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Journal de Mycologie Mdicale (2014) 24, e35e42

Available online at

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ORIGINAL ARTICLE/ARTICLE ORIGINAL

Bioassays guided isolation of compounds


from Chaetomium globosum
s de Chaetomium globosum base
e sur des
Isolation des compose
tests biologiques
N.E. Awad a, H.A. Kassem b, M.A. Hamed c,*, M.A.A. El-Naggar d,
A.M.M. El-Feky a
a

Pharmacognosy Department, National Research Center, Dokki, Cairo, Egypt


Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
c
Therapeutic Chemistry Department, National Research Center, El-Tahrir Street, 12311 Dokki, Cairo, Egypt
d
Plant pathology Department, National Research Center, Dokki, Cairo, Egypt
b

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

N.E. Awad et al.


Re
sume
Le but de cette tude tait dvaluer les diffrentes activits biologiques du
champignon Chaetomium globosum (famille Chaetomiaceae). Lvaluation a t faite en testant
les effets antibactriens, antioxydants et anticancreux. C. globosum a t isol dun concombre du sol (rhizosphre) et inhibe la croissance myclienne de Fusarium solani, Rhizoctonia
solani et Sclerotium rolfsii dans un test de biculture. Les produits dextraction du liquide de
culture de C. globosum par lther de ptrole et lactate dthyle ont montr in vitro une forte
activit antioxydante. C. globosum a exerc une puissante activit antibactrienne contre
Bacillus subtilis, Escherichia coli et Pseudomonas fluorescens. Une nette activit antifongique a
galement t note contre Candida albicans, F. solani, Fusarium oxysporum, R. solani et
Pythium ultimum. Les extraits avaient une action cytotoxique sur une ligne cellulaire dhpatocarcinome humain (HepG2). Les substances insaponifiables et saponifiables issues de lextraction par lther de ptrole ont rvl la prsence dhydrocarbones, de strols et dacides gras.
Les extraits thyle actate ont montr la prsence de prnisatine, chrysophanol, chrysazine,
chaetoviridine A et B. Les mtabolites secondairement isols ont montr une activit significative antioxydante et antibactrienne sur B. subtilis, E. coli et R. solani. En conclusion : ce
champignon possde plusieurs des activits biologiques diffrentes. Des tudes complmentaires doivent tre menes pour son utilisation en agriculture et en mdecine.
# 2013 Elsevier Masson SAS. Tous droits rservs.

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

biologically active secondary metabolites from the liquid


culture of C. globosum will be done and the antioxidant
and antimicrobial activities of the isolated compounds will
be tested.

Results and discussion


Fungal isolation and identification
The isolate was identified as C. globosum based on the
criteria of Kirk et al. [13]. The colony was grayish white
with a cottony texture, having black dots on the surface.
The microscopic examination of C. globosum revealed
that the hyphae were septate with pale brown color. Perithecia were oval brown to black color and surrounded by long
helical filamentous appendages. The asci were stalked clavate and the ascus walls were evanescent. The ascospores
were dark brown, oval shape with single cell of no
septum.

Successive extracts of C. globosum


The dried petroleum ether and ethyl acetate extracts of
liquid culture of C. globosum yield 0.25 and 0.4 g/L,
respectively.

Antioxidant screening of all the successive


extracts
Petroleum ether extract of the liquid culture of C. globosum
recorded inhibitory effect of DPPH radicals by 66.7, 88.9 and
92% at 10, 50 and 100 mg/mL (absolute ethanol), respectively, while the ethyl acetate extract of the liquid culture of
C. globosum recorded 53, 81.9 and 93.9% inhibition at 10, 50
and 100 mg, respectively. These results are very close to that
of Vit. C, as a standard antioxidant agent. As a result,
petroleum ether and ethyl acetate extracts of the liquid
culture of C. globosum were considered as the most active
extracts showed antioxidant effect in comparison with the
other successive extracts (Table 1).

Bioassays guided isolation of compounds from Chaetomium globosum


Table 1 In vitro inhibition of DPPH after treatment with
the successive extracts of liquid culture of Chaetomium
globosum in serial concentrations.
` s traitement avec les extraits
Inhibition in vitro de DPPH apre
successifs du liquide de culture de Chaetomium globosum en
rie
es.
dilutions se
Group

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

Data are inhibition percentages (%) of DPPH free radicals at


different concentrations.

e37

Inhibition %

Data are mean  S.D. of triplicate reading of the colony diameter.


a
Significantly different from the control at P  0.05 using one
way ANOVA test.

