Escolar Documentos
Profissional Documentos
Cultura Documentos
Available online at
ScienceDirect
www.sciencedirect.com
Received 20 June 2013; received in revised form 10 October 2013; accepted 28 October 2013
Available online 19 December 2013
KEYWORDS
Antioxidant;
Antibacterial;
Antifungal;
Hepatocellular
carcinoma;
Chaetomium
Summary The aim of the present study was to evaluate different biological activities of the
fungus Chaetomium globosum (family Chaetomiaceae). The evaluation was done through testing
its antimicrobial, antioxidant and anticancer effects. C. globosum was isolated from the
Cucumber soil (rhizosphere) and caused inhibition of the mycelial growth of Fusarium solani,
Rhizoctonia solani and Sclerotium rolfsii in the biculture test. Petroleum ether and ethyl acetate
extracts of the liquid culture of C. globosum showed potent in vitro antioxidant activity.
C. globosum proved potent antibacterial activity against Bacillus subtilis, Escherichia coli
and Pseudomonas fluorescens. It also recorded significant antifungal activity against Candida
albicans, F. solani, Fusarium oxysporum, R. solani and Pythium ultimum. It exerted cytotoxic
effect on human hepatocellular carcinoma cell line (HepG2). Unsaponifiable and saponifiable
matters of the petroleum ether extract showed the presence of hydrocarbons, sterols and fatty
acids. The ethyl acetate extract showed the presence of prenisatin, chrysophanol, chrysazin,
chaetoviridin A and B. The isolated secondary metabolites proved significant antioxidant and
antimicrobial activity on B. subtilis, E. coli and R. solani. In conclusion, this fungus showed
different biological activities. Further studies must be done to apply its use in the agricultural
and medicinal field.
# 2013 Elsevier Masson SAS. All rights reserved.
* Corresponding author.
E-mail address: manal_hamed@yahoo.com (M.A. Hamed).
1156-5233/$ see front matter # 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.mycmed.2013.10.005
e36
MOTS CLS
Effet antioxydant ;
Effet antibactrien ;
Effet antifongique ;
Carcinome
hpatocellulaire ;
Chaetomium
Introduction
Endophytes are considered as one of the main topics in the
field of natural product chemistry [6]. They produce a large
number of bioactive compounds, which have a vital role
against different diseases during the last century [6]. They
have been recognized as important sources of a variety of
structurally novel active secondary metabolites with anticancer, antimicrobial and other biological activities [10]. An
endophyte, Chaetomium is the largest genera of saprophytic
ascomycetes [16]. Chaetomium is a genus of fungi in the
Chaetomiaceae family. Members of this filamentous fungal
genus are widespread, commonly found in soil, air and plant
debris [22].
Several secondary metabolites had been isolated from
Chaetomium cochliodes such as benzoquinone derivatives,
tetra-S-methyl derivatives, azaphilones, cytochalasans,
chaetoglobosin analogs, anthraquinone chromanone, orsellinic acid, globosumones [16] and epipolythiodioxopiperazines [23]. Rotiorinols, rotiorin, epi-isochromophilone II and
rubrorotiorin were isolated from Chaetomium cupreum [12].
Chaetoglobosins [19], cytoglobosins, azaphilones, chaetoviridins, pyrones, orsellides and globosumones were isolated
from Chaetomium globosum [10].
Phytopathogenic fungi are fungi that cause disease in
plants, obtain nutrients from living host cells, infect and/
or kill host tissue, affecting the quality and the quantity of
the economically important plants and leading to loss of the
crop yield [11]. Therefore, the demand for introduction of
natural biological controls using antagonistic products isolated from other microorganisms is increasing. This natural
control is considered safe, economic and reduces crop losses
caused by various plants pathogenic. C. globosum had been
reported to be a potential antagonist of various plant pathogens through three modes of action: competition, mycoparasitism and antibiosis [15].
