October 2014⎪Vol. 24⎪No. 10
J. Microbiol. Biotechnol. (2014), 24(10), 1377–1381http://dx.doi.org/10.4014/jmb.1405.05030 Research Article jmbReview
Inhibition of the Calcineurin Pathway by Two Tannins, ChebulagicAcid and Chebulanin, Isolated from Harrisonia abyssinica Oliv.Won Jeong Lee1, Jae Sun Moon1, Sung In Kim1, Young Tae Kim1, Oyekanmi Nash2, Yong-Sun Bahn3, and
Sung Uk Kim1*
1Division of Biosystems Research, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea2Southwest Biotechnology Center of Excellence, Institute for Advanced Medical Research and Training, College of Medicine, University of
Ibadan, Ibadan, Nigeria3Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea
In the past 20 years, the incidences of fungal infections,
particularly opportunistic fungal infections, have significantly
increased owing to the introduction of organ transplantation
and cancer chemotherapy, and prevalence of AIDS [15, 18].
Less pathogenic species of Candida such as Candida
dubliniensis and Saccharomyces cerevisiae, previously considered
as clinically insignificant, are emerging as opportunistic
pathogens affecting a variety of different body sites [5, 8,
10]. An outbreak of meningitis due to the zoonotic fungus
Cryptococcus neoformans on the east coast of Vancouver
Island in Canada attracted public attention in the year 2000
because this strain causes life-threatening meningocephalitis
in immunocompromised and immunocompetent individuals
[9, 12]. However, most available antifungal compounds
exhibit high toxicity and various harmful effects on human
cells; moreover, pathogenic fungi are resistant to these
antifungal compounds. Therefore, demand for the development
of novel antifungal agents that have few side effects and a
broad-spectrum activity against various fungi is greater
than ever before.
C. neoformans, a group of ubiquitous human and animal
pathogen, is widely distributed in the environment and
commonly isolated from avian excreta, soil, and trees.
Disease is thought to progress upon inhalation of its
basidiospores into the lungs, and then spreads from the lungs
to the central nervous system to cause meningoencephalitis
[17].
Harrisonia abyssinica Oliv. (Simaroubaceae) is a small tree
or shrub widely used in African folk remedies for the
treatment of gonorrhea, dysentery, skin diseases, fever,
bubonic plague, hemorrhoids, snakebite, and tuberculosis
[1, 16]. The extracts of the bark and root of H. abyssinica
have been found to exhibit in vitro antimicrobial, cytotoxic,
insect antifeedant, antiviral, and molluscicidal activities [1,
16], and stimulatory effects on Striga hermonthica seeds [2].
Various steroids, limonoids, chromone [2, 3, 14], and
Received: May 14, 2014
Revised: July 4, 2014
Accepted: July 7, 2014
First published online
July 7, 2014
*Corresponding author
Phone: +82-42-860-4554;
Fax: +82-42-861-2675;
E-mail: [email protected]
pISSN 1017-7825, eISSN 1738-8872
Copyright© 2014 by
The Korean Society for Microbiology
and Biotechnology
In order to discover and develop novel signaling inhibitors from plants, a screening system
was established targeting the two-component system of Cryptococcus neoformans by using the
wild type and a calcineurin mutant of C. neoformans, based on the counter-regulatory action of
high-osmolarity glycerol (Hog1) mitogen-activated protein kinase and the calcineurin
pathways in C. neoformans. Among 10,000 plant extracts, that from Harrisonia abyssinica Oliv.
exhibited the most potent inhibitory activity against C. neoformans var. grubii H99 with
fludioxonil. Bioassay-guided fractionation was used to isolate two bioactive compounds from
H. abyssinica, and these compounds were identified as chebulagic acid and chebulanin using
spectroscopic methods. These compounds specifically inhibited the calcineurin pathway in
C. neoformans. Moreover, they exhibited potent antifungal activities against various human
pathogenic fungi with minimum inhibitory concentrations ranging from 0.25 to over 64 µg/ml.
Keywords: Signaling pathway, Cryptococcus neoformans, two-component system, calcineurin
inhibitor, antifungal activity
1378 Lee et al.
J. Microbiol. Biotechnol.
prenylated polyketides [2] have been isolated from H.
abyssinica, and a very recent study showed that the novel
prenylated acetophenones, harronin I and harronin II,
isolated from this plant have potent antibacterial and
antifungal activities [13].
In the course of searching for fungal signaling modulators
derived from plant extracts, calcineurin inhibitors of C.
neoformans were found in the methanol extract of the whole
body of H. abyssinica Oliv. Although H. abyssinica extract
has been recognized as a useful folk remedy in Africa,
there are no reports to date that suggest that the extract can
be used as a specific inhibitor of the signaling pathway of
C. neoformans by targeting calcineurin. Here, we describe
the isolation and structure determination of two tannin
compounds, chebulagic acid and chebulanin, and their
inhibitory activities against various human fungi.
