.2
0.2)* 7.6
0)* 56.3
Rhizoma dryopter is
crassirhizomae - - - 9.2
Rhizoma polygoni
cuspidati - - - 12.1
Spica prunellae vulgaris - - - P
*“P” stands for partial inhibitory effect; *“-” indicates for no inhibitory
effect ; *Values in p arenthesis in dicate standard deviation; *“*” indicate signifi-
cant differences (p < 0.05)
Galla ChinensisRhizoma Coptidis Control
TCM tested
Inhibition zone (mm)
Figure 1. Sensitivity of S. mitis to 2 out of 20 TCM tested
shown in mean value.
Copyright © 2011 SciRes. CM
S. sanguis
Galla ChinensisRhizoma Coptidis Control
TCM tested
Inhibition zone (mm)
Figure 2. Sensitivity of S. sanguis to 2 out of TCM tested
shown in mean value.
S. mutans
Fructus CrataegiGalla ChinensisRhizoma Coptidis Cont rol
TCM tested
Inhibition zone (mm)
Figure 3. Sensitivity of S. mutans to 3 out of 20 TCM tested
shown in mean value.
3.3. Periodontal Disease Causing Bacteria
(Porphyromonas Gingivalis)
Chlorhexidine was measured to have 23.7 mm in average
mean against Porphyromonas gingivalis. Nine of the
twenty TCM extracts also demonstrated consistent antim-
icrobial activity with zones of growth inhibition ranging
from 7 mm to 57 mm against Porphyromonas gingivalis.
The sequences of drug in ascending orders of effectiveness
towards Porphyromonas gingivalis were Folium artemisiae
argyi (7.2 mm), Fructus crataegi (7.3 mm), Rhizoma
dryopteris crassirhizomae (9.2 mm), Flos magnoliae (9.6
mm), Rhizoma polygoni cuspidati (12.1 mm), Radix scro-
phulariae ningpoensis (19.4 mm), Galla chinensis (20.2
mm), Radix scutellariae baicalensis (25.3 mm) and Rhi-
zoma coptidis (56.3 mm). Galla chinensis (20.2 mm) and
Radix scutellariae baicalensis (25.3 mm) demonstrated
comparable effective to chlorhexidine (28.7 mm), while
Rhizoma Coptidis shows a significant antimicrobial effect
compares with chlorhexidine against Porphyromonas gin-
givalis (Table 1, Figure 4).
3.4. Partial Effects
For the remaining TCMs tested, Fructus crataegi, Radix
scutellariae baicalensis, Radix scrophulariae ningpoen-
sis, Radix sophorae and Herba taraxaci mongolici cum
radice showed weak antimicrobial effects against Strep-
tococcus sanguis. While Cortex fraxini, Herba artemi-
siae. Folium isatidis, Spica prunellae vulgaris, Radix
arnebia and Herba taraxaci mongolici cum radice dem-
onstrated weak antimicrobial effects against Porphyro-
monas gingivalis.
3.5. Minimum Inhibitory Concentrations
The MIC and MBC values for Rhizoma Coptidis against
S. mutans were 0.039 g/mL and 0.156 g/mL respectively.
It stated as 0.020 g/mL and 0.156 g/mL while against S.
mitis. And 0.039 g/mL for both MIC and MBC values
when against S. sanguis (Tables 2 and 3).
Galla chinensis when hold against S. mutans had the
MIC value as 0.0391 g/mL and 0.078 g/mL as MBC
value. For S. mitis, the MIC value was 0.010 g/mL and
MBC value was 0.078 g/mL. Both MIC and MBC v alues
were the same when Galla chinensis against S. sanguis,
which is 0.039 g/mL (Tables 2 and 3).
P. gingivalis
01020 3040 5060 70
Radix Scrophulariae Ningpoensis
Rhizoma Coptidis
Radix Scutellariae Baicalensis
Rhizoma Polygoni Cuspidati
Galla Chinensis
Flos Magnoliae
Rhizoma Dryopteris crassirhizomae
Fructus Crataegi
Artemisia Argyi
TCM tested
Inhibition zone (mm)
Figure 4. Sensitivity of P. gingivalis to 9 out of 20 TCM
tested show n in mean value.
Table 2. MIC of Rhizoma copiditis and Galla chinensis
against oral micro-organisms.
Rhizoma copiditis
Galla chinensis
S. mutans
S. mitis
S. sanguis 0.039 0.039
P. gingivalis
Copyright © 2011 SciRes. CM
Table 3. MBC of Rhizoma copiditis and Galla chinensis
against oral micro-organisms.
Rhizoma copiditis
Galla chinensis
S. mutans 0.156 0.078
S. mitis 0.156 0.078
S. sanguis 0.039 0.039
P. gingivalis <0.001 <0.001
Both MIC and MBC for Rhizoma Coptidis and Galla
chinensis against P. gingivalis were below <0.001 g/mL,
which reached the lowest boundary for both MIC and
MBC (Tables 2 and 3).
4. Discussion
In this study, twenty TCM extracts were evaluated for
their antimicrobial activities against four common bacte-
rial species presented in oral cavity which had been con-
sidered as important in biofilm formation (Streptococcus
mitis, Streptococcus sanguis), or causing dental caries
(Streptococcus mutans) or causing periodontal disease
(Porphyromonas gingivalis).