The present study is focusing on testing different biological activities of C. globosum fungi, as antioxidant, antibacterial and antifungal agent. The work was extended to
examine its cytotoxic effect on human hepatocellular carcinoma cell line. Isolation and identification of the most
Standard (Vit. C)
Petroleum ether extract
Diethyl ether extract
Chloroform extract
Ethyl acetate extract
Table 2 The percentage of inhibition and the colony diameter of the tested pathogenic fungi.
` tre des colonies des chamPourcentage dinhibition et diame
` nes teste
s.
pignons pathoge
Treatment
Colony diameter
(mm)
mean S.D.
Pathogens alone
Fusarium solani
in biculture
Rhizoctonia solani
in biculture
Sclerotium rolfsii
in biculture
8.97
6.15 1.15 a
31.44
6.50 1.15 a
27.54
7.00 1.00 a
21.96
DPPH concentrations
10 mg
50 mg
100 mg
43.5
66.7
6.9
19
53
80.8
88.9
42
42
81.9
95.63
92
52
53.5
93.9
e37
Inhibition %
organisms (bacteria/fungi), while 100 mg/disc of the petroleum ether and ethyl acetate extracts inhibited the growth
of the given bacteria/fungi. The zone of inhibition was
determined and this concentration was considered as the
minimum inhibitory concentration (MIC). Concentration of
100 mg/disc of the petroleum ether extract exhibited noticeable antibacterial activity, giving remarkable inhibition
zone on Bacillus subtilis, Escherichia coli and Pseudomonas
fluorescens but less than the standard ampicillin. Maximum
antifungal effect against Candida albicans, R. solani and
Pythium ultimum were seen, while moderate antifungal
effect against F. solani and Fusarium oxysporium were
recorded in comparison to the standard antifungal fluconazole drug.
Concentration of 100 mg/disc of the ethyl acetate extract
gave noticeable antibacterial effect and highest antifungal
effect against C. albicans, F. solani and P. ultimum, while
moderate effect against F. oxysporium and R. solani were
seen in comparison to the standard antifungal fluconazole
drug.
With increasing the concentration of the petroleum ether
and ethyl acetate extracts to 200, 300 and 400 mg/disc, the
zones of the inhibition were increased as shown in Tables 3
and 4.
[(Figure_1)TD$IG]
Figure 1 Biculture antagonistic test between Chaetomium globosum and Fusarium solani (a), Rhizoctonia solani (b) and Sclerotium
rolfsii (c).
Test de biculture antagoniste entre Chaetomium globosum et Fusarium solani (a), Rhizoctonia solani (b) et Sclerotium rolfsii (c).
e38
Table 3 Antibacterial effects of petroleum ether and ethyl acetate extracts of the liquid culture of Chaetomium globosum.
riens des extraits de liquide de culture de Chaetomium globosum.
Effets antibacte
Treatment
Concentration
(mg/disc)
Escherichia coli
Pseudomonas fluorescens
100
200
300
400
14 0.56
14 0.584 a
15 0.54 a
16 0.58 a
13 1.12
15 0.85 a
16 1.00
18 1.92
14 0.92 a
14 1.05 a
15 1.01 a
17 1.62
100
200
300
400
15 0.75 a
16 1.05 a
17 1.26
20 1.42 a
15 1.03 a
16 0.80
17 0.97
19 1.69 a
15 0.87 a
15 0.96 a
16 0.93
20 1.62 a
Standard drug
100
18
17
17
Data are mean S.D. of triplicate reading of the inhibition zone diameter.
a
Significantly different from the control at P 0.05 using one way ANOVA test.
Table 4 Antifungal effects of petroleum ether and ethyl acetate extracts of the liquid culture of Chaetomium globosum.
Effets antifongiques des extraits de liquide de culture de Chaetomium globosum.
