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In vitro antifungal activity of Calocybe gambosa extracts against yeasts and filamentous fungi

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Three strains of Calocybe gambosa (Fr.) Donk were investigated as possible sources of biologically active substances with antifungal activity. After submerged cultivation of C. gambosa mycelium, biologically active materials were isolated as ethyl acetate extracts from culture liquids. The in vitro activity of these C. gambosa extracts (Cg1, Cg2, Cg3) against yeasts and filamentous fungi was evaluated using the Clinical and Laboratory Standards Institute (CLSI) M27-A2 and M38-A2. The minimum inhibitory concentration (MIC) against most of the tested clinical fungal strains for Cg1 and Cg2 extracts ranges from 12.5 to 50 µg/ml while Cg3 ranged from 1.56 to 12.5 µg/ml. Candida albicans (DBVPG 4268) were the most sensitive fungal strain to C. gambosa extracts, with MIC ranges of 1.56 to 12.5 µg/mL, while the strains of Aspergillus tubingensis showed the least sensitivity to the extracts. The high performance liquid chromatography (HPLC) fingerprint of the isolates shows four principal compounds produced by the cultured mycelium. Considering the potential use of natural antifungal compounds in medicine, we are currently working on a small-scale extraction, isolation and structural characterization of compounds produced from the C. gambosa.
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African Journal of Microbiology Research Vol. 6(8), pp. 1810-1814, 29 February, 2012
Available online at http://www.academicjournals.org/AJMR
DOI: 10.5897/AJMR11.1384
ISSN 1996-0808 ©2012 Academic Journals
Full Length Research Paper
In vitro antifungal activity of Calocybe gambosa
extracts against yeasts and filamentous fungi
P. Angelini1*, B. Tirillini2 and R. Venanzoni1
1Department of Applied Biology, University of Perugia, Borgo XX Giugno, 74 06121 Perugia, Italy.
2Institute of Botany, University of Urbino, Via Bramante, 28- 61028 Urbino (PU), Italy.
Accepted 30 November, 2011
Three strains of Calocybe gambosa (Fr.) Donk were investigated as possible sources of biologically
active substances with antifungal activity. After submerged cultivation of C. gambosa mycelium,
biologically active materials were isolated as ethyl acetate extracts from culture liquids. The in vitro
activity of these C. gambosa extracts (Cg1, Cg2, Cg3) against yeasts and filamentous fungi was
evaluated using the Clinical and Laboratory Standards Institute (CLSI) M27-A2 and M38-A2. The
minimum inhibitory concentration (MIC) against most of the tested clinical fungal strains for Cg1 and
Cg2 extracts ranges from 12.5 to 50 µg/ml while Cg3 ranged from 1.56 to 12.5 µg/ml. Candida albicans
(DBVPG 4268) were the most sensitive fungal strain to C. gambosa extracts, with MIC ranges of 1.56 to
12.5 µg/mL, while the strains of Aspergillus tubingensis showed the least sensitivity to the extracts. The
high performance liquid chromatography (HPLC) fingerprint of the isolates shows four principal
compounds produced by the cultured mycelium. Considering the potential use of natural antifungal
compounds in medicine, we are currently working on a small-scale extraction, isolation and structural
characterization of compounds produced from the C. gambosa.
Key words: Anti-fungal, fungal metabolites, minimal inhibitory concentration (MIC), high performance liquid
chromatography (HPLC), thin layer chromatography (TLC).
INTRODUCTION
During the last two decades, there has been a dramatic
increase in the incidence of life-threatening systemic
fungal infections. The challenge has been to develop
effective strategies for treating fungal diseases, to treat
opportunistic fungal infections in human
immunodeficiency virus-positive patients and others who
are immunocompromised due to cancer chemotherapy or
the indiscriminate use of antibiotics (Pfaller and Diekema,
2004; Singh, 2001). Most clinically-used antifungal drugs
have various drawbacks in terms of toxicity, efficacy and
cost, and their frequent use has led to the emergence of
resistant strains (Fridkin, 2005). Hence, there is a great
need for novel antifungals that belong to a wide range of
structural classes, that can selectively act on new targets
*Corresponding author. E-mail: paola.angelini@unipg.it. Tel:
+39 075 5856423. Fax: +39 075 5856404.
with fewer side effects.
