Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2012, Article ID 106583, 6 pages
Potent AntifungalActivity of Pure Compoundsfrom
TraditionalChineseMedicineExtracts against Six Oral
Candida Speciesand theSynergywithFluconazoleagainst
ZhiminYan,1Hong Hua,1YanyingXu,2and LakshmanP. Samaranayake3
1Department of Oral Medicine and Traditional Chinese Medicine, Peking University School and Hospital of Stomatology,
22 South Zhongguancun Street, Beijing 100081, China
2Department of Health Sciences, National Natural Science Foundation of China, 83 Shuangqing Road, Haidian Distract,
Beijing 100085, China
3Department of Oral BioSciences, Faculty of Dentistry, Prince Philip Dental Hospital, The University of Hong Kong,
34 Hospital Road, Hong Kong
Correspondence should be addressed to Yanying Xu, firstname.lastname@example.org and Lakshman P. Samaranayake, email@example.com
Received 20 August 2011; Revised 8 November 2011; Accepted 17 November 2011
Academic Editor: Jairo Kenupp Bastos
Copyright © 2012 Zhimin Yan et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This study was designed to evaluate the in vitro antifungal activities of four traditional Chinese medicine (TCM) extracts. The
inhibitory effects of pseudolaric acid B, gentiopicrin, rhein, and alion were assessed using standard disk diffusion and broth
microdilution assays. They were tested against six oral Candida species, Candida albicans, Candida glabrata, Candida tropicalis,
Candida krusei, Candida dubliniensis, and Candida guilliermondii, including clinical isolates from HIV-negative, HIV-positive,
and Sj¨ ogren’s syndrome patients. It was found that pseudolaric acid B had the most potent antifungal effect and showed similar
antifungal activity to all six Candida spp, and to isolates from HIV-negative, HIV-positive, and Sj¨ ogren’s syndrome patients. The
MIC values ranged from 16 to 128µg/mL. More interestingly, a synergistic effect of pseudolaric acid B in combination with
fluconazole was observed. We suggest that pseudolaric acid B might be a potential therapeutic fungicidal agent in treating oral
Candida species are commensal fungi that are found in 30–
50% of human oral cavities. However, under certain con-
ditions, commensal fungi can transform into opportunistic
pathogens that cause both superficial mucosal and systemic
mycoses. As the most common opportunistic infection, the
incidence of oral candidiasis has increased rapidly for several
decades due to the growing number of compromised hosts,
such as patients with organ transplants or HIV infection or
those undergoing chemotherapy or misusing antibiotics. In
particular, oral candidiasis has much greater prevalence in
immunocompromised individuals, such as patients with
[2, 3]. In addition, an important trend that can be detected
with the incidence of oral candidiasis is an increase in the
number of infections related to the non-albicans Candida
species such as Candida glabrata, Candida krusei, and Can-
dida tropicalis. This situation poses a clinical challenge
because some non-albicans Candida species are inherently
resistant to first-line antifungals such as fluconazole, espe-
cially in medically complex groups of patients [4, 5]. There-
fore, novel fungal therapies for effective management of
Candida infections are urgently required.
Research on traditional Chinese medicines (TCMs) or
their extracts is known to be one important means of devel-
oping new drugs. Although some TCMs have already been
demonstrated to possess activity against Candida albicans
[6–8], their true anticandidal properties against most non-
albicans Candida species remain unknown. In this study, we
2Evidence-Based Complementary and Alternative Medicine
evaluated the anticandidal effect of four active constituents
(pseudolaric acid B, gentiopicrin, rhein, and alion) extracted
from TCMs that have been used to treat infectious diseases
in China for thousands of years.
2.1. Organisms and Culture Conditions. Salivary specimens
were collected from various groups of patients with oral can-
imens were obtained from Peking University Department of
Oral Medicine. The study was approved by the Ethics Com-
mittee of Peking University Health Science Center.
