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: kimsu@kribb.re.kr

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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