Treatment
Concentration
(mg/disc)
Fusarium
solani
Fusarium
oxysporium
Rhizoctonia
solani
Pythium
ultimum
100
200
300
400
16 0.97 a
17 1.11 a
18 1.12 a
20 1.59 a
14 1.00 a
15 5.78 a
18 1.16 a
18 5.78 a
14 1.83 a
15 1.38 a
16 1.092 a
17 0.55 a
13 0.49 a
14 0.51 a
16 0.99 a
16 0.87 a
11 1.28 a
12 1.28 a
15 1.13 a
17 0.75 a
100
200
300
400
15 1.40 a
17 1.70 a
17 1.08 a
20 1.40 a
15 1.42 a
16 1.63 a
17 1.00 a
18 1.28 a
13 1.33
14 1.54 a
14 1.28 a
15 1.51 a
12 1.34
13 1.54 a
17 1.23 a
18 1.43 a
13 1.35 a
15 1.46 a
16 1.17 a
19 1.44 a
Standard drug
100
12 0.5574
13 0.6345
12 1.5956
11 1.3455
Data are mean S.D. of triplicate reading of the inhibition zone diameter.
a
Significantly different from the control at P 0.05 using one way ANOVA test.
8 1.4649
e39
Table 5 Cytotoxic effects, LC50 and LC90 of the petroleum ether and ethyl acetate extracts of the liquid culture of Chaetomium
globosum.
Effets cytotoxiques, CL50, CL90 des extraits de liquide de culture de Chaetomium globosum.
Group
LC50 (mg/mL)
LC90 (mg/mL)
Cytotoxicity effect
(100 mg/mL) (%)
11.8
45.9
21.6
25.0
76.8
37.8
100
96.3
100
LC50: lethal concentration of the sample, which causes the death of 50% of cells in 48 hrs; LC90: lethal concentration of the sample, which
causes the death of 90% of cells in 48 hrs.
[(Figure_2)TD$IG]
Figure 2 Antioxidant (a) and antimicrobial screening of all the eluted fractions from the column of the ethyl acetate extract on
Bacillus subtilis (b), Escherichia coli (c), Fusarium solani (d) and Rhizoctonia solani (e).
lution de la colonne dextrait en ace
tate de
thyle sur
Test de screening antioxydant (a) et antimicrobien de toutes les fractions de
Bacillus subtilis (b), Escherichia coli (c), Fusarium solani (d) et Rhizoctonia solani (e).
e40
the antibacterial effect of chrysophanol isolated from the
leaves of Aloe excels against B. subtilis, S. epidermidis and
E. coli.
Chrysazin is obtained as orange solids, Rf value 0.85 in
(benzene: ethyl acetate 8:2) as solvent system and the
molecular formula is assigned as C14H8O4 as deduced from
electron impact (EI) mass spectrum which exhibited a molecular ion at m/z 240 and major fragment ions at m/z values
of 212, 197, 169, 139, 121, 113, 83, 71, 57. The UV spectrum
displayed an absorption maximum lmax (MeOH) at: 225, 255,
285, 430 nm; The IR spectrum in (KBr) showed characteristic
bands at 3347, 3078, 1647, 1590, 1465, 1443, 1296, 1263,
1217, 1030, 638 cm1. In a previous study by Phonkerd et al.
[16], chrysazin was also isolated from the ethyl acetate
extract of C. globosum. On the other hand, two major
compounds, chaetoviridin A and B are isolated from fraction
3 and purified on preparative TLC using benzene: ethyl
acetate (8:2) as solvent system.
Chaetoviridin A is obtained as brown solid, Rf value 0.84 in
(benzene: ethyl acetate 8:2) and the molecular formula is
assigned as C23H25O6Cl as deduced from the electron impact
(EI) mass spectrum which exhibits a molecular ion at m/z 432
and major fragment ions at m/z values of 389, 345, 332, 316,
289 and 234.
Chaetoviridin B is obtained as yellow solid, Rf value 0.79 in
(benzene: ethyl acetate 8:2) as solvent system and the
molecular formula was deduced as C23H27O6Cl, The EI mass
spectrum displayed a molecular ion at m/z 434 and major
fragment ions at m/z 419, 400, 390, 355, 289 and 263. In
2005, chaetoviridins were isolated and identified [15]. These
compounds contain a pyrone-quinone structure (azaphilones) that recorded antifungal activity with more potent
inhibitory effect of chaetoviridin A than chaetoviridin B [15].
