Anti-Dermatophytes Activity of some Algal Extracts Isolated From Baghdad City-Iraq

 

Maarb S. Al-maoula Ahmed S. Dwaish

Biology Department, College of Science, AL-Mustansiriyah University, Baghdad, Iraq.

*Corresponding Author E-mail: ahmedsahi@uomustansiriyah.edu.iq

 

ABSTRACT:

In this study, the methanolic andethanolic extracts of species algae (Division: Chlorophyta) Cladophoraglomerata and Oedogoniumsp were examined for antifungal (Dermatophytes) efficiency in vitro at concentration (100mg/ml) against two isolates of Dermatophytes fungi (Trichophyton rubrum and Trichophytonmentagrophytes) by measuring percentage of inhibition growth. The results indicated that the hot methanolic and ethanolic extracts of Cladophoraglomerata was more efficient (100%) inhibition growth on both fungal isolates and the lowest effect was (63.4%) at hot ethanolic extracts against the tested pathogenic fungi. The quantity chemical analyses showed that the active chemical compounds for hot ethanolic and methanolic algae (Cladophoraglomerata and Oedogoniumsp) extracts were contains alkaloids, phenols, Terpenes, Steroids, Flavones, Resins, Saponines and tannins. Finally the result of GC mass analysis for Cladophoraglomerata and Oedogoniumsp extracts proved the presence of many antimicrobial activity compounds.

 

KEYWORDS: Fresh water algae, Anti-Dermatophytes activity, hot ethanolic and methanolic extracts.

 

 


INTRODUCTION:

Algae a varied groups of plant kingdom, have diverse bioactive composites. The active constituents made up through actively developing cells of algae which are proteins, fats, phenol, flavonoid, lipids, carbohydrates, vitamins, free amino acids, enzymes, growth regulators, pigments, toxins and antibiotics. Algae are good resources of antibiotics which killed of bacteria and fungi which causes illnesses of humans [1]. Fungi are causes of superficiality mycoses source, widespread of sicknesses in humans and animals, including outside surface in the stratum corneal of skin and commonly making chronic infections. The main etiological causes of these mycoses are dermatophytes and Candida sp., global fungi which capable of influence lower side of the skin and mucous or tissues in weakened persons. As the people of immune-compromised remains to evaluate, the opportunistically fungal pathogens infect a patients continue to rise too [2].

 

 

 

Resistance Pathogen to synthetic drugs, and antibiotics used makes searcher to more plants have antimicrobial action are very essential, also they can modification for formation antibiotics and drugs. Phycopharmacology is a new word first used by,[3], means the study of new naturalistic materials and chemical components appeared inside algal from a biological point of view.

 

It mostly researches the spreading of secondary metabolites in many parts body of algae in many seasons and diverse of habitat situations. Everywhere algalogists studied the changed kinds of natural yields appearing inside sea algae. Many fatty acids, (unsaturated and saturated), sterols, sugars and terpenes have separated from algae. so limited volume of phycopharmacological understanding is existing around river algae, in contrast with complete work approved out on sea algae.  The capacity 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 Trichophytonmentagrophytes in order to discover new natural antifungal compound.

MATERIALS AND METHODS:

Algal isolates:

The two species algae Cladophoraglomerata and Odeogonium sp. were collected from water cannel in Baghdad University-Baghdad-Iraq, during April 2016. The algae were taken to the laboratory, in plastic bags, in its sea water to stop vaporization. Algae after that washed from epiphytes, and rock remains and given a rapid new water clean to eliminate layer salts. The algal identified by [8]. The samples were air dried in the shade at room temperature 25°C: 30ºC on absorbent paper, cut into small pieces and grounded to fine powder.

 

Algal extracts:

Alcoholic hot extract ready through Soxhelet were extracted as said by[9]. In this method a dried powder of algae substance were extracted via ethanol and methanol, alcohol. After, process where completed the focused active components of algae kept in sterilized tubes stored in refrigerator till it use. A few of ethanol and methanol isolated by putting the flasks at 50ºC for 1 hr in oven. The gotten remains (crude extracts) suspended in own diluters for last concentration of 100 mg/ml. The extract was kept at -20ºC in airtight glass flask for the antifungal assay.