Macromycetes used in traditional medicine usually
constitute an important source of new biologically active
products (Liu, 2005). Calocybe gambosa (Fr.) Donk
(Lyophyllaceae, Agaricales, Basidiomycota), commonly
known as St. Georges mushroom, is widespread and
found in temperate regions throughout much of Europe
including parts of Russia, and Asia, including Korea and
Japan. It is quite common in various grassy habitats, on
roadsides and wood edges or in pastureland (Hall et al.,
2010). C. gambosa has been reported to have important
medicinal properties including antioxidant activity
(Palacios et al., 2011), antibacterial activity towards
Bacillus subtilis and Escherichia coli (Keller et al., 2002)
and an ability to reduce blood sugar levels (Brachvogel,
1986). Earlier studies on the secondary metabolites
produced by C. gambosa, demonstrated that in culture,
this fungal species synthesized an orange crystalline
metabolite which was shown to be the phenoxazine
Figure 1. Basidiocarps of C. gambosa (Fr.) Donk (Giancarlo
Bistocchi photographer).
Table 1. Mycelial dry weights and yields of ethyl acetate extracts
(EtOAc) from six-weeks-old C. gambosa culture liquid.
C. gambosa
Mycelial dry weight*
EtOAc extract*
strains
(mg)
(mg)
1
47.2±0.6
1.1±0.3
2
31.8±0.2
0.7±0.3
3
56.6±0.8
1.6±0.1
*The values are the means of three repetitions ± standard error.
derivatives (phenoxazone and a-amino-phenoxazone)
(Clemencon, 1987; Moncalvo et al., 1991; Schlunegger et
al., 1976). These derivatives are used extensively in
medicine, biology and chemistry (Eregowda et al., 2000;
Kalpana et al., 1999; Shimizu et al., 2004; Sridhar et al.,
1999).
Considering the potential use of natural antifungal
compounds in medicine, the aim of the present work was
to evaluate the antifungal potential of ethyl acetate
extract of C. gambosa cultural liquid on several fungal
strains of medical importance.
MATERIALS AND METHODS
Mushroom
Three fruiting bodies of C. gambosa were collected in nature during
field trips between 2009 and 2010, in the regions of Umbria and
Abruzzo (Italy). The specimens were identified according to their
macroscopic and microscopic features and the related literature
(Watling, 1979; Moser, 1978) (Figure 1). Voucher cultures (Cg1,
Cg2 and Cg3) are maintained in the DBA (Department of Applied
Biology) culture collection (University of Perugia, Italy) and are
accessible.
Pieces of the fruiting bodies were isolated and placed on solid
medium in test tubes. After being cultivated for 3 weeks at room
temperature, the mycelium was removed from the agar tubes and
transferred into a liquid medium (Angelini et al., 2008). The culture
Angelini et al. 1811
was then incubated for 6 weeks at 25°C. The evaluation of growth
by dry weights determination of fungal mycelia from liquid culture
was examined (Lilly and Barnett, 1951).
Extraction and isolation
After submerged cultivation, the culture liquids were separated from
the mycelia by filtration and extracted with ethyl acetate using a
separating funnel. The ethyl acetate fractions were evaporated to
dryness in vacuo.
The dried extracts were analysed with a high-performance liquid
chromatography (HPLC) Jasco 880-PU using a C18 column (250 x
4 mm) eluent water/methanol in a gradient from 60 to 0% water in
30 min. The chromatographic profiles were recorded at 254 nm.
The chromatographic analysis of the culture media extract of before
inoculation did not show any peaks. The dried extracts were also
subjected to thin layer chromatography (TLC) analysis
(chloroform/toluene 1:1) on silicagel G.
Antifungal assay
Thirty-three clinical strains, five of yeasts (obtained from Industrial
Yeast Collection DBVPG) and twenty-eight filamentous fungi
(moulds), were used in this study (Table 2) while two ATCC
(American Type Culture Collection) referente strains [Candida
parapsilosis (ATCC 22019) and C. albicans (ATCC 90028)] served
as controls as recommended by Clinical and Laboratory Standards
Institute, CLSI (formerly the National Committee for Clinical
Laboratory Standards). The filamentous fungi (provided by the
Clinical Microbiology and Parasitology Section, R. Silvestrini
Hospital, Perugia, Italy), were identified by morphological and
molecular analyses (Pagiotti et al., 2011). Stock cultures were
maintained on Sabouraud Dextrose Agar (SDA; Oxoid, Milan, Italy)
slants at 4°C.
Antifungal agents
The dried extracts of C. gambosa strains (Cg 1, Cg 2 and Cg 3)
were dissolved in 10% methanol and diluted with sterile double-
distilled water. Amphotericin B (Sigma-Aldrich, Milan, Italy) were
used as reference drug.