Chlorhexidine was showed to be effective against all
four tested bacteria. Of these, two TCM extracts, Rhi-
zoma copiditis and Galla chinensis were shown to have
similar effects. Although the first one has gained much
attention due to its wide range of antimicrobial activities
[5,7-9], ours is the first to show their effects specifically
on oral bacteria. It has shown antimicrobial activity
against periodontopathogenic bacteria including Por-
phyromonas gingivalis, Prevotella intermedia, Prevo-
tella nigrescens and Actinobacillus actinomycetemcomi-
tans Actinomyces naeslundii, but had less inhibitory ef-
fect on the growth of Streptococcus and Lactobacillus
[5]. One active component, berberine alkaloid has been
shown to have potent activ ity against all tested strains of
methicillin-resistant Staphylococcus aureus (MRSA) [7].
Other studies demonstrated the antibacterial effect of
Rhizoma copiditis and its alkaloids against Propionibac-
terium and Helicobacter pylori [8,9]. Results of the Con-
trol Group were published in a contemporary study eva-
luating another group of TCMs [10].
Results showed that both Rhizoma copiditis and Galla
chinensis have comparable effects with chlorhexidine.
When comparing the two TCMs, both Galla chinensis and
Rhizoma Coptidis demonstrated the same MBC values
against S. sanguis (Tables 2 and 3). Galla chinensis
showed a lower MBC value a ga inst S. mitis and S. sanguis
compared to Rhizoma Coptidis. This demonstrated that
Galla chinensis has a higher bacteriocidal activity than
Rhizoma Coptidis against the two bacteria. For P. gin-
givalis, both TCMs reached the lowest concentration
boundary for the MIC and MBC values, further investiga-
tions are needed to determine the exact MIC and MBC
values for both TCMs. This study is the first study that
investigates TCMs specifically on oral bacteria. Therefor e
these TCMs are p romising agents that can develop to new
antibacter ia ls f or or al micro -organ isms. Fu rt her research is
needed to identify the specific active components that are
related to the antibacterial action, to determine the range
of action, and to investigate the mechanisms involved.
5. Conclusions
Both Rhizoma copitidis and Galla chinensis had inhibi-
tory effects on Streptococcus mitis, Streptococcus san-
guis, Streptococcus mutans and Porphyromonas gin-
givalis in vitro.
6. Acknowledgements
We thank Ms Joyce Yau on her technical assistance. This
study was supported by the University Research Grant
No.: 10207346.15633.08003.323.01, The University of
Hong Kong.
7. Conflict of Interest Statement
The authors declare that there are no potential conflicts
exist between the authors and the products mentioned in
the paper.
8. References
[1] L. P. Samaranayake, “Essential Microbiology for den-
tistry,” Churchill Livingstone, Churchill, 2006.
[2] T. M. Auschill and N. Hein, “Effect of Two Antimicro-
bial Agents on early in Situ Biofilm Formation,” Journal
of Clinical Periodontology, Vol. 32, No. 2, February
2005, pp. 147-152.
[3] D. M. Eisenberg, “Trends in Alternative Me dicine Use in
the United States,” Journal of the American Medical As-
sociation, Vol. 280, No. 18, 1998, pp. 1569-1575.
[4] L. Wang, G. B. Zhou, P. Liu, J. H. Song, Y. Liang, X. J.
Uan, F. Xu, B. S. Wang, J. H. Mao, Z. X. Shen, S. J.
Chen and Z. Chen, “Dissection of Mechanisms of Chi-
nese Medicinal Formula Realgar-Indigo Naturalis as an
Effective Treatment for Promyelocytic Leukemic,” Pro-
ceedings of the National Academy of Sciences, Vol. 105,
No. 12, March 2008, pp. 4826-4831.
[5] J. P. Hu, N. Takahashi and T. Yamada, “Coptidis Rhi-
zoma Inhibits Growth and Proteases of Oral Bacteria,”
Oral Disease, Vol. 6, No. 5, September 2000, pp. 297-
302. doi:10.1111/j.1601-0825.2000.tb00142.x
[6] M. Levison, “Antibacterial Therapy,” In: Vitro Testing,
Copyright © 2011 SciRes. CM
Pharmaco-Dynamics, Pharmacology, New Agents, Infec-
tious Disease Clini cs of North America, Vol. 9, 1995, pp.
[7] H. H. Yu, K. J. Kim, J. D. C h a, H. K . Kim , Y. E. Lee, N. Y.
Choi and Y. O. You, “Antimicrobial Activity of Berberine
Alone and in C om bination with Ampicillin or Oxacillin
against Methicillin-Resistant Staphylococcus aureus,”
Journal of Medical Food, Vol. 8, No. 4, December 2005,
pp. 454-461. doi:10.1089/jmf.2005.8.454
[8] E. A. Bae, M. J. Han, N. J. Kim and D. H. Kim, “Anti-
Helicobacter Pylori Activity of Herbal Medicines,”
Biological Pharmarcy Bulletin, Vol. 21, No. 9, Sep-
tember 1998, pp. 990-992.
[9] S. Higaki, M. Nakamura, M. Morohashi, Y. Hasegawa
and T. Yamagishi, “Anti-Lipase Activity of Kampo For-
mulations, Coptidis Rhizoma and Its Alkaloids against
Propionibacterium Acnes,” Journal of Dermatology, Vol.
23, No. 5, 1996, pp. 310-314.
[10] R. W. K. Wong, U. Hagg, L Samaranayake, M. K. Z.
Yuen, C. J. Seneviratne and R. Kao, “Antimicrobial Ac-
tivity of Chinese Medicine Herbs against Common Bac-
teria in Oral Biofilm: A Pilot Study,” International Jour-
nal of Oral and Maxillofacial Surgery, Vol. 39, No. 6,
2010, pp. 599-605. doi:10.1016/j.ijom.2010.02.024