Antifungal Activity of Methanolic Extract of Calliandra calothyrsus
against Storage Fungi Isolated from Dried-stored Spices
Kiki Nurtjahja
1
and Albert Pasaribu
2
1
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia
2
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia
Keywords: Antifungal, Calliandra calothyrsus, Postharvest Fungi, Spices.
Abstract: Methanolic extract of Calliandra calothyrsus (Leguminoceae) was conducted. The aim was to investigate
the presence of flavonoid and the ability of the crude extract to inhibit mycelial growth and conidial
germination of storage fungi. Eight fungal species were isolated from ten kinds of dried spices obtained
from traditional markets in Medan North Sumatera. Fungal isolation was conducted using dilution method
in dichloran 18% glycerol agar (DG18) medium. Plant methanolic extract obtained from 4 kg fresh leaves of
Calliandra calothyrsus. The leaves were dried under shade to constant weight and ground using electrical
grinder to a 1.2 kg fine powder and dissolved in ±15 l of methanol (MeOH) for 48 hours. and dried using
rotary evaporator. For qualitative flavonoid determination, 10 g of fine powder of the leaves was dissolved
100 ml methanol and suspended for 12 hours, then acidified with 6% hydrochloric acid (HCl). The presence
of flavonol was identified using Ultra Violet Visible Spectroscopy (UV-Vis). Results showed methanolic
extract of Calliandra calothyrsus contain flavonoids group of flavonol. The crude extract have no antifungal
activity againts storage fingi. However, all of the concentration inhibit conidial germination to all fungi
tested.
1 INTRODUCTION
Spices are one of commodities that susceptible
infected by fungi (Škrinjar et al. 2012; Toma and
Abdulla 2013). Most genera of storage fungi are able
to colonize stored spices at low water activity (a
w
) (≤
0.85) (Pitt and Hocking 2009). The infection
occurred during preharvest, harvesting and
postharvest handling. Field fungi such as Fusarium,
Alternaria, Helmintosporium, Cladosporium cause
damage on crops during harvesting while moisture
content of the commodities still high, while storage
fungi such as Aspergillus and Penicillium infect
during storage with low a
w
(Nurtjahja et al. 2017).
The damage of spices by storage fungi cause
moldiness, color change, aroma, taste and
contaminated by mycotoxins. Flavonoids are
secondary metabolite derivatives of 2-phenyl-
benzo[α]pyrane synthesized in all parts of the plants
(Seleem et al. 2017). They play a role to provide
color to attract pollinator in flower, fragrance and
taste of the fruit and protect plant from UV radiation
and pathogenic fungi Mierziak et al. (2014).
The toxicity of flavonoids inhibit mycelial
growth and spore germination is an alternative to
control fungal infection and mycotoxin
contamination on dried-stored spices. Molds control
using chemicals such as fumigant such as ethylen
oxide dan sulphur dioxide commonly used, however,
the chemicals leave residue that are toxic to human.
Fungal biological control using phytochemical is
potential as an alternative to prevent fungal infection
on spices during storage. The purpose of the study
was to investigate antifungal activity crude
methanolic extract of Calliandra calothyrsus in
inhibiting mycelial growth and fungal population of
storage fungi isolated from dried spices.
2 MATERIALS AND METHOD
2.1 Isolation of Fungi
Ten kinds of dried spices were obtained from
traditional markets in Medan, North Sumatera. Each
fungal species on each kind of spice was isolated by
538
Nurtjahja, K. and Pasaribu, A.
Anti-fungal Activity Methanolic Extract of Calliandra calothyrsus against Storage Fungi Isolated from Dried-stored Spices.
DOI: 10.5220/0010612600002775
In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 538-541
ISBN: 978-989-758-556-2
Copyright
c
 2022 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved

dilution method followed by pour plate method in
dichloran 18% glycerol agar (DG18 medium) (5 g/l
peptone, 10 g/l glucose, 1 g/l KH
2
PO
4
, 220 g/l
glycerol, 0.5 g/l MgSO
4
.7H
2
O, 15 g/l bacto agar, 2.0
mg/l dichloran and 100 mg/l chloramphenicol (Pitt
and Hocking 2009). Twenty five gram of each
sample in 500 ml erlenmeyer was diluted in 250 ml
sterilized distilled water. The suspension was
homogenized using shaker (Gallenkamp, orbital
shaker SG92, England) 100 rpm for 2 minutes.
Four dilutions, 10
-1
, 10
-2
, 10
-3
and 10
-4
were made.