Antifungal susceptibility tests
Evaluation of the susceptibility of yeasts and filamentous fungi was
performed using the broth microdilution method (BMM) according to
CLSI M38-A2 (for filamentous fungi) and M27-A2 (for yeast)
guidelines (NCCLS, 2002; CLSI, 2008). Yeast strains were grown
aerobically overnight at 35°C on SDA plates. Yeasts were
harvested and suspended in 1% sterile saline and the turbidity of
the supernatants measured spectrophotometrically at 625 nm with
an absorbance of 0.08-0.1 equivalent to the No. 0.5 McFarland
standard following the NCCLS M27-A2 guideline (NCCLS, 2002).
The working suspension was diluted 1:20 in a mixture containing
RPMI 1640 medium (with L-glutamine, without bicarbonate;
Cambrex Bio Science, Verviers, Belgium) and 0.165 M
morpholinepropanesulfonic acid (MOPS; Sigma-Aldrich, Milan,
Italy) buffered to pH 7.0. The working suspension was further
diluted with the medium (1:50) to obtain the final test inoculum (1-
5x103 CFU ml). The working inoculum suspension (100 µl) were
added to each well of 96-well flat-bottom microdilution plates
containing 100 µl of drug dilution incubated in an aerobic
environment at 35°C for 24 h. Growth was observed visually with
the aid of a concave mirror; MICs (Minimal Inhibitory
1812 Afr. J. Microbiol. Res.
Table 2. Minimal inhibitory concentrations (MICs) of C. gambosa extracts against yeasts and filamentous fungi.
Yeasts and filamentous fungi
MIC (µg/mL)
Cg1*
Cg2*
Amphotericin B
Candida albicans (DBVPG 6133)
25
12.5
> 8.0
Candida albicans (DBVPG 4268)
12.5
12.5
4.0-8.0
Candida albicans (DBVPG 3908)
12.5
12.5
2.0-8.0
Filobasidiella neoformans var. neoformans (DBVPG 6010)
25
25
>8.0
Filobasidiella neoformans var. bacillospora (DBVPG 6225)
25
25
>8.0
Penicillium chrysogenum (3)**
50-100
25-50
>8.0
Verticillium sp. (3)**
25-50
25-50
>8.0
Aspergillus tubingensis (6)**
100-200
100-200
>8.0
Aspergillus minutus (4)**
12.5-50
12.5-50
4.0-8.0
Beauveria bassiana (5)**
25-50
25-50
>8.0
Microsporum gypseum (7)**
50-100
25-50
>8.0
*ethyl acetate extracts from culture liquids of C. gambosa strains. **Values in brackets indicate number of strains that were screened.
Concentrations) were taken on a growth or no-growth (100%
visible-growth inhibition) scale.
The activity of the ethyl acetate extracts of C. gambosa strains
against filamentous fungi was also determined using the BMM,
according to the CLSI M38-A2 guideline (CLSI, 2008). Cultures
were grown on Potato Dextrose Agar (PDA; Oxoid, Milan, Italy) at
35°C until sporulation (48 h to 7 days). Spores were harvested and
suspended in 1% sterile saline, allowed to settle and the upper
layer aspirated. The turbidity was measured spectrophotometrically
at 625 nm, and optical density was adjusted to yield a stock
suspension of 0.4-5x106 sporangiospores per millilitre. A working
suspension was prepared by diluting 1:50 of the conidia stock
suspension in a standard medium (RPMI 1640, MOPS). The fungal
inocula (100 µl) were added to each well of 96-well flat-bottom
microdilution plates containing 100 µl of drug dilution and incubated
for 48 h in an aerobic incubator at 35°C. After incubation, potential
antimicrobial activity (MICs) was assessed as described previously.
RESULTS
Analysis of C. gambosa extract by high-performance
liquid chromatography (HPLC) and thin layer
chromatography (TLC)
The mycelial dry weights and the yields of ethyl acetate
extracts from 6 week old C. gambosa culture liquids are
shown in Table 1.
The biosynthetic activity of the dried extract obtained
from C. gambosa culture medium involves at least twenty
chemical compounds that absorb at 254 nm (Figure 2).
The TLC analysis showed that the coloured fraction of
the culture media is due to four components.
Antifungal activity
The MIC values of ethyl acetate extracts from C.
gambosa relative to the yeasts and filamentous fungi
tested are reported in Table 2. C. albicans (DBVPG
4268) were the most sensitive fungal strain to C.
gambosa extracts, with MIC ranges of 1.56 to 12.5 µg/ml,
while the strains of A. tubingensis showed the least
sensitivity to the extracts.
