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[beta]-Asarone, an active principle of Acorus calamus rhizome, inhibits morphogenesis, biofilm formation and ergosterol biosynthesis in Candida albicans.


Keywords: [beta]-Asarone Acorus calamus rhizome Candida albicans Ergosterol biosynthesis Dimorphism Biofilm formation


Anti-Candida potential of Acorus calamus rhizome and its active principle, [beta]-asarone, was evaluated against the human fungal pathogen, Candida albicans. [beta]-Asarone exhibited promising growth inhibitory activity at 0.5 mg/ml and it was fungicidal at 8 mg/ml. Time dependant kill curve assay showed that MFC of [beta]-asarone was highly toxic to C. albicans, killing 99.9% inoculum within 120 min of exposure. [beta]-Asarone caused significant inhibition of C albicans morphogenesis and biofilm development at subinhibitory concentrations. Our data indicate that the growth inhibitory activity of p-asarone might be through inhibition of ergosterol biosynthesis. Hemolytic assay showed that [beta]-asarone is non-toxic, even at concentrations approaching MIC value. Our results suggest that [beta]-asarone may be safe as a topical antifungal agent.

[c] 2012 Elsevier GmbH. All rights reserved.


Acorus calamus Linn., popularly known as "sweet flag" is native to Central Asia, North America and Eastern Europe (Gilani et at. 2006). Rhizome of this plant is widely used in the Indian systems of medicine, such as Ayurveda, Siddha and Unani (Meena et al. 2010). The rhizomes, roots and essential oil of A. calamus have been reported to possess several biological activities including antifungal (Lee et al. 2004; Lee 2005), antibacterial (McGaw et al. 2002; Phongpaichit et at. 2005) and immunosuppressive activities (Mehrotra et at. 2003). Studies on chemical composition of Acorus spp. have revealed that [alpha]- and [beta]-asarones are the major active components (Raina et al. 2003; Lee et at. 2010; Geng et al. 2010). Various bioactivities are attributed to [beta]-asarone, like antibacterial, anti helmintic, and antifungal properties (McGaw et at. 2002; Devi and Ganjewala 2009; Lee et al. 2004).

Candida albicans is a major fungal pathogen of the humans causing a variety of infections (Kim and Sudbery 2011). Many of the available antifungal drugs have undesirable side effects or are very toxic (amphotericin B), show drug-drug interactions (azoles) or lead to the development of resistance (fluconazole, 5-flucytosine) (White et at. 1998). Therefore it is necessary to search for more effective and less toxic novel antifungal agents that would overcome these disadvantages. Plants are rich sources of bioactive molecules and are being explored for novel antimicro-bial/antifungal properties (Wagner and Ulrich-Merzenich 2009). Essential oils as well as their components exhibit good anti-Candida activities (Devkatte et al. 2005; Zore et at. 2011).

In This Communication, we report the antl-Candida albicans. properties of Acorus calamus rhizome extract and its active principle, [beta]-asarone. For the first time we are reporting the effect of [beta]-asarone on dimorphism, biofilm development and ergosterol biosynthesis in Candida albicans.

Materials and methods

Candida albicans, ATCC 90028 (MTCC 3017), strain was obtained from the IMTECH, Chandigarh, India, and maintained on Yeast-Peptone-Dextrose (YPD) agar slants at 4 [degrees] C. RPMI-1640 medium.

Extract preparation and identification of [beta]-asarone

The Acorus calamus rhizome powder (Yogesh Pharmacy Pvt. Ltd., Maharashtra state, India) was sequentially extracted with solvents of varying polarity, i.e. n-hexane, ethyl acetate, methanol and water. [beta]-Asarone (Sigma-Aldrich Chemicals, Pvt. Ltd., Bangalore, India) in n-hexane extract was identified and quantified by standard HPTLC and HPLC methods described elsewhere (Wagner and Bladt 1996; Waghmode et al. 2012; Singh et al. 2010).

Growth, viability and kill-curve assay

The susceptibility study was carried out by the standard methodology M27-A2 as per CLSI guidelines (Routh et al. 2011). Minimum fungicidal concentration (MFC) was considered as the lowest concentration killing 99.9% cells (Zore et al. 2011). Kinetics of the anti-Candida activity of [beta]-asarone was studied by time-dependent kill-curve assay using C albicans MTCC 3017 (ATCC 90028) strain (Zore et al. 2011).

Hemolytic activity

The hemolytic activity of the [beta]-asarone on human red blood cells was determined as described by Ahmad etal. (2010).

Ergosterol extraction and quantitation

Ergosterol levels were assayed using sterol quantitation method of Arthington-Skaggs et al. (1999).

Germ tube formation assay

Inhibition of in vitro RPMI-1640 induced germ tube formation by [beta]-asarone was studied by 96-well microtiter plate based morphological assay (Chauhan et al. 2011).

Adhesion assay

Effect of P-asarone on adherence of C albicans to a solid surface (i.e. polystyrene) was studied using micro plate based assay (Camacho et al. 2007).

Biofilm formation and quantitation

Candida albicans biofilms were developed on polystyrene surface of 96-well plates as per standard methodologies (Hawser and Douglas 1994). 100[mu]l of 1 x [10.sub.7] cells/ml cell suspension in PBS was inoculated and plates were incubated at 37 [degrees] C for 90 min to allow attachment of cells on the surface. Non-adhered cells were removed by washing the wells with sterile PBS, two to three times. To observe effect on development of biofilms, RPMI-1640 medium with various concentrations of p-asarone was added immediately after adhesion phase and the plates were incubated for 24 h at 37 [degrees] C. After incubation, wells were washed to remove any released cells, and biofilms were observed using an inverted light microscope (Metzer, India). Biofilm growth was analyzed with MIT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromidel-metabolic assay as described by Hawser and Douglas (1994).