Antifungal screening of C. globosum fungi


Biculture of C. globosum, Fusarium solani, Rhizoctonia
solani and Sclerotium rolfsii had been done to determine
the possible interaction between the biological antagonist of
C. globosum and F. solani, R. solani and S. rolfsii fungi
(Fig. 1). Table 2 presented the colony diameter and the
percent of inhibition. In the control plate, the pathogenic
fungi grow faster and significantly formed larger colony
diameter with a mean of 8.97 cm, while those in the biculture plate produced smaller colony with a mean diameter of
6.15 cm for F. solani, 6.5 cm for R. solani and 7.00 cm for
S. rolfsii. C. globosum; the fungal antagonist caused 31.44,
27.54 and 21.96% inhibition of mycelial growth of F. solani,
R. solani and S. rolfsii, respectively.
This result suggests that C. globosum can exert inhibitory
effect on the mycelial growth of F. solani, R. solani and
S. rolfsii.

Antimicrobial activity of the noticeable and


remarkable extracts
Both petroleum ether and ethyl acetate extracts of the
liquid culture of C. globosum were tested in 50, 100, 200,
300 and 400 mg/disc. Concentration of 50 mg/disc showed no
significant inhibition zone on the tested pathogenic

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

N.E. Awad et al.

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)

Diameter of inhibition zone (mm)


mean  S.D.
Bacillus subtilis

Escherichia coli

Pseudomonas fluorescens

Petroleum ether extract

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

Ethyl acetate extract

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.

Cytotoxic effect of the noticeable and


remarkable extracts
Petroleum ether and ethyl acetate extracts of the liquid
culture of C. globosum exhibited significant cytotoxic
effect on human hepatocellular carcinoma cell line
(HepG2) (Table 5). Petroleum ether extract showed
100% killing of the HepG2 cell line, while ethyl acetate
extract recorded a ratio of 96.3% as compared to the
standard reference natural drug; Adrinamycin.

Identification of the petroleum ether extract


The yields of the unsaponifible matter and the saponifible
matter were 57.5% and 27.5% w/w of the dried petroleum
ether extract of the liquid culture of C. globosum, respectively. Identification of the constituents was carried out by

comparison of their spectral fragmentation patterns with


those of the available database libraries Wiley (Wiley Int.,
USA) and NIST (Nat. Inst. St. Technol., USA) and/or published
data [2]. GC/MS analysis of the unsaponifiable matter of
liquid culture of C. globosum revealed the identification of
27 compounds, which amounting 94.31% of the total unsaponifiable matter. Undecyl benzene and ergosterol were
presented as major compounds of the unsaponifiable matter
of C. globosum.
GC/MS analysis of the saponifiable matter of the liquid
culture of C. globosum revealed the identification of nineteen compounds, representing 79.48% of the total saponifiable matter, saturated fatty acids constituted 68.75%,
while unsaturated fatty acids constituted 10.73%. Methyl
tetradecanoate (17.11%) was the major saturated fatty acid,
while Methyl 9-tetradecanoate (4.34%) was the major unsaturated fatty acid.

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)

Diameter of inhibition zone (mm)


mean  S.D.
Candida
albicans

Fusarium
solani

Fusarium
oxysporium

Rhizoctonia
solani

Pythium
ultimum

Petroleum ether extract

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

Ethyl acetate extract

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

Bioassays guided isolation of compounds from Chaetomium globosum

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) (%)

Petroleum ether extract


Ethyl acetate extract
Positive control (Adrinamycin)

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.

Isolation of the secondary metabolites from the


ethyl acetate extract
The screening of all the eluted fractions from the column of
the ethyl acetate extract revealed that the fractions 2 and 3
eluted by chloroform: methanol (9.5:0.5 and 9:1) gave
significant antioxidant and antimicrobial activity against
B. subtilis, E. coli, F. solani and R. solani more (Fig. 2).
Therefore, both fractions 2 and 3 had been monitored on TLC
separately, using benzene: ethyl acetate (8:2) as solvent
system and the major secondary metabolites were isolated.