The Cg1 and Cg2 extracts displayed MIC in the
concentration range of 15.5 to 50 µg/ml against some of
the fungal strains while Cg1 extract was found to possess
a MIC in a concentration range of 1.56 to 12.5 µg/ml
against most of the tested fungal strains. The Cg3 extract
showed MIC in a range of 1.56 to 6.25 µg/ml against all
Candida albicans and Filobasidiella neoformans strains.
Of the three extracts employed in this study, Cg3 was the
most potent against majority of fungal strains.
Amphotericin B showed a low activity in vitro against
the fungal species tested. C. albicans (DBVPG 4268 and
3908) and Aspergillus minutus had an amphotericin MIC
range of 2.0 to 8.0 µg/mL, whereas the other fungal
species exhibited amphotericin MIC values >8 µg/ml.
The MIC values of amphotericin B, for the strains
Candida parapsilosis (ATCC 22019) and C. albicans
(ATCC 90028) were within the established ranges
(NCCLS, 2002; CLSI, 2008).
DISCUSSION
Five clinical strains of yeast and twenty-eight clinical
strains of filamentous fungi were employed in evaluating
the antifungal potency of the ethyl acetate extracts from
C. gambosa strains (Cg1, Cg2 and Cg3). Most of the
filamentous fungi strains tested in this study were
resistant to amphotericin B, according to the recently
proposed in vitro breakpoints; susceptible: MIC ≤1 µg/ml,
intermediate, MIC = 2 µg/ml, and resistant, MIC ≥4 µg/ml
(CLSI, 2008). Breakpoints with proven clinical relevance
are not available for yeasts vs. amphotericin B due to
methodology problems (Espinel-Ingroff, 2008).
The antifungal activity of the C. gambosa extracts (Cg1,
Cg2 and Cg3) against fungal species varied markedly,
probably due to differences in mycelial growth and
metabolite yield but this activity was more effective
Angelini et al. 1813
Figure 2. High-performance liquid chromatography (HPLC) of ethyl acetate extract of C. gambosa culture liquid.
than that of the commercial drug, amphotericin B. It is not
clear why yeasts were more sensitive than filamentous
fungi to the metabolites produced by C. gambosa
mycelium culture. However, it is possible that the
biosynthesis of fungal cell wall including pathways,
regulation and assembly of cell wall components may
differ among the various fungi, resulting in different
sensitivities to the C. gambosa metabolites. In liquid
medium, high levels of C. gambosa metabolites were
produced after mycelium growth has occurred. The
highest mycelial growth of C. gambosa strains in liquid
culture, increased the yields of ethyl acetate extracts and
their antifungal activity (Tables 1 and 2). The production
of secondary metabolites can be delayed until the end of
the trophophase (mycelium growth phase) by repressing
the enzymes of secondary metabolism during growth by
removing the sources of carbon, nitrogen and
phosphorus (Demain, 1986).
In light of the results of this study, it can be concluded
that C. gambosa is a prospective candidate from which
new antifungal compounds or chemical substances can
be isolated. Considering the potential use of natural
antifungal compounds in medicine, we are currently
working on a small-scale extraction, isolation and
structural characterization of compounds produced from
the C. gambosa.
REFERENCES
Angelini P, Granetti B, Bellini M (2008). Effetti di alcune sorgenti di
azoto sull’accrescimento del micelio in coltura pura di Calocybe
gambosa. Mic. Ital., 37: 50-57.
Brachvogel R (1986). Reduction of blood sugar by Calocybe gambosa
Fr. Donk. Zeitsch. Mykol., 52: 445.
Clemencon H (1987). Phenoxazone in Mycelkulturen von Calocybe
Arten (Agaricales, Basidiomycetes). Beitrage zur Kenntnis der Pilze
Mitteleuropas, 3: 107-115.
CLSI (2008). Clinical and Laboratory Standards Institute. Reference
method for broth dilution antifungal susceptibility testing of
filamentous fungi, approved standard M38-A. Clinical and Laboratory
Standards Institute, Wayne, PA, 2002. 22, 22: 16.
Demain AL (1986). Regulation of secondary metabolism in fungi. Pure
Appl. Chem., 58(2): 219-226.
Eregowda GB, Kalpan HN, Ravi Hegde, Thimmaiah KN (2000).
Synthesis and analysis of structural features of phenoxazine
analogues needed to reverse vinblastine resistence in multidrug
resistant (MDR) cancer cells. Indian J. Chem., Sect. : Org. Chem.
Incl. Med. Chem., 39: 243-259.