From the 10
-3
or 10
-4
dilution, 1 ml was transfered
onto petridish (diameter 9 cm) and pour plated in
DG18 medium. The plates were incubated at
ambient temperature (29±2ºC) for 6 days. All
colonies were counted as colony forming unit per
gram (cfu/g) of the sample. Each single separate
colony was isolated and cultured on czapex yeast
extract agar (CYA) or CYA+20% sucrose
(CYA20S) and identified. Macroscopic and
microscopic identification of each fungal species
were conducted according to Pitt and Hocking
(2009).. All of the storage fungi are culture
collection of Microbiology Laboratory, Biology
Department, Sumatera Utara University. Each fungal
isolates was kept on potato dextrose agar (PDA)
slants and subcultured before used.
2.2 Plant Material
Calliandra calothyrsus was obtained from
abandoned area 1300 meter above sea level at
Berastagi, Karo Regency, North Sumatera Province,
Indonesia.
2.3 Plant Extraction
Methanol extract of Calliandra calothyrsus was
prepared according to Larson et al. (2016). Four
kilogram of the fresh leaves were air dried to
constant weight and ground using electrical grinder
to 1.2 kg fine powder and dissolved in 15 l 96%
methanol (MeOH) for 48 hours. The simplisia was
then filtered using Whatman’s No.1 filter paper,
removed to clean vessel and concentrated using
rotary evaporator (Heidolph, Instruments GmbH and
Co, KG Walpersdorfer Str, Germany). This was
repeated three times. The extract appears as semi-
solid greenish paste.
2.4 Flavonoid and Flavonol Test
For test flavonoid, 10 g fine powder of the leaves
was extracted with 100 ml 96% methanol and
suspended for 12 hours, the extract was filtered
using Whatman’s No.1 filter paper and hydrolized
by hydrochloric acid (HCL) 6% in warm condition
for 1 hour. Immediate development of red color
indicates the presence of flavonoids. The flavonol in
methanol extract was identified both using
Ultraviolet-Visible spectroscopy (UV-Vis) (200 to
500 nm) and nuclear magnetic resonance proton
spectrum (
1
H-NMR).
2.5 Antifungal Test
Paper disc diffusion inhibition method was used to
determine antifungal activity of the extract in petri
dish 9 cm diameter. Agar plugs (5.0 mm in
diameter) of potato dextrose agar plate containing 5
days fungal mycelia were made. Plant extract was
dissolved in DMSO to make concentration 0, 20, 40,
60, 80 and 100%. Antifungal ketoconazole (10
µl/mL) were used as the reference standard. Steril
paper discs (5.0 mm) were impregnated with each
extract concentration and allowed to dry before
being placed in PDA plate. The agar plugs and discs
were placed in petridishes (9 cm diameter)
containing PDA plates. This was repeated three
times. All plates were incubated at 29°C for 6 days
and observed for zone inhibition. The diameter of
the zones were determine in millimeter (mm).
2.6 Germination Inhibition Test
Ten mL of semi-solid potato dextrose agar medium
containing plant extract at concentration 0, 20, 40,
60 and 80% and ketoconazole (10 µl/mL) were
made. Conidial suspension of each storage fungi
were prepared by washing 7 days old PDA cultures
with 10 mL steril distilled water containing 0.05%
Tween 80. The concentration of conidia was
adjusted to 1×10
7
and 1 mL of the conidial
suspension was mixed thoroughly with the semi-
solid PDA containing plant extract. Three drops of
conidial suspension of each fungal species were
cultured on sterilized microscope glass slide. All of
the glass slides were covered with cover glass then
were incubated for 20 h at ambient temperature
(29°C). Each was repeated three times. The
percentage of germinating conidia was observed
microscopically after 20 h by counting 100 conidia.
Germinating conidia were considered based on the
presence of germ tubes at the germ pore.
Anti-fungal Activity Methanolic Extract of Calliandra calothyrsus against Storage Fungi Isolated from Dried-stored Spices
539

3 RESULTS AND DISCUSSION
3.1 Inhibition Zone of Plant Extract
against Postharvest Fungi
The presence of the secondary metabolites C.
calothyrsus showed no inhibition on the growing
mycelia except for mycelia of Aspergillus candidus
that inhibited begin at 40% extract concentration
(Table 1). The highest crude leaves extract (80%)
have no effect on the mass growing mycelia of the
other fungal species. In contast to ketoconazole, all
mycelia of storage fungi were inhibited and
Aspergillus tamarii was the most inhibited.
However, the effect of the extract reduced fungal
population particularly on A. chevalieri, A. flavus, A.
niger and A. tamarii (Table 2).
Flavonoids are group of polyphenolic
compounds having a benzo-γ-pyrone that commonly
found on plants. They are synthesized in response
microbial infection (Dixon et al. 1983). Flavonoids
consist of chalcone, flavon, isoflavon, flavonol,
flavanon, flavonol, and antocyanidin (Andrae-
Marobela et al. 2013) The ability of flavonoid as
antimicrobe was reported by Yigit et al. (2009).