 

Fungal isolates:

In this study the Dermatophytes fungi i.e., (Trichophyton rubrum and Trichophytonmentagrophytes) were gotten 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 solution of algae with Potato Dextrose Agar (PDA) medium to acquire focuses of extract (100 mg/ml) and fungal mycelia inoculated to grow. Inhibition Percentage of mycelial, growth in every case which calculated through this formula:

 

% inhibition = 100×(A-B/) A

Where

A=mycelial, biomass of fungi / dry weight in control,.

B=mycelial, biomass of fungi / dry weight in control in many test, concentration [10].

 

Evaluation some of the active compounds in the algal extracts:

The existence or absence of groups active composites in all algae extracts were decided in using standard methods  [9].

 

Gas Chromatography-Mass Spectrophotometry:

To GC-MS Spectrophotometry examination, a in height-temperature column, was used from Agilent Technologies, (SHIMADZU—Japan), by doing in high-temperature, column. The injector, and detector, temperatures fixed at 280°C whereas the beginning column temperature fixed at 100°C. A 5 μL tester volume inserted inside the column and start by split (1:10) mode Next 1 min, oven temperature elevated to 225°C at a ramp ratio, of 12.5°C/min(hold time, 4 min). Oven temperature elevated to 300°C at a ramp level, of 7.5°C/min (hold time, 5 min). Helium transporter gas set to get flow level, of 17.5 mL/min and mass spectra gained and doing by both Agilent GC-Mass. Solution, (SHIMADZU—Japan) and post run, software. A recognized composites were by comparison of the mass with NIST, library search, and authentic criterion which found in.

 

RESULTS AND DISCUSSION:

Morphological Structure of algae:

Cladophora is a benthic, branching, linked, filamentous green algae which forms a hair similar organization (Figure 1). This alga a wide-reaching found in sea and river habitations. Filaments frequently longer, making “streamers” that can be 1-2 m in length, Rough, dark-green to brownish-green in color, branched, hair-like threads with cross walls splitting parts; every part has multi nucleus. Cladophora development and growth generally wants solid substrates to connection, for example rocks, a additional physical necessity for Cladophora growth is movement water, and reach agreement with [11,12].

 

 

Figure 1: Filaments of Cladophoraglomerata showing the branching  (40X).

 

Oedogoniumsp. is green algae no branched filamentous. has a thick chloroplast, fill the cell, bypyrenoids Figure (2). Cells are broader on one side than other; sometimes one bulbous, exactly globular cells. Main indicative property is occurrence of rings in broader end, that arise as result of cell division, a ring for division which cell has submit, that can be noticed in the filament with focusing attention under suitable lighting. This study agree with [13] which indicated a presence of Cladophoraglomerata and Oedogonium sp. in Tigris Riverin AL- Jadria Site in Baghdad.

 

 


Figure 2: Filaments of Oedogoniumsp showing the arrow symbols a cell is creating a zoospore  (40X) .

 

 

 

 

Algal extracts as Anti- Dermatophytes:

Cladophoraglomerata and Odeogonium sp.ethanolic and methanolic hot extracts were tested to control biomass of dermatophytic (Trichophytonrubrum and Trichophytonmentegrephytes ). The validity results of experiments for inhibition percentage were obtained at Concentration (100mg/ml),checked by statistical parameters and tabulated in Table 1 and 2 . Statistically significant increase has been recorded in the percentage inhibition of the clinical fungal species with different algae kinds extracts. It was observed that every the fungi, maximum inhibition affected by the inhibition growth of Trichophyton rubrum and Trichophytonmentegrephytes  when we used ethanolic and methanolic hot extracts of Cladophoraglomerata (100% inhibition growth) and minimum of that by Trichophyton rubrum, in case of used Ethanolic hot extracts of Odeogonium sp. (63.4%) at ten day of incubation at 100mg/ml concentration.


Table 1: Mean percentage of growth inhibition Trichophyton rubrumby hot ethanolic and methanolic extracts of algae.