Identification of the secondary metabolites from


the ethyl acetate extract
Three major compounds have been isolated from the fraction 2 and purified on preparative TLC using benzene: ethyl
acetate (8:2) as solvent system. These compounds were
prenisatin, chrysophanol and chrysazin.
Prenisatin (5-(3-methyl-2-butenyl)-indole-2,3-dione) is
obtained as orange solids, Rf value 0.67 in (benzene: ethyl
acetate 8:2), the molecular formula has been assigned as
C13H13NO2 as deduced from electron impact (EI) mass spectrum which exhibited a molecular ion at m/z 215 and major

fragment ions at m/z values of 200, 187, 172, 158, 144,132,


129, 117, 91, 77, 56, 42. The IR spectrum in (KBr) showed
characteristic bands at 3267, 2923, 1747, 1730, 1617,
1498 cm1. In 1996, preisatin was isolated from the ethyl
acetate extract of C. globosum and proved to have antifungal
activity [3]. It was reported that the compounds with an
indole moiety had considered as radical scavengers agent
and had antioxidant [9] and antitubercular activity [1].
Chrysophanol (1,8-dihydroxy-3-methyl anthraquinone) is
obtained as reddish orange solid, Rf value 0.80 in (benzene:
ethyl acetate 8:2) as solvent system and the molecular
formula is assigned as C15H10O4 as deduced from electron
impact (EI) mass spectrum which exhibited a molecular ion at
m/z 254. The major fragment ions at m/z values of 139, 121,
112, 104, 83, 71, 57. The UV spectrum displayed an absorption maximum lmax (MeOH) at: 225, 253, 276 nm. The IR
spectrum in (KBr) showed characteristic bands at 3448, 3020,
2923, 1647, 1510, 1396, 1117, 1030, 990 and 855 cm1. In
2011, chrysophanol was isolated from the ethyl acetate
extract of Aspergillus and recorded in vitro protective effect
against ethanol-induced toxicity [17]. In addition, GarcaSosa et al. [7] recorded the antibacterial and antifungal
activities of chrysophanol isolated from the root of Colubrina
greggii. Moreover, Coopoosamy and Magwa [5] postulated

[(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].

Antioxidant and antimicrobial screening of the


isolated compounds
The compounds isolated from the ethyl acetate extract of
the liquid culture of C. globosum gave significant antioxidant
activity where it acted as free radicals scavenging agent. It
was also had antimicrobial activity on B. subtilis, E. coli and
R. solani (Table 6 and Fig. 3).

Materials and methods


Fungal isolation and identification
C. globosum was isolated from the Cucumber soil (rhizos[(Figure_3)TD$IG]phere), Omar Makram farm (Beheara Governorate, Egypt).

N.E. Awad et al.


Table 6 Antimicrobial activity of the compounds isolated
from the ethyl acetate extract of the liquid culture of
Chaetomium globosum.
s antibacte
rienne des extraits de liquide de culture
Activite
de Chaetomium globosum.
Treatment

Prenisatin
Chrysophanol
Chrysazin
Chaetoviridin A
Chaetoviridin B

Diameter of inhibition zone (mm)


Bacillus
subtilis

Escherichia
coli

Rhizoctonia
solani

13
12
12
15
14

13
13
12
15
14

13
14
13
15
14

The isolate was incubated on potato dextrose agar (PDA;


Becton and Dickinson Co., MA, USA) at 28 8C for 15 days with
12 h of illumination daily. Mycelial fragments and spores
were collected, placed on glass slides and observed. The
isolate was identified based on the criteria described by Kirk
et al. [13].

Preparation of the fungal successive extracts:


Erlenmeyer flasks (1 liter) containing 200 mL potato dextrose
broth were autoclaved at 121 8C for 15 min and then inoculated with mycelium plugs from a 5-day-old culture of
C. globosum on PDA. The flasks were incubated for 2 weeks
at 25 8C. After filtration through Whatman No. 1 filter paper,
the culture filtrate was successively extracted with petroleum ether, diethyl ether, chloroform and ethyl acetate. The
successive extracts were concentrated to dryness to be
tested for antioxidant, antimicrobial and anticancer
activities.

Antioxidant screening of the successive extracts


The antioxidant activity of serial concentrations of the
different successive extracts of liquid culture of
C. globosum (10, 50, 100 mg) was estimated by the method
of Chen et al. [4], using DPPH free radical (40 mg of DPPH in
10 mL of 70% ethanol). The in vitro inhibition percentages of
DPPH radical is assayed spectroscopically based on the
measurement of the color intensity at 518 nm against blank
solution and compared with standard Vit. C of the same serial
concentrations. The decrease in optical density of DPPH
was calculated in relation to control as follows:

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

Bioassays guided isolation of compounds from Chaetomium globosum

% 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:

% inhibition A  B=A  100


where: A = colony diameter of pathogen in control;
B = colony diameter in biculture.

e41

the experiment was done under aseptic conditions. Bacterial


plates were incubated at 30 8C for 24 hours, while fungal
plates were incubated at 28 8C for 48 hours. The diameter of
the inhibition zone was recorded for each replicate and the
average diameter was calculated.