Candida strains were isolated and stored in sterile water
at 4◦C and passaged two times on Sabouraud dextrose agar
(SDA) before use. The identity of Candida species was con-
firmed using standard methodology: yeast-like growing
colonies on SDA were routinely Gram-stained and, if found
to be yeast cells by microscopy, were identified with the stan-
dard carbohydrate assimilation tests and with the API 20C
AUX (bioM´ erieux, France) complemented with germ tube
test and growth assessment at 45◦C. The API 20C yeast iden-
tification system was inoculated with the samples, and the
results were interpreted by following the manufacturer’s
Six Candida species, Candida albicans (15 strains),
Candida glabrata (4 strains), Candida tropicalis (4 strains),
Candida krusei (4 strains), Candida dubliniensis (4 strains),
and Candida guilliermondii (4 strains), were isolated and in-
cluded in this study. C. albicans ATCC 90028 and C. krusei
ATCC 6258 were used as quality control strains in all sus-
ceptibility testing experiments.
2.2. Preparation of TCM Compounds Suspensions. A total
of four constituents of TCMs were obtained from the Na-
tional Institute for the Control of Pharmaceutical and
Biological Products of China: pseudolaric acid B (purity
99.1%), gentiopicrin, rhein, and alion (purity, ≥98%). The
powder was dissolved in DMSO or distilled water at the
appropriate concentrations. Fluconazole (kindly provided
by Pfizer Pharmaceuticals, Pfizer-Roerig, Groton, CT, USA,
purity, 99.7%) was dissolved in distilled water as a control.
All drug solutions were stored in sterile tubes at −70◦C and
were further diluted in the assay medium before use.
2.3. Antifungal Susceptibility Testing. Two standard methods
were employed to evaluate the fungicidal effect of TCM
2.4. Disk Diffusion Testing. All clinical strains were subcul-
tured on SDA overnight, and inocula were adjusted to 0.5
McFarland standards (1 × 106–5 × 106CFU/mL). Mueller-
Hinton agar supplemented with 2% glucose and 0.5µg/mL
were incubated automatically with a Spiral plater (Autoplate
4000; Spiral Biotech, Inc., Bethesda, MD, USA). Subse-
quently, filter paper discs (9mm in diameter) containing
25µg of preprepared drugs were placed on the agar plates
and incubated at 35◦C for 24–48 hours. Meanwhile, discs
containing 25µg fluconazole and solvent were included as
positive and negative controls, respectively. Afterwards, the
diameter of the inhibition zone was measured and zone
inhibition interpretive criteria for fluconazole disc testing
were based on NCCLS M44P recommended categories. The
isolates with zone diameters ≥ 19mm were reported as
sensitive to fluconazole, whereas those with diameters of
15–18mm and ≤14mm were reported to have intermediate
sensitivity and resistance to fluconazole, respectively .
Quality controls were performed with each batch of clinical
isolates by testing C. krusei ATCC 6258 and C. albicans ATCC
90028 with a recommended acceptable performance range.
The experiment was repeated on three separate occasions.
2.5. Broth Microdilution Susceptibility Testing. The TCM
extract that showed antifungal activity in the agar diffusion
assay was subsequently selected to determine the MIC. Broth
used to prepare the inoculum to a concentration equivalent
to 0.5 McFarland standard at 520nm. The suspensions were
further diluted in RPMI 1640 medium (Life Technologies,
New York, NY, USA) to yield an inoculum concentration
of approximately 104CFU/mL. The microdilution test was
performed in presterilized, polystyrene, flat-bottom, 96-
well microtiter plates, and each of the Candida species
was exposed to a double dilution of each TCM extract.
The microtiter plates were then incubated for 24 hours
at 35◦C, and the endpoints were read with a microtiter
plate reader (SpectraMAX 340 Tunable Microplate Reader;
Molecular Devices Ltd., Sunnyvale, CA, USA) at 520nm.
Testing of these isolates was performed in quadruplicate.
The MIC of each TCM drug was defined as the lowest
concentration at which there was 100% inhibition of yeast
growth. According to CLSI criteria 100% growth inhibition
is defined as clear wells. Therefore, the OD readings at MIC
value were similar to the negative control without Candida.