Espinel-Ingroff A (2008). Mechanisms of resistance to antifungal
agents: Yeasts and filamentous fungi. Rev. Iberoam. Micol., 25: 101-
106.
Fridkin SK (2005). The changing face of fungal infections in health care
settings. Clin. Infect. Dis., 41(10): 1455-1460.
Hall IR, Stephenson SL, Buchanan PK (2010). Edible and poisonous
mushrooms of the world. Timber Press, United States, p. 372.
Kalpana HN, Eregowda GB, Jagadeesh S, Thimmaiah KN (1999).
Effect of phenoxazine MDR modulators on photoaffinity labeling of P-
glycoprotein by (3H) azidopine: An approach to understand drug
resistance in cancer chemotherapy. Indian J. Pharm. Sci., 16: 168.
1814 Afr. J. Microbiol. Res.
Keller C, Maillard M, Keller J, Hostettmann K (2002). Screening of
European fungi for antibacterial, antifungal, larvicidal, molluscicidal,
antioxidant and free-radical scavenging activities and subsequent
isolation of bioactive compounds. Pharm. Biol., 40(7): 518-525.
Lilly VG, Barnett HL (1951). Physiology of the fungi. McGraw-Hill Book
Company, Inc., New York.
Liu JK (2005). N-Containing Compounds of Macromycetes. Chem.
Rev., 105(7): 2723-2744.
Moncalvo JM, Toriola D, Clémençon H (1991). Analyse taxonomique du
complexe Lyophyllum decastes sensu latu (Agaricales,
Basidiomycetes) sur la base des caractères culturaux. Mycol.
Helvetica, 3: 397-415.
Moser M (1978). Keys to Agarics and Boleti (Polyporales, Boletales,
Agaricales, Russulales). In: G. Kibby (ed.), Roger Phillips, London, p.
118.
NCCLS (2002). Clinical and Laboratory Standards Institute. Reference
method for broth dilution antifungal susceptibility testing of yeasts,
approved standard M27-A2. 2nd edn, CLSI Document. Clinical and
Laboratory Standards Institute, Villanova, PA, 2002. 22, 22: 15.
Pagiotti P, Angelini P, Rubini A, Tirillini B, Granetti B, Venanzoni R
(2011). Identification and characterisation of human pathogenic
filamentous fungi and susceptibility to Thymus schimperi essential oil.
Mycoses, 54(5): e364-e376.
Palacios I, Lozano M, Moroa C, D’Arrigo M, M.A. R istagno MA,
Martínez JA, García-Lafuente A, Guillamóna E, Villaresa A (2011).
Antioxidant properties of phenolic compounds occurring in edible
mushrooms. Food Chem., 128(3): 674-678.
Pfaller MA, Diekema DJ (2004). Rare and emerging opportunistic fungal
pathogens: concern for resistance beyond Candida albicans and
Aspergillus fumigatus. J. Clin. Microbiol., 42: 4419-4431.
Schlunegger UP, Kuchen A, Clemencon H (1976). Mycelium products in
higher fungi. I. phenoxazine derivatives in Calocybe gambosa. Helv.
Chim. Acta., 59(4): 1383-1388.
Shimizu S, Suzuki M, Tomoda A, Arai S, Taguchi H, Hanawa T, Kamiya
S (2004). Phenoxazine compounds produced by the reactions with
bovine hemoglobin show antimicrobial activity against non-
tubercolosis mycobacteria. Tohoku. J. Exp. Med., 203(1): 47-52.
Singh N (2001). Trends in the epidemiology of opportunistic fungal
infections: predisposing factors and the impact of antimicrobial use
practices. Clin. Infect. Dis., 33(10): 1692-1696.
Sridhar MA, Ramegowda M, Lokanath NK, Shashidharaprasad J,
Eregowda GB, Thimmaiah KN (1999). Structural studies of some
phenoxazine derivatives. Mol. Cryst. Liq. Cryst., 312: 189-214.
Watling R (1979). The morphology, variation and ecological significance
of anamorphs in the Agaricales. In: The Wole Fungus (B. Kendrick,
ed.). National Museum of Natural Sciences, Ottawa, pp. 453-472.
... These derivatives are used extensively in medicine, biology, and chemistry (Shimizu et al. 2004). Considering the potential use of natural antifungal compounds in medicine, three strains of C. gambosa were investigated as possible sources of biologically active substances with antifungal activity (Angelini et al. 2012). After submerged cultivation of C. gambosa mycelium, biologically active materials were isolated as ethyl acetate extracts from culture liquids. ...
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