Cytotoxic effect of the noticeable and


remarkable extracts
Cytotoxic effect of the most active extracts was performed
on human hepatocellular carcinoma cell line (HepG2). Cell
viability was assessed by the mitochondrial dependent
reduction of yellow MTT (3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyl tetrazolium bromide) to purple formazan according to Mosmann [14]. A positive control (Adrinamycin) was
used as a known cytotoxic natural agent [20]. The experiment was performed in a sterile area using a laminar flow
cabinet biosafety class II level (Baker, SG403INT and Sanford,
ME, USA). The percentage of change in viability was calculated according to the formula:

1-reading of extract=
reading of negative control  100:
A probit analysis was carried for IC50 and IC90 determination using SPSS 11 program.

Identification of the petroleum ether extract


Antimicrobial activity of the noticeable and
remarkable extracts
The antimicrobial activity of the most active extracts was
performed using antibiotic assay method [20]. The antibacterial activity was tested against B. subtilis (Gram-positive
bacteria), E. coli and P. fluorescens (Gram-negative bacteria) on nutrient agar medium. The antifungal activity was
tested against the yeast C. albicans and the phytopathogenic
fungi (F. solani, F. oxysporium, R. solani and P. ultimum)
using Sabouraud dextrose agar medium.
Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) of the most
active extracts was determined by antibiotic assay method.
Nutrient agar media were prepared and sterilized, then
distributed in sterile petri dishes, each of 12 cm diameters.
Each suspension of the test organisms was separately inoculated into the surface of a number of petri dishes. Each
antibiotic assay disc (6 mm) was loaded with 50, 100, 200,
300 and 400 mg/disc of the tested extracts of the liquid
culture of C. globosum in dimethyl sulfoxide (DMSO) and
firmly applied to the surface of the inoculated agar plates.
The plates were observed for the growth of microorganisms.
The lowest concentration of the extracts inhibit the growth
of the given bacteria/fungi was determined and considered
as the minimum inhibitory concentration (MIC).
Antibiotic assay method
The most active extracts (100 mg/disc) were screened in
vitro for their antimicrobial activity against the microorganisms under study (bacteria and fungi) by antibiotic assay
method [8]. Ampicillin (100 mg/disc) standard antibacterial
and fluconazole (100 mg/disc) standard antifungal were used
as reference drugs. This assay was replicated three times. All

Saponification of the petroleum ether extract of the liquid


culture of C. globosum was performed according to Tsuda
et al. [21].
Study of the unsaponifiable and saponifiable matters of
the petroleum ether extract was carried out to identify the
sterols and/or terpenes, as well as the fatty acids using GC/
MS technique [Gas chromatograph coupled with a mass
spectrometer was performed on a model Shimadzu GC/
MSQP5050A].

Isolation of the secondary metabolites from the


ethyl acetate extract
Three grams of dried ethyl acetate extract of the liquid
culture of C. globosum was dissolved in the least amount
of ethyl acetate, loaded onto a silica gel column (120 cm
height  2.5 cm i.d.) containing 175 g activated silica (70
230 mesh; E. Merck, Darmstadt, Germany). Elution was
successively carried out by absolute chloroform, chloroform:
methanol (9.5:0.5, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8,1:9)
and absolute methanol. Fractions each of 15 mL were successively collected and concentrated to 5 mL and screened
by TLC, using benzene: ethyl acetate (8:2) as solvent system.
All the fractions were tested for antioxidant screening on
TLC using DPPH reagent and antimicrobial activity using
antibiotic assay method on B. subtilis, E. coli, F. solani and
R. solani. The most active fractions were monitored on TLC,
using benzene: ethyl acetate (8:2) as solvent system and the
major secondary metabolites were isolated.
Identification of the isolated compounds was determined
by spectroscopic analysis. Mass spectra were recorded on a
double-focusing high-resolution (HR) mass spectrometer
(Finnigan Model 3200 at 70 eV). UV was obtained on a

e42
UV-VIS double beam UVD-3500 spectrophotometer, Labomed, Inc. IR spectra (KBr) were recorded on a Jasco FTIR4100.

Antioxidant and antimicrobial screening of the


isolated compounds
The isolated compounds were tested for antioxidant screening on TLC using DPPH reagent and antimicrobial activity
using antibiotic assay method on B. subtilis, E. coli, F. solani
and R. solani.

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.

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