The MIC of fluconazole was determined for each species in
parallel as a control, and antibiotic-free solvent was included
as a negative control. MICs of fluconazole were determined
at 50% inhibition (MIC50). To ensure reliable results, the
MICs of the quality control strains C. krusei ATCC 6258 and
C. albicans ATCC 90028 were monitored within the reference
2.6. Interaction of Pseudolaric Acid B and Fluconazole In Vitro.
The interaction between pseudolaric acid B and fluconazole
against five resistant C. albicans strains was tested using a
microdilution checkerboard technique according to CLSI
(formerly NCCLS) document M27-A2 (2002). Each drug
wasseriallydiluted twofold in RPMI1640 medium. The final
drug concentrations ranged from 128 to 0.125µg/mL for
fluconazole and for pseudolaric acid B. Plates were incubated
at 35◦C for 24 hours. For pseudolaric acid B, the endpoint
was determined as the lowest concentration to produce
optically clear wells (no growth, MIC-0) or a cell count
Evidence-Based Complementary and Alternative Medicine3
Figure 1: Agar diffusion assay of effect of pseudolaric acid B on
C. albicans showing significant fungicidal activity. Note the circular
zone is clearer compared to that of Fluconazole. PSE: pseudolaric
acid B; FLZ: fluconazole; DMF: dimethylformamide; H2O: distilled
of ≤5% of the control well. For fluconazole, considering
that the trailing phenomenon often occurs, the MIC was
defined as the lowest concentration showing prominent 50%
growth inhibition compared with the growth control. For
the combination test, the MIC was defined as the lowest
concentration showing prominent 100% growth inhibition.
The fractional inhibitory concentration (FIC) for each drug
was calculated by dividing the MIC in the presence of the
second drug by the MIC in its absence, with the sum of
the two FICs giving the FIC index (FICI). FICI values were
interpreted as follows: FICI ≤ 0.5, synergy; 0.5 < FICI ≤ 4,
no interaction; FICI > 4, antagonism.
2.7. Statistical Analysis. Statistical analysis was performed
with SPSS for Windows version 10.0 (SPSS, Chicago, IL,
USA). Continuous variables were analyzed by ANOVA test.
All values were two tailed, and P < 0.05 was considered to
indicate statistical significance.
3.1. Disk Diffusion Assay. From the initial screening with
disk diffusion assay, only pseudolaric acid B exhibited
considerable anticandidal activity. It had zones of growth
inhibition ranging from 8 to 25mm against C. albicans,
C. glabrata, C. krusei, C. tropicalis, C. dubliniensis, and C.
parapsilosis (Figure 1). Gentiopicrin, rhein, and alion did not
exert anticandidal activity and had no inhibitory zone. Thus,
pseudolaric acid B had potent antifungal activity against all
six evaluated Candida species.
3.2. Microdilution Susceptibility Test. The M-27A broth
microdilution assay indicated that pseudolaric acid B had a
strong inhibitory effect on all six Candida species, and the
MIC ranged from 16 to 128µg/mL, with a mean of 40.58 ±
22.46µg/mL (geometric mean titer ± standard deviation)
similar level of antifungal activity against all the Candida
species tested compared with that of fluconazole. ANOVA
tests indicated no significant difference in MICs against
different Candida species (P = 0.58 > 0.05). Also of in-
terest were the clear, nontrailing endpoints for pseudolaric
acid B against all Candida strains. This might be evidence
that pseudolaric acid B is fungicidal, rather than fungistatic
against Candida species. We evaluated the antifungal effect
of pseudolaric acid B against C. albicans isolated from
various sources including patients with Sj¨ ogren’s syndrome
(5 strains), HIV-negative patients (5 strains), and HIV-
positive patients with oral candidiasis (5 strains) (Table 2).