In order to discover and develop novel signaling
modulators derived from plant extracts, we established a
screening system for signaling modulators targeting the
two-component system of wild-type C. neoformans var. grubii
H99 and a calcineurin mutant cna1 of the C. neoformans H99
strain, based on the counter-regulatory action of high-
osmolarity glycerol (Hog1) mitogen-activated protein kinase
(MAPK) and the calcineurin pathways in C. neoformans
[11].
C. neoformans var. grubii H99 (MATα) and C. neoformans
cna1 (MATα cna1∆::NAT-STM#117) mutant strains [11]
were cultured on YPD (1% yeast extract, 2% peptone, and
2% dextrose) medium at 30°C, and various human
pathogenic fungi were cultured in Sabouraud broth (Difco)
at 30°C for 24 h.
The discovery of signaling modulators against C. neoformans
was performed using the agar diffusion method [6]. The
agar plates for the bioassay were prepared as two separate
layers. The C. neoformans H99 and cna1 mutant suspensions
were added into the overlay media containing 0.8% YPD
agar. The base media with solidified YPD agar were then
overlaid with each medium containing the C. neoformans
H99 or cna1 mutant strains. After solidification of the
overlay media, the two plates seeded with each strain were
used in bioassays as follows. The A plate (known
antifungal agent): C. neoformans H99 plus test sample; B
plate (calcineurin inhibitor): C. neoformans H99 plus sample
and fludioxonil; C plate (Hog1 inhibitor): C. neoformans
cna1 mutant plus test sample and fludioxonil; D plate
(Hog1 activator): C. neoformans cna1 mutant plus test
sample. Sterile paper disks (8 mm in diameter) were placed
on the surface of the four agar plates seeded with the
C. neoformans H99 and cna1 mutant strains. Aliquots of the
test samples with or without fludioxonil, which is a known
Hog 1 activator [11], were loaded onto the paper disks and
incubated for 24 h at 30°C. The diameters of the inhibitory
Fig. 1. HPLC chromatograms (left) and the biological activities (right) of chebulagic acid (A, top) and chebulanin (B, bottom),
isolated from Harrisonia abyssinica Oliv.
Bioassays of chebulagic acid and chebulanin were performed on plates containing C. neoformans var. grubii H99 and fludioxonil.
Chebulagic Acid and Chebulanin Isolated from Harrisonia abyssinica 1379
October 2014⎪Vol. 24⎪No. 10
zones observed on the four plates were measured and the
samples showing a large clear zone on A, B, and D plates
were primarily selected, while those with a smaller clear
zone on the C plate compared with the control, fludioxonil,
were chosen. Finally, the plant extracts simultaneously
showing inhibitory activities on each plate were excluded,
and the samples with the desired clear zone only on B, C,
or D plates were selected.
In addition, the minimum inhibitory concentrations
(MICs) of the isolated compounds against various human
pathogenic fungi were determined using the broth dilution
method of the Clinical and Laboratory Standards Institute
(CLSI, formerly NCCLS) [4].
Large-scale screening of signaling pathway modulators
was performed using 10,000 foreign plant extracts supplied
by the International Biological Material Research Center
(IMBRC), Korea Research Institute of Bioscience and
Biotechnology. The change in the inhibition zone of the
same sample on the four bioassay plates containing wild
type and a calcineurin mutant of C. neoformans was analyzed
with the naked eye in comparison with the control
compound, fludioxonil; three candidates showing inhibitory
activities only on B or C plate were selected from the 10,000
plant extracts. Among these, bioactive compounds from
H. abyssinica Oliv. with a clear zone only on the B plate
were isolated and purified using ethyl acetate extraction,
silica gel column chromatography, Sephadex-LH20 column
chromatography, thin-layer chromatography, and high-
performance liquid chromatography (HPLC). The retention
times of the purified bioactive compounds in an HPLC
analysis were 19.5 and 16.8 min (Fig. 1). Structural analyses
of the isolated compounds with electrospray ionization
mass spectrometry (ESI-MS) and various nuclear magnetic
resonance (NMR) techniques, including 1H-1H COSY,
HMQC, DEPT, and HMBC, revealed that these compounds
had the molecular formulas C41H30O27 and C27H24O19 with
molecular weights of 954 and 652, respectively (Fig. 2).