Growth curve assay

Growth curve studies were performed as described by Sheikh et al. (2011).

Scanning electron microscopy (SEM) of C. albicans biofilms

Candida biofilms were developed by seeding silicon disc in 12-well plates and SEM analysis was done as per Chauhan etal. (2011).

Statistical analysis

All the experiments were performed on three independent occasions. Values mentioned are the mean of triplicate observations.

Results and discussion

Content of [beta]-asarone in n-hexane extract calculated was 0.148 [mu]g/[mu]l (v/v). Hexane fraction and its active principle [beta]-asarone inhibited C albicans growth in a concentration dependent manner, exhibiting minimum inhibitory concentration (MIC) at 10 mg/ml and 0.5 mg/ml respectively (Fig. 1). Fungicidal effect was observed at 8 mg/ml of p-asarone and 20 mg/ml of n-hexane fraction which killed the planktonic cells. [beta]-Asarone was highly toxic to Candida cells and killed 99.9% inoculum within 120 min of exposure (Fig. 4).[beta]-Asarone inhibited RPM1-1640 induced hyphae in C. albicans at sub-MIC values. Significant inhibition (90% and >50%) of yeast to hyphal form was observed at MICR and MIC/4 respectively. In addition to germ tube inhibition, budding was also inhibited considerably at sub-inhibitory concentrations (Fig. 2). Ability to inhibit virulence factor (dimorphism), without killing the pathogen, may be valuable to avoid natural selection and thereby emergence of drug resistant population of microbes. Adherence of C. albicans cells to polystyrene surface was not influenced by P-asarone up to MFC value. Previous works in our laboratory and by various other researchers have shown that Candida biofilms are resistant to antifungal antibiotics, including the most commonly prescribed drug, fluconazole (Ramage et al. 2005; Shinde et al. 2011). Methanolic fraction of A. calamus rhizome is reported to exhibit significant inhibition of biofilms compared to conventional antifungals (Subha and Gnanamani 2008). MIC of biofilm formation was achieved even at MIC/2 value, while, complete inhibition in biofilm development at sub-MFC was found in C albicans. At MIC, most of the cells in the biofilm were in the yeast form and filamentous forms were absent (Fig. 3). Inhibition of biofilm development might be due to prevention of germ tube formation which is a crucial step in biofilm formation. The effect of [beta]-asarone on the growth pattern of C. albicans was observed to be concentration dependent (Fig. 5). An increase in the concentration of [beta]-asarone lead to a significant decrease in growth with suppressed and delayed exponential phases. However, control cells showed a normal pattern of growth. [beta]-Asarone has an oral [LD.sub.50] of 1010 mg/kg in rats (JECFA 1981). Considering the toxicity of [beta]-asarone, caution need to be taken when using it as an anti-Candida agent. It may be safe as a topical agent. In present study, [beta]-asarone showed negligible hemolytic activity compared to the conventional antifungal, fluconazole, even at concentration values approaching MIC. Use of antifungals in combination with natural products or molecules is reported by various workers (Han 2007; Zore et al. 2011). The potential of using low concentrations of [beta]-asarone in combination with amphotericin-B or other antifungals need to be explored in animal models. MIC obtained at low concentration of [beta]-asarone and low cytotoxicity encouraged us to explore its mode of action. [beta]-Asarone caused complete inhibition of ergosterol synthesis at NC value. Average decrease in total cellular ergosterol content of Candida cells after exposure to their respective MIC, MIC/2, MIC/4, MIC/8 and MIC/16 of [beta]-asarone was 100%, 92%, 45%, 32% and 20% respectively (Fig. 6). Our data suggest that P-asarone, like fluconazole, may exert its antifungal activity through inhibition of ergosterol biosynthesis. The selective cytotoxic behavior of [beta]-asarone hints its affinity for this particular specific target and hence, its biosynthetic pathway.







Docking experiments indicated that alpha-asarone binds in the active site of human HMG-CoA reductase, where, methoxy groups play a key role in the binding and probably also in its biological activity, as shown by extensive SAR studies reported for analogues of alpha-asarone (Medina-Franco et al. 2005). Additionally, catalytic portion of EIMG-CoA reductase of C albicans possesses amino acid sequences with high degree of homology to the human HMG-CoA reductase (NCBI-BLAST). Based on these similarities, there is a possibility that [beta]-asarone would be having a similar target in C. albicans. Our data supports this hypothesis, as HMG-CoA reductase is involved in pre-lanosterol step of ergosterol biosynthesis pathway (Macreadie et al. 2006). Unanswered concerns in this aspect may be resolved by identification of target site and in vivo studies.

Conflict of interest

Authors have no conflict of interest to disclose.


SBR is thankful to DST, New Delhi, for providing DST-INSPIRE fellowship, Ref No. DST/INSPIRE FELLOWSHIP/2010/(290). We are thankful to Prof. S.B. Nimse, Hon'ble Vice Chancellor, SRTM University, for his kind support.


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* Corresponding author. Tel.: +91 09028528438; fax: +91 2462229245. E-mail address: (S.M. Karuppayil).

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Sandeep B. Rajput, S. Mohan Karuppayil *

DST-FIST and UGC-SAP Sponsored School of Life Sciences, SRTM University, Nanded 431606, MS, India
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Title Annotation:Short communication
Author:Rajput, Sandeep B.; Karuppayil, S. Mohan
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Jan 15, 2013
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