tested (P = 0.34 > 0.05). More importantly, it demonstrated
a strong anticandidal effect against fluconazole-resistant
3.3. Interaction of Pseudolaric Acid B and Fluconazole. A
synergistic phenomenon was observed in the agar diffusion
assay (Figure 2), which was confirmed by the checkerboard
method. It was most pronounced after 24 hours incubation
and was sustained through 48 hours. The results for the
tested drug alone and in combination against the 14 isolates
are summarized in Table 3. Pseudolaric acid B had in vitro
antifungal activity against orally isolated Candida spp. More
importantly, FICI showed a synergism of pseudolaric acid B
and fluconazole against azole-resistant clinical isolates of
C. albicans, whereas there was no such reaction with other
In recent years, candidiasis has reemerged with higher pre-
valence and mortality rates that are nearly 45% among
compromised population groups . Moreover, clinicians
have encountered the new challenge of failure of treatment
with existing antifungal agents. This may be due to either
emergence of non-albicans species such as C. krusei and C.
glabrata, which are more resistant to commonly used anti-
agents for the efficient management of candidal infections.
Research on the active constituents of natural or traditional
medicines, which are a potential source for new drugs, is
drawing more attention. The aim of our study was to
which have been used for antifungal treatment for thousands
of years in China.
In the initial screening with the disc diffusion assay,
pseudolaric acid B showed considerable zones of growth in-
hibition. The remaining three TCM extracts, gentiopicrin,
rhein, and alion, did not exert a significant anticandidal
effect, although the plants (Gentian, Radix et Rhizoma Rhei,
and Aloe) from which they were isolated have been tradition-
ally used to treat fungal skin infections. Pseudolaric acid is
date, these natural products and their derivatives have been
reported to exhibit antifungal, antifertility, cytotoxic, and
4Evidence-Based Complementary and Alternative Medicine
Table 1: MIC of pseudolaric acid B against Candida species.
C. k. ATCC 6258
C. a ATCC 90028
MIC: minimum inhibitory concentration; MFC: minimum fungicidal concentration; PSE: pseudolaric acid B; FLZ: fluconazole.∗Data were demonstrated as
geometric mean titer ± SD.
32 ∼ 64
0.5 ∼ 1
Table 2: MIC of pseudolaric acid B against C. albicans strains isolated from different sources.
MIC: minimum inhibitory concentration; MFC: minimum fungicidal concentration; PSE: pseudolaric acid B; FLZ: fluconazole; SS: Sj¨ ogren’s syndrome.
Figure 2: Agar diffusion assay of effect of pseudolaric acid B and fluconazole on Candida albicans demonstrated synergistic antifungal
activity. Note that there is no microcolony growth in the cross-sectional area of pseudolaric acid B and fluconazole. PSE: pseudolaric acid B;
Evidence-Based Complementary and Alternative Medicine5
Table 3: In vitro interaction of pseudolaric acid B and fluconazole (1:1) against oral Candida spp.
C. albicans (M3)FLZ
C. krusei (K2)0.5–4 No interaction
C. tropicalis (T1) 0.5–4No interaction
C. dubliniensis (D4)0.5–4 No interaction
C. glabrata (gp)0.5–4No interaction
C. guilliermondii (gm3)0.5–4 No interaction
MIC: Minimum inhibitory concentration; FIC: fractional inhibitory concentration; FICI: fractional inhibitory concentration index; PSE: pseudolaric acid B;
FLZ: fluconazole; M, K2, T1, D4, gp, gm3: strain no.∗Concentration in micromolar.
antiangiogenic activities . Although tujingpi has been
used in China for the treatment of fungal skin infection since
the 17th century, recent, studies on TCM extracts have led to
the identification of the pseudolaric acids as the main
antifungal constituents, in which pseudolaric acid B is one of
the major antifungal components. Subsequent in vitro
studies have shown that pseudolaric acid B is active against
C. albicans, Torulopsis petrophilum, Trichophyton menta-
grophytes, and Microsporum gypseum [14, 15]. However,
the anticandidal effect of pseudolaric acid B against non-
Candida species has rarely been reported, and there have
been no reports of its activity against orally isolated Candida
species. The purpose of the present study was therefore to
investigate activity of pseudolaric acid B against oral Can-
dida, with an emphasis on non-albicans and Candida spp.
from different sources.
