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In the present study, the metabolic andethanolic extracts of specie (Division: Chlorophyta) Chara vulgaris was tested for antifungal (Dermatophytes) activity for in vitro at concentration (100,50 and 25 mg/ml) against two clinical isolates of Dermatophytes fungi (Trichophyton rubrum and Trichophyton mentagrophytes) by measuring percentage of inhibition growth. The results indicated that the hot ethanol extract of Chara vulgaris was more efficient(89.8 %) inhibition growth at concentration 100 mg/ml and the lowest effect was (20.4%) at concentration 25 mg/ml against the tested pathogenic fungi. Chemical analyses showed that the active chemical compounds for hot ethanolic and methanolic alga (Chara vulgaris)extracts were contains alkaloids, phenols, Terpenes, Steroids, Flavones, Resins, Saponines and tannins. Finally the result of GC mass analysis for Chara vulgaris extracts proved the presence of many antimicrobial activity compounds.
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ISSN: 0975 -8542
Journal of Global Pharma Technology
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RESEARCH ARTICLE
©2009-2018, JGPT. All Rights Reserved 759
Anti-Dermatophytes Activity of Macroalgal Extracts (Chara
vulgaris) Isolated from Baghdad City-Iraq
Ahmed S. Dwaish*, Dina Y. Mohammed Yousif, Adel Hamdan Alwan, Siham
N. Lefta
Department of Biology, College of Science, University of Mustansiriyah Baghdad-Iraq.
*Corresponding Author: Ahmed S. Dwaish
Abstract
In the present study, the metabolic andethanolic extracts of specie (Division: Chlorophyta) Chara
vulgaris was tested for antifungal (Dermatophytes) activity for in vitro at concentration (100 ,50 and
25 mg/ml) against two clinical isolates of Dermatophytes fungi (Trichophyton rubrum and
Trichophyton mentagrophytes) by measuring percentage of inhibition growth. The results indicated
that the hot ethanol extract of Chara vulgaris was more efficient(89.8 %) inhibition growth at
concentration 100 mg/ml and the lowest effect was (20.4%) at concentration 25 mg/ml against the
tested pathogenic fungi. Chemical analyses showed that the active chemical compounds for hot
ethanolic and methanolic alga (Chara vulgaris )extracts were contains alkaloids, phenols ,Terpenes ,
Steroids, Flavones, Resins ,Saponines and tannins. Finally the result of GC mass analysis for Chara
vulgaris extracts proved the presence of many antimicrobial activity compounds.
Keyword: Fresh water algae, Anti-Dermatophytes activity, Hot ethanolic and methanolic extracts,
Baghdad City-Iraq.
Introduction
Algae a diverse group of plant kingdom,
contains different bioactive compounds. The
bioactive substances produced by actively
growing cells of algae includes proteins, fats,
lipids, carbohydrates, phenol, flavonoid,
vitamins, free amino acids, enzymes, growth
regulators, pigments, toxins and antibiotics.
Algae are admirable sources of antibiotics
which in laboratory tests, inhibited bacteria
and fungi that incite diseases of humans [1].
Fungi as agents of superficial mycoses cause
a wide range of diseases in humans and
animals, involving the outer layer in the
stratum corneal of the skin and frequently
causing chronic infections.
The major etiological agents of these
mycoses are dermatophytes and Candida
species, cosmopolitan fungi that are able to
affect deeper layers of the epidermis and
mucous or organs in debilitated individuals.
As the population of immune-compromised
continues to arise, the opportunistic fungal
pathogens infecting these patients continue
to increase as well [2].
Pathogen resistance to synthetic drugs and
antibiotics already in use makes search for
plants with antimicrobial activity more
important, as they can substitute for
synthetic antibiotics and drugs.
Phycochemistry is a new term first used by
[3], which is actually the study of natural
products and chemical constituents
occurring within algal thallus from a
biological point of view.
It primarily investigates the distribution of
secondary metabolites in different body
parts of algae under different seasons and
variety of habitat conditions.