Algal

Extracts

Agal Extract Concentration  (100mg/ml)

Control

LSD

Ethanolic

Methanolic

Cladophoraglomerata

100 ± 0.1

100 ± 0.2

0.00 ± 0

7.500 *

Odeogonium sp.

63.4 ± 0.1

64.0 ± 0.2

0.00 ± 0

6.479 *

LSD

8.273 *

8.051 *

0.00 NS

----

* (P<0.05).

 

Table 2: Mean percentage of growth inhibition in Trichophytomentegrephytesby hot ethanolic and methanolic extracts of algae.

Algal

Extracts

Agal Extract Concentration (100mg/ml)

Control

LSD

Ethanolic

Methanolic

Cladophoraglomerata

100 ± 0.2

99.1 ± 0.05

0.00 ± 0

7.428 *

Odeogonium sp.

65.7 ± 0.1

66.9 ± 0.2

0.00 ± 0

6.921 *

LSD

7.394 *

7.583 *

0.00 NS

----

* (P<0.05).

 


There are many information of compounds resulting from algae by a wide-ranging of biological effects, for example antibiotics, (antibacterial and antifungal characteristics)[14,15,16]. Actually, the testing of microorganisms vary inhibition action in relative with their capability to algal extracts antimicrobial elements, The reason return to cell wall of fungi contains polysaccharides, for instancehitchin and glucan, [17] or because of the permeability barrier providing through the cell wall or the membrane assemblage mechanism [18]. Also the connections of antimicrobial substance with the cell membrane. The antifungal action of the extract can be return for the existence of diverse substances that may contain flavonoids and triterpenoids also phenolic. They could have an stimulating or inhibitory influence on microbial growing in relation to their composition and concentration, composites and free hydroxyl group [19] amides and alkaloids [20].

 

Evaluation of Phyto – active compounds:

Several papers have been discussed the techniques of product and the structure of algal extract that the structure of extracts toughly determined by the raw substantial which found in the extraction manner. The active biological composites which are converted from inside of algae to the liquid phase, they have good explained th evaluable, influence on humans, animals, and plants, basically through defense of an organism from biotic and a biotic pressure (antimicrobial action, scavenging of free radicals, and host resistance motion and others.[21,22].

 

The primary observed(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 (Cladophoraglomerata) extracts were comprises alkaloids, Terpenes, Steroids, Flavones, Resins, Saponines, tannins and the mean of pH extracts was 6, also the Outcomes presented that active chemical compounds for hot ethanolic and methanolic alga (Oedogoniumsp) extracts were holds many active chemical compounds for example phenols ,tannins, alkaloids, Terpenes , Steroids, Flavones, Resins and the mean of pH extracts was 6. This Outcomes supports the findings of many authors[23,24,25],and disagree with [26] who isolated the same algae from Baher AL-Najaf, that may be because variation in water environmental factors  and time collection.

 

Evaluations of Gas Chromatography-mass Spectrometry for algal extracts:

Both Hot crud methanolic and ethanolic extract of  Cladophoraglomerata and were exposed to GC-MS analysis for its high activity against fungi .The chromatogram showed that Cladophoraglomeratam ethanolic extract was a mixture of at least 7 compounds (Fig. 4) and their effects (Table 4). However, there were only seven major components which accounted for 95.53% of the total mass. From mass spectral analysis seven compounds were identified and they together accounted for at least 95.53% of the total mass, composition of the remaining 4.47% could not be ascertained due to their low abundance.

 

Table 3: Presence or absence of active compounds in algal extracts.

 

Cladophoraglomerata

Oedogonium sp.

Group of Active Compound

Hot Methanol

Hot Ethanol

Hot Methanol

Hot Ethanol

Glycosides

_

_

_

_

Phenols

_

_

+

+

Alkaloids

+

+

+

+

Terpenes and Steroids

+

+

+

+

Resins

+

+

+

+

Saponines

+

+

_

_

Tannins

+

+

+

+

Flavones

+

+

+

+

Coumarines

_

_

_

_

pH

6

6

6.1

5.9

 


 


 

Figure 4: The chromatogram of GC-Mass spectrophotometery showed that hot methanolic extract of Cladophoraglomerata

 


The chromatogram showed that Cladophoraglomerata ethanolic extract was a