It is well known that fungal infection is much more
common in immunocompromised individuals, such as HIV-
are increasing reports on C. albicans that is resistant to anti-
fungal medications especially in HIV-infected hosts and
Sj¨ ogren’s syndrome patients who have undergone repeated
courses of antifungal therapy [4, 16]. Therefore, C. albicans
strains isolated from HIV-infected and Sj¨ ogren’s syndrome
patients were included as part of our study. We found that
pseudolaric acid B was effective against C. albicans isolated
from HIV-positive, HIV-negative, and Sj¨ ogren’s syndrome
patients, including both fluconazole-sensitive and -resistant
strains. This potent nonselective effect of pseudolaric acid B
implies that it has potential as a novel antifungal agent,
especially against clinically resistant infections.
More interestingly, pseudolaric acid B demonstrated ap-
proximately similar antifungal activity against C. albicans
and non-albicans Candida. This phenomenon is of great
importance, because susceptibility to antifungal medication
differs significantly among Candida spp. For example, C.
krusei has natural resistance to fluconazole, a standard anti-
fungal agent commonly used in the clinic. Another example
is C. glabrata, which possesses a low level of intrinsic resis-
tance to the azole drugs and fluconazole and ketoconazole.
The antifungal activity of pseudolaric acid B against different
Candidaspp. demonstrated in thepresentstudy mayprovide
a possible answer to the frustrating drug resistance and war-
rants further research.
In this study, the phenomenon of the interactions
between pseudolaric acid B and fluconazole was observed by
agar diffusion assay and determined qualitatively by checker-
board microdilution method. The FICI model is the most
commonly used approach to study the interaction between
antifungal drugs and has been used to interpret the data.
It was found that a combination of pseudolaric acid B and
fluconazole exhibited good synergism against oral azole-
resistant isolates of C. albicans, which has not been reported
fungal drug resistance in C. albicans follows from pro-
longed exposure to azole antifungals [17, 18]. In patients
with advanced AIDS, oral candidiasis continues to be a
common presenting illness associated with significant mor-
bidity. In recent years, oral fluconazole, given its low toxicity,
has become the most common form of treatment for symp-
tomatic oral candidiasis . The widespread use of flu-
conazole has, however, led to an increased incidence of
clinically resistant oral candidiasis due to infection with fluc-
onazole-resistant organisms. The finding of synergy in the
present study was significant because azole resistance is an
emerging issue and it is necessary to find an effective and
novel therapeutic method to overcome the problem of drug
In conclusion, the present study provided strong evidence of
the potent antifungal activity of pseudolaric acid B against a
6Evidence-Based Complementary and Alternative Medicine Download full-text
wide range of Candida species, and the synergistic effect of
pseudolaric acid B in combination with fluconazole. Further
studies are warranted to explore the mechanism behind the
antifungal activity of pseudolaric acid B.
The authors thank Professor C. J. Seneviratne and Ms. J. Y.
Yau from the University of Hong Kong for their kind help.
 F. J. Owotade, C. H. Shiboski, L. Poole et al., “Prevalence of
oral disease among adults with primary HIV infection,” Oral
Diseases, vol. 14, no. 6, pp. 497–499, 2008.
 N. L. Rhodus, C. Bloomquist, W. Liljemark, and J. Bereuter,
“Prevalence, density, and manifestations of oral Candida
ryngology, vol. 26, no. 5, pp. 300–305, 1997.
 L. Radfar, Y. Shea, S. H. Fischer et al., “Fungal load and can-
didiasis in Sj¨ ogren’s syndrome,” Oral Surgery, Oral Medicine,
Oral Pathology, Oral Radiology, and Endodontics, vol. 96, no. 3,
pp. 283–287, 2003.