Prenisatin
Chrysophanol
Chrysazin
Chaetoviridin A
Chaetoviridin B
Escherichia
coli
Rhizoctonia
solani
13
12
12
15
14
13
13
12
15
14
13
14
13
15
14
Figure 3 Antioxidant (a) and antimicrobial screening of the compounds isolated from the ethyl acetate extract of the liquid culture
of Chaetomium globosum on Bacillus subtilis (b), Escherichia coli (c) and Rhizoctonia solani (d).
Test de screening antioxydant (a) et antimicrobien de tous les produits de lextrait du liquide de culture de Chaetomium globosum en
tate de
thyle sur Bacillus subtilis (b), Escherichia coli (c) et Rhizoctonia solani (d).
ace
% inhibition percentages
control sample=control 100
Antifungal screening of C. globosum fungi
Biculture test
Biculture test was done following the methods of Soytong
and Quimio [18]. A virulent isolate of C. globosum was used in
biculture test with different antagonistic fungi.
An agar disc taken from the edge of radial growth of
F. solani, R. solani, or S. rolfsii separately in PDA plate was
obtained using a sterile cork borer and placed in one side of a
potato dextrose agar (PDA) plate about 2.0 cm from the
center. An agar disc of C. globosum, the antagonistic fungus,
was placed on the other side of the plate. For control
treatment, the agar plug of only pathogen was placed on
PDA plates. The biculture plates were incubated at room
temperature until colony of control grew to full plate. At this
point, colony diameter was measured using ruler. Percentage of the growth inhibition was calculated using the formula below:
e41
1-reading of extract=
reading of negative control 100:
A probit analysis was carried for IC50 and IC90 determination using SPSS 11 program.
e42
UV-VIS double beam UVD-3500 spectrophotometer, Labomed, Inc. IR spectra (KBr) were recorded on a Jasco FTIR4100.
Conclusion
C. globosum exerted an inhibitory activity against the mycelial growth of F. solani, R. solani and S. rolfsii. So, it can be
used clearly as a potential antagonist of various plant pathogens. Petroleum ether and ethyl acetate extracts of the
liquid culture of C. globosum proved significant antioxidant,
antimicrobial activity and cytotoxic effect on human hepatocellular carcinoma cell line. Undecyl benzene and ergosterol are the major compounds of the unsaponifiable
fraction, while methyl tetradecanoate and methyl 9-tetradecanoate are identified as the major saturated and unsaturated fatty acids in the saponifiable fraction respectively.
Prenisatin, chrysophanol, chrysazin, chaetoviridin A and
chaetoviridin B are isolated from the ethyl acetate extract
of the liquid culture of C. globosum and proved significant
antioxidant and antimicrobial activity on B. subtilis, E. coli
and R. solani.
Disclosure of interest
The authors declare that they have no conflicts of interest
concerning this article.
References
[1] Aboul-Fadl T, Bin-Jubair FAS. Anti-tubercular activity of isatin
derivatives. Int J Res Pharm Sci 2010;1:11326.
[2] Adams RP. Identification of essential oils by ion trap mass
spectroscopy. New York: Academic Press, Inc.; 1989.
[3] Breinholt J, Demuth H, Heide M, Jensen GW, Mller IL, Nielsen
RI, et al. Prenisatin (5-(3-methyl-2-butenyl)-indole-2, 3-dione):
an antifungal isatin derivative from Chaetomium globosum.
Acta Chem Scand 1996;50:4435.
[4] Chen Y, Wang M, Rosen RT, Ho CT. 2,2-Diphenyl-2-picrylhydrazyl radical-scavenging active components from Polygonum multiflorum Thunb. J Agric Food Chem 1999;47:22268.
[5] Coopoosamy RM, Magwa ML. Antibacterial activity of chrysophanol isolated from Aloe excelsa (Berger). Afr J Biotechnol
2006;5:150810.