Based on the data, these compounds were identified as
chebulagic acid and chebulanin (Fig. 3). Chebulagic acid: a
white powder; ESI-MS (negative), m/z 953.10 [M-H]-; 1H
NMR (acetone-d6, 500 MHz) (J in Hz): δ = 2.18 (2H, m, che-
5’-H), 3.87 (1H, m, che-4’-H), 4.39 (1H, dd, J = 8.1, 5.4, glc-6-
H), 4.76 (1H, d, J = 9.4, glc-6-H), 4.79 (1H, d, J = 5.2, glc-5-
H), 4.95 (1H, d, J = 7.1, che-2’-H), 5.11 (1H, dd, J = 7.1, 1.5,
Fig. 2. ESI-MS spectra of chebulagic acid (top) and chebulanin (bottom), isolated from Harrisonia abyssinica Oliv.
1380 Lee et al.
J. Microbiol. Biotechnol.
che-3’-H), 5.21 (1H, d, J = 3.7, glc-4-H), 5.50 (1H, br s, glc-2-
H), 5.94 (1H, br s, glc-3-H), 6.49 (1H, br s, glc-1-H), 6.64
(1H, s, HHDP-H), 7.07 (1H, s, HHDP-H), 7.18 (2H, s,
galloyl-H), 7.51 (1H, s, che-3’’-H). Chebulanin: an off-white
powder; ESI-MS (negative), m/z 651.0834 [M–H]-; 1H NMR
(acetone-d6, 500MHz) (J in Hz): δ = 2.18 (2H, m, che-5’-H),
3.89 (1H, m, che-4’-H), 4.00 (1H, dd, J = 11.3, 5.7, glc-6-H),
4.13 (1H, dd, J = 11.3, 6.7, glc-6-H), 4.30 (1H, t, J = 6.4, glc-5-
H), 4.82 (1H, br s, glc-3-H), 4.88 (1H, m, glc-4-H), 4.92 (1H,
d, J = 7.1, che-2’-H), 5.17 (1H, dd, J = 7.1, 1.5, che-3’-H), 5.23
(1H, dd, J = 2.7, 1.5, glc-2-H), 6.35 (1H, d, J = 2.7, glc-1-H),
7.19 (2H, s, galloyl-H), 7.49 (1H, s, che-3’’-H). The MS and
NMR data for the purified compounds were in good
agreement with the spectral data for both compounds
previously published [7].
The antifungal activities of the isolated chebulagic acid
and chebulanin against human pathogenic fungi were
determined by MIC evaluations using the broth dilution
method of the CLSI. Chebulagic acid showed potent
antifungal activities against various human pathogenic
fungi, such as C. albicans, C. krusei, C. lusitaniae, C. tropicalis,
C. neoformans, and C. neoformans var. grubii, with MICs of
0.25-4 µg/ml. The strongest antifungal activities were noted
against C. neoformans and C. lusitaniae at concentrations of
0.25 and 0.5 µg/ml, respectively, whereas relatively weak
antifungal activities against A. fumigatus and Trichophyton
mentagrophytes were observed (Table 1). Chebulanin also
showed potent antifungal activities against C. neoformans,
C. lusitaniae, and C. neoformans var. grubii, albeit weaker
than that of chebulagic acid.
Although a number of compounds isolated from
H. abyssinica Oliv. have been reported [2, 3, 13, 14], only
limited information is available on their biological activities.
In particular, little is known of the in vitro antifungal
activities of tannin compounds against various human
pathogenic fungi. Taken together, this is the first report
that chebulagic acid and chebulanin inhibited a calcineurin
pathway of C. neoformans and showed potent antifungal
activities in vitro against human pathogenic fungi. Therefore,
chebulagic acid and chebulanin may serve as lead compounds
for the development of inhibitors of the signaling pathway
Fig. 3. Structures of chebulagic acid (A) and chebulanin (B)
isolated from Harrisonia abyssinica Oliv.
Table 1. In vitro antifungal activities of chebulagic acid and chebulanin against various human pathogenic fungi.
Test microorganismsMIC (µg/ml)
Chebulagic acid Chebulanin Amphotericin B
Candida albicans ATCC 10231 2 > 64 0.25
Candida lusitaniae ATCC 42720 0.5 2 0.25
Candida krusei ATCC 6258 1 8 0.5
Candida tropicalis ATCC 13803 4 > 64 0.25
Cryptococcus neoformans ATCC 36556 0.25 0.5 < 0.12
Cryptococcus neoformans var. grubii H99 1 2 0.25
Aspergillus fumigatus ATCC 16424 > 64 > 64 0.5
Trichophyton mentagrophytes ATCC 9533 > 64 > 64 0.25
Chebulagic Acid and Chebulanin Isolated from Harrisonia abyssinica 1381
October 2014⎪Vol. 24⎪No. 10
of C. neoformans. The detailed mode of action of these
compounds against C. neoformans remains to be investigated.
Acknowledgments
This work was supported by a grant from Procurement
and Development of Foreign Biological Resources funded
by the Ministry of Science, ICT and Future Planning of the
Korean government (NRF-2011-00495).
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