 M. Ruhnke, A. Eigler, I. Tennagen, B. Geiseler, E. Engelmann,
and M. Trautmann, “Emergence of fluconazole-resistant
strains of Candida albicans in patients with recurrent oropha-
ryngeal candidosis and human immunodeficiency virus infec-
tion,” Journal of Clinical Microbiology, vol. 32, no. 9, pp. 2092–
 M. Ruhnke, A. Schmidt-Westhausen, and J. Morschh¨ auser,
“Development of simultaneous resistance to fluconazole in
Candida albicans and Candida dubliniensis in a patient with
AIDS,” Journal of Antimicrobial Chemotherapy, vol. 46, no. 2,
pp. 291–295, 2000.
 D. Pellati, C. Fiore, D. Armanini, M. Rassu, and G. Bertoloni,
“In vitro effects of glycyrrhetinic acid on the growth of clinical
isolates of Candida albicans,” Phytotherapy Research, vol. 23,
no. 4, pp. 572–574, 2009.
 M. Tomczykowa, M. Tomczyk, P. Jakoniuk, and E. Trynis-
zewska, “Antimicrobial and antifungal activities of the extracts
and essential oils of Bidens tripartita,” Folia Histochemica et
Cytobiologica, vol. 46, no. 3, pp. 389–393, 2008.
 C. Ferez and C. Suarez, “Antifungal activity of plant extracts
against Candida albicans,” American Journal of Chinese Med-
icine, vol. 25, no. 2, pp. 181–184, 1997.
 National Committee for Clinical Laboratory Standards,
“Method for antifungal disk diffusion susceptibility testing of
yeasts: approved guideline M44-A,” National Committee for
Clinical Laboratory Standards, Wayne, Pa, USA, 2004.
 National Committee for Clinical Laboratory Standards, “Ref-
erence method for broth dilution antifungal susceptibility
testing of yeasts. Approved standard M27-A2,” National Com-
mittee for Clinical Laboratory Standards, Wayne, Pa, USA,
 L. D. Liebowitz, H. R. Ashbee, E. G. V. Evans et al., “A two
year global evaluation of the susceptibility of Candida species
to fluconazole by disk diffusion,” Diagnostic Microbiology and
Infectious Disease, vol. 40, no. 1-2, pp. 27–33, 2001.
 R. P. Wenzel and C. Gennings, “Bloodstream infections due to
Candida species in the Intensive Care Unit: identifying espe-
cially high-risk patients to determine prevention strategies,”
Clinical Infectious Diseases, vol. 41, supplement 6, pp. S389–
 P. Chiu, L. T. Leung, and B. C. B. Ko, “Pseudolaric acids:
isolation, bioactivity and synthetic studies,” Natural Product
Reports, vol. 27, no. 7, pp. 1066–1083, 2010.
pseudolaric acid B, a major constituent of Pseudolarix kaemp-
feri,” Journal of Natural Products, vol. 58, no. 1, pp. 57–67,
 S. P. Yang, L. Dong, Y. Wang, Y. Wu, and J. M. Yue, “Antifungal
diterpenoids of Pseudolarix kaempferi, and their structure-
activity relationship study,” Bioorganic and Medicinal Chem-
istry, vol. 11, no. 21, pp. 4577–4584, 2003.
 J. Xu, A. R. Ramos, R. Vilgalys, and T. G. Mitchell, “Clonal and
spontaneous origins of fluconazole resistance in Candida
albicans,” Journal of Clinical Microbiology, vol. 38, no. 3, pp.
 L. E. Cowen, D. Sanglard, D. Calabrese, C. Sirjusingh, J. B.
Anderson, and L. M. Kohn, “Evolution of drug resistance in
experimental populations of Candida albicans,” Journal of
Bacteriology, vol. 182, no. 6, pp. 1515–1522, 2000.
albicans to fluconazole during treatment of oropharyngeal
candidiasis in a patient with AIDS: documentation by in
Infectious Diseases, vol. 18, no. 2, pp. 240–242, 1994.
 S. G. Revankar, O. P. Dib, W. R. Kirkpatrick et al., “Clinical
evaluation and microbiology of oropharyngeal infection due
virus-infected patients,” Clinical Infectious Diseases, vol. 26,
no. 4, pp. 960–963, 1998.