All over the world phycologists studied the
different types of natural products occurring
within marine algae. A variety of fatty acids
(both saturated and unsaturated), sterols,
terpenes and sugars have been isolated from
Ahmed S. Dwaish et. al.| Journal of Global Pharma Technology| 2018; 10(03):759-766
© 2009-2016, JGPT. All Rights Reserved 760
them. A very limited amount of
phycochemical knowledge is available about
freshwater algae, in comparison with the
detailed work carried out on seaweeds.
The ability of algae to produce secondary
metabolites of antimicrobial value, such as
volatile components (phenols and terpenes)
[4; 5], steroids [6], phlorotannins [7] and
lipids [6] has been already studied.
This work aims to evaluate the antifungal
activity of three species of freshwater algae
with tow extraction solvents (methanol and
ethanol) against Trichophyton rubrum and
Trichophyton mentagrophytes in order to
discover new natural antifungal compound.
Materials and Methods
Algal Isolates
The algae Chara vulgaris was collected from
water cannel in Baghdad University -
Baghdad-Iraq, during April 2016.
The alga was brought to the laboratory in
plastic bags containing water to prevent
evaporation.
Algae was then cleaned from epiphytes and
rock debris and given a quick fresh water
rinse to remove surface salts. The algal was
identified by [8].
The samples were air dried in the shade at
room temperature 25 °C: 30 ºC on absorbent
paper, cut into small pieces and grounded to
fine powder.
Algal Extracts
The hot alcoholic extract was prepared by
Soxhelet extraction according to [9]. In this
process the dried powder form of algae
material extracted by using ethanol and
methanol alcohol.
After completion the process the
concentrated active constituents from algae
were kept in sterilized test tubes stored in
refrigerator till further use. The traces of
ethanol and methanol were removed by
keeping the tubes at 50˚c for 1 hr.
The obtained residues (crude extracts) were
suspended in the respective solvents to final
concentration of 100,50 and 25 mg/ml. The
extract was stored at -20 ºC in airtight glass
bottle for the antifungal assay.
Fungal Isolates
In this study the Dermatophytes fungi i.e.,
(Trichophyton rubrum and Trichophyton
mentagrophytes) were obtained from
Culture Collections of the health Center
Laboratories (Ministry of health) Baghdad
Iraq.
Assessment of Antifungal
Anti fungal activity of algae extracts were
preceded by mix the crude extracts of the
macro algae with Potato Dextrose Agar
(PDA) medium to get concentrations of the
extract (100 mg/ml) and the fungal mycelia
were inoculated to grow.
Percentage inhibition of mycelial growth in
each case was calculated by using following
formula:
% inhibition = 100×(C-T/) C
Where
C= Fungal mycelial biomass / dry weight in
control.
T= Fungal mycelial biomass / dry weight in
control in various test concentration [10].
Evaluation Some of the Active
Compounds in the Algal Extracts
The presence of active compounds in the
studied algae was determined by adopting
standard protocols [11].
Gas Chromatography-Mass
Spectrometry
For GC-MS analysis, a high-temperature
column (Inert cap 1MS; 30 m × 0.25 mm id ×
0.25μm film thickness) was purchased from
Agilent Technologies (SHIMADZUJapan),
by employing a high-temperature column.
Derivatization of each sample was
eliminated. The injector and detector
temperatures were set at 280°C while the
initial column temperature was set at
100°C. A 5 μL sample volume was injected
into the column and ran using split (1:10)
mode After 1 min, the oven temperature was
raised to 225°C at a ramp rate of
12.5°C/min(hold time 4 min). The oven
temperature was then raised to 300°C at a
ramp rate of 7.5°C/min (hold time 5 min).
Ahmed S. Dwaish et. al.| Journal of Global Pharma Technology| 2018; 10(03):759-766
© 2009-2016, JGPT. All Rights Reserved 761
The helium carrier gas was programmed to
maintain a constant flow rate of 17.5
mL/min and the mass spectra were acquired
and processed using both Agilent GC-Mass.
Solution (SHIMADZUJapan) and postrun
software. The compounds were identified by
comparison of their mass with NIST library
search and authentic standards.
Results and Discussion
Morphological Structure of Algae
Chara vulgaris macroalgae attached to rock
and plant, distinguished to root and shoot
system further comprising of well-developed
vegetative and reproductive structures.