Flavonoids namely 3.7.5’-trihidroksi anthocyanidin
and 3.5-dihidroksi-7-metoksi antocyanidin isolated
from Monanthotaxis littoralis (Annonaceae) inhibit
mycotoxigenic fungi such as Aspergillus, Fusarium
and Penicillium isolated from maize (Clara et al.
2014).
Based on analysis using UV-Vis spectroscopy
and
1
H-NMR, we assumed the methanolic extract of
C. calothyrsus contain flavonoid group of flavonol.
Flavonoids including flavanones, flavones and
flavans has antifungal activity. Purified flavon
inhibit mycelial growth and conidial germination
was reported by Cotoras et al. (2011). The used of
the crude extract and low concentration the
flavonoid compounds in the plant might the
antifungal have no effect on growing mycelia.
Our results is in corcordance to Steinkellner and
Mammerler (2007), they report that low flavonoid
concentration exhibit slight antifungi properties
againts Fusarium oxysporum f. sp. lycopersici. The
effect of purified flavon on mycelial growth and
conidial germination of Botrytis cinerea was studied
by Cotoras et al. (2011), they found that the mycelia
of B. cinerea stop growing after six days incubation
without reaching maximum radial growth. Dorta et
al. (2005) reported that the presence of flavonoid
surrounding fungal mycelia and conidia interact with
the plasma membrane and it affects mitochondrial
respiratory chain. Low concentration of the
secondary metabolites was indicated with no
inhibitory effect on mycelial growth of storage
fungi,
However, both of the compounds inhibit fungal
population to most of all storage fungal tested. The
use of the crude extract prevent fungal population,
therefore, spoilage and mycotoxin contamination of
foodstuffs during storage can be prevented. Basil
leave with concentration lower than 85 % has no
effect on all fungi tested. The minimum inhibition of
the extract begin to inhibit fungal mycelia at
concentration 85 % particularly on Aspergillus
candidus and Aspergillus tamarii. Some species of
fungi such as A. oryzae, A. niger, A. fumigatus and
Penicillium have no effect at the highest
concentration (100%).
Table 1: Antifungi activity of crude methanol extract of Calliandra calothyrsus on mycelial growth of storage fungi.
Fungal species Extract concentration / fun
g
al population (cfu/ml)
0 20 40 60 80 Ketoconazole
A
sper
g
illus candidus 0- 0- 1.5 2.3 3.5 3.13
A
. chevalieri 0- 0- 0- 0- 0- 0.30
A
.
f
lavus 0- 0- 0- 0- 0- 3.40
A
.
f
umi
g
atus 0- 0- 0- 0- 0- 5.13
A
. ni
g
e
r
0- 0- 0- 0- 0- 5.43
A
. or
y
zae 0- 0- 0- 0- 0- 5.23
A
. tamarii 0- 0- 0- 0- 0- 7.23
Penicillium sp. 0- 0- 0- 0- 0- 2.86
IMC-SciMath 2019 - The International MIPAnet Conference on Science and Mathematics (IMC-SciMath)
540

Table 2: Antifungi activity of crude methanolic extract of Calliandra calothyrsus on conidial germination.
Fungal species Extract concentration / fun
g
al population (cfu/ml)
0 20 40 60 80 Ketoconazole
A
spergillus candidus 0 2.8×10
6
2.7×10
6
2.2×10
6
2.5×10
6
0
A
. chevalieri 4.2×10
6
13.2×10
6
4.5×10
6
6.6×10
6
4.1×10
6
0
A
.
f
lavus 1.0×10
6
1.0×10
6
3.5×10
6
4.2×10
6
2.5×10
6
0
A
. fumigatus 3.2×10
6
2.6×10
6
4.5×10
6
4.8×10
6
6.1×10
6
0
A
. ni
g
e
r
0 2.2×10
7
2.7×10
6
2.4×10
6
1.9×10
6
0
A
. or
y
zae 0.5×10
7
8.3×10
7
3.4×10
7
4.5×10
7
4.6×10
7
0
A
. tamarii 1.1×10
6
0.4×10
7
1.8×10
6
0.9×10
6
0.6×10
6
0
Penicillium sp. 31.6×10
6
27.2×10
6
14.2×10
6
5.1×10
6
7.0×10
6
0
4 CONCLUSION
The presence of secondary metabolites in
methanolic extract of C. calothyrsus reduce fungal
population of storage fungi.
ACKNOWLEDGEMENT
The research was supported by TALENTA-
Sumatera Utara University, Grant no.
322/UN5.2.3.1/ PPM/KP-TALENTA USU/2019.
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