Vegetative parts have been studied in detail
under light microscope but it had helped
very little to study the minor details
particularly with regard to the reproductive
organs especially the mature oospore which
exhibit peculiar patterns of ornamentation
in the fossa (Figure 1), this findings agreed
with [12] and [13] .
Figure 1: Filaments of Chara vulgaris showing the branch and Oogonium (40X)
Algal Extracts as Anti- Dermatophytes
Chara vulgaris ethanolic and methanolic hot
extracts were tested to control biomass of
dermatophytic (Trichophyton rubrum and
Trichophyton mentegrephytes ). The validity
of experimental results of percentage
inhibition obtained at concentrations
(100,50 and 25mg/ml) and checked by
statistical parameters and tabulated in
Table 1 and 2 . Statistically significant
increase has been recorded in the
percentage inhibition of the target fungal
species with different algal concentrations
extract. It was observed that among all the
fungi, maximum inhibition was caused by
the inhibition growth of Trichophyton
rubrum and Trichophyton mentegrephytes
when we used ethanolic and methanolic hot
extracts (88.9 and 89.8 % inhibition growth
respectively) Figure (2) and minimum
inhibition was caused in case of used
Methanolic hot extracts of algae (20.4%)
against Trichophyto mentegrephytes at 25
mg/ml concentration.
Table 1: Mean percentage of growth inhibition Trichophyton rubrum by hot ethanolic and methanolic extracts of
Chara vulgaris at different concentrations
Concentrations
Agal Extract
Control
LSD
Ethanolic
Methanolic
100 mg/ml
88.9± 0.3
88.3 ± 0.1
0.00 ± 0
0.424
50 mg/ml
46.2± 0.3
43.2± 0.2
0.00 ± 0
2.121
25 mg/ml
23.2± 0.3
21.2± 0.1
0.00 ± 0
1.414
LSD
33.34
34.21
0.00 NS
* (P<0.05).
Table 2: Mean percentage of growth inhibition in Trichophyto mentegrephytes by hot ethanolic and methanolic
extracts of Chara vulgaris at different concentrations
Agal Extract
Control
LSD
Ethanolic
Methanolic
89.8 ± 0.2
87.9 ± 0.05
0.00 ± 0
1.344
45.3 ± 0.2
42.1 ± 0.3
0.00 ± 0
2.263
22.9 ± 0.2
20.4 ± 0.2
0.00 ± 0
1.768
34.053
34.46
0.00 NS
----
* (P<0.05).
Ahmed S. Dwaish et. al.| Journal of Global Pharma Technology| 2018; 10(03):759-766
© 2009-2016, JGPT. All Rights Reserved 762
Figure 2: Photographs showing fungicidal effect of ethanolic hot extracts of Chara vulgaris
(100mg/ml) against Trichophyton rubrum
There are numerous reports of compounds
derived from algae with a broad range of
biological activities, such as antibiotics
(antibacterial and antifungal properties) [14,
15 and 16].
In fact, the test micro organism’s different
inhibition activity in relation to their
susceptibility to algal extracts antimicrobial
substances, Gram positive bacteria were low
sensitive than the Gram negative bacteria.
The reason for different sensitivity between
the fungi and bacteria can be found in
different transparency of the cell wall.
The cell wall of the Gram positive bacteria
consist of peptidogly can (murein) and
teichoic acids, while the cell wall of the
Gram negative bacteria consists of lipo
polysaccharides and lipopoliproteins [17].
Whereas, the cell wall of fungi consists of
polysaccharides such as hit chin and glucan
[18] and or due to the permeability barrier
provided by the cell wall or to the membrane
accumulation mechanism [19]. Among them
are the interactions of antimicrobial
compound with the cell membrane.
The antifungal activity of the extract could
be due to the presence of different chemicals
that may include flavonoids and
triterpenoids besides phenolic that may
affect growth and metabolism of bacteria.
Also, they could have an activating or
inhibitory effect on microbial growth
according to their constitution and
concentration, compounds and free hydroxyl
group [20] amides and alkaloids [21].
It indicates that the Chlorophyceae member
also has some medicinal properties against
tested pathogens, in view of this in future
the study is required further to identify the
bioactive compounds and their purification
and identification to know the chemical
properties this alga, these finding agree
with [22] .
Evaluation of Phyto active
Compounds
Many papers have been published which
discuss the methods of manufacture and the
composition of algal extract the general that
the composition of extracts strongly
depends on the raw material ( geographical
location of harvested algae and algae species
)as well as on the extraction method.
The biologically active compounds which
are transferred from the biomass of algae to
the liquid phase ,they have well documented
beneficial effect on humans, animals and
plants, mainly by protection of an organism
from biotic and a biotic stress (antimicrobial
activity, scavenging of free radicals and
host defence activity etc .[23; 24].
The primary detected(Presence or absence)
for the active compounds shown in Table (3)
for hot ethanolic and methanolic algal
extract, the results showed that the active
chemical compounds for hot ethanolic and
methanolic alga (Chara vulgaris)extracts
were contains many active chemical
compounds such as phenols ,tannins,
Saponines, alkaloids, Terpenes , Steroids,
Flavones, Resins and the mean of pH
extracts was 5.8.
This results supports the findings of authors
.This results supports the findings of many
authors [25 , 26 and 27] who find same this
compounds in macro and microalgae.
Ahmed S. Dwaish et. al.| Journal of Global Pharma Technology| 2018; 10(03):759-766
© 2009-2016, JGPT. All Rights Reserved 763
Table 3: Presence or absence of active compounds in Chara vulgaris Hot Methanol and Ethanol Extracts
Chemicals Compound
Hot Methanol Extract
Hot Ethanol Extract
Glycosides
-
-
Phenols
+
+
Alkaloids
+
+
Terpenes and Steroids
+
+
Resins
+
+
Saponines
+
+
Tannins
+
+
Flavones
+
+
Coumarines
-
-
pH
5.8
6
Evaluations of Gas Chromatography-mass Spectrometry for Algal Extracts
Both Hot crud methanolic and ethanolic
extract of Chara vulgaris was subjected to
GC-MS analysis. GC-MS analysis of the
methanolic extract Chara vulgaris showed
six compounds, which together accounted for
94.47 % of the total mass (Fig. 3) and their
effects ( Table 4).
Composition of the remaining 5.53% could
not be ascertained due to their low
abundance. While the GC-MS analysis of
ethanolic extract of Chara vulgaris showed
that the a mixture of at least eight
compounds (Fig. 4) and their effects ( Table
5). However, there were only eight major
components which accounted for 83.9% of
the total mass, composition of the remaining
16.1 % could not be ascertained due to their
low abundance. The present study provides
data to show the appreciable antifungal
activity of studied algae.
The result presumes that the long chain
hydrocarbons may act as potential bioactive
substance and can be exploited in
pharmaceutical preparations.
The cultivable nature of seaweeds is an
added advantage for mass production of
potential antimicrobial products, our finding
agreed with [28 and 29] who reported the
most similarly compound in algae where
isolated from green algae.
Figure 3: The chromatogram of GC-Mass spectrophotometery showed that hot methanolic
extract of Chara vulgaris
Table 4: The major identified compounds of hot Methanolic extract of Chara vulgarisby using GC-Mass
spectrophotometer
No.
Rt
Area%
The compounds
Antimicrobial Activity
Reference
1
2.153
2.16
Tridecyne
antibacterial activity
[30]
2
15.571
10.51
Hexadecanoic acid
Anti-inflammatory, Antioxidant,
hypocholesterolemicnematicide,pesticide,
anti androgenicflavor, hemolytic, 5-
Alpha reductase inhibitor , potent
mosquito larvicide
[31,32 and 33]
3
17.436
47.59
Octadecynoic acid
antioxidant, and antibacterial activities
[34]
4
18.693
4.46
Hexadecanoic acid
Antibacterial and Antifungal
[35]
5
20.299
25.59
Pentadecadien-1-
Antimicrobial Activity
[36]
6
22.058
4.16
Tetradecenal
antibacterial activity
[30]
Ahmed S. Dwaish et. al.| Journal of Global Pharma Technology| 2018; 10(03):759-766
© 2009-2016, JGPT. All Rights Reserved 764
Figure 4: The chromatogram of GC-Mass spectrophotometery showed that hot ethanolic
extract of Chara vulgaris
Table 5: The major identified compounds of hot ethanolic extract of Chara vulgarisby using GC-Mass
spectrophotometer
Reference
Biological Activity
The Compounds
Area%
Rt.
No.
[37]
antidiuretic , anticancer, anti-
inflammatory, antioxidant,
antimicrobial
Hexadecen-1-ol
2.25
14.616
1
[38]
Antimicrobial Activity and
pharmacological Activity
Phthalic acid
18.65
15.566
2
[39]
Antimicrobial Activity and Antifungal
Octadecenoic acid
1.61
16.875
3
[34]
antioxidant, and antibacterial activities
Octadecynoic acid
43.85
17.351
4
[35]
Antibacterial and Antifungal
Hexadecanoic acid
1.95
18.697
5
[36]
Antimicrobial Activity
Pentadecadien
12.01
20.245
6
[30]
antibacterial activity
Tetradecenal
2.09
22.044
7
[40]
anti-bacterial activities for food and
cosmetics industries.
Octadecadien-1-ol
1.49
24.635
8
Conclusion
The present study data indicates that the
different solvent extracts of Chara vulgaris
have strongest antimicrobial properties and
may be considered as alternative source for
synthetic substances, these substances are
potential source of bioactive compounds
against various pathogens which can be
used as natural non-toxic preservative and
may be more acceptable to consumers.
Finally, it is concluded from this research
that crude extracts of Chara vulgaris proved
to be worthy by showing antimicrobial
character in different solvents used against
the different Dermatophytes in the present
investigation.
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Chemical Composition and Anti-
bacterial Activity of Essential oil from
Cedrela sinensis (A. Juss.) Roem. seed.
African Journal of Biotechnology,
11(7):1789-1795.
Ahmed S. Dwaish et. al.| Journal of Global Pharma Technology| 2018; 10(03):759-766
© 2009-2016, JGPT. All Rights Reserved 767
... Although the different extractions of C. vulgaris cells with solvents like methanol, ethanol, chloroform and diethyl ether, show their antibacterial activity against gram negative and gram positive human pathogenic bacteria (Dineshkumar et al., 2017). Biological treatment of phenol solution using the green macroalga Chara sp and Chlorella vulgaris used in wastewater treatment reveal to their ability to remove phenol from aqueous solution beside they have strongest antimicrobial properties and may be considered as alternative source for synthetic substances, these substances are potential source of bioactive compounds (Dwaish et al., 2018). ...
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... A Sahi/Department of Biology/Mustansiriyah University. As a fine powder Soxhelet extraction was used to prepare extract as explained in 9 . Dried powder form of Cladophora glumerata material has been extracted by using chloroform. ...
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... The results of the FTIR spectrum for C. vulgaris (Fig. 3 A) confirmed the presence of Cis Alkene at the peak 2925.41 cm -1 [29]; additionally, the peak at 2856.66 cm -1 corresponded to the HC=CH stretching, which means that some alkene compounds are phenols and alcohols while the peak at 1750-1480 cm -1 corresponded to C=O and/or C=C, which refers to the presence of celluloses and hemicelluloses [30]. The high alkenes and carboxylic compounds content in addition to the moderate amounts of phenolic compounds present in C. vulgaris refer to the antioxidant potentials of this plant [27][31] [32]. ...
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... This explains that water extracts are less soluble than most active substances (phenols, terpenes, flavonoids, etc.), which have inhibitory effect on the growth of pathogenic fungi, while organic solvents have a great ability to dissolve many of the active substances of this plant. This corresponds to (Pornphan et al., 2016;Dwaish et al., 2018). ...
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... This explains that water extracts are less soluble than most active substances (phenols, terpenes, flavonoids, etc.), which have inhibitory effect on the growth of pathogenic fungi, while organic solvents have a great ability to dissolve many of the active substances of this plant. This corresponds to (Pornphan et al., 2016;Dwaish et al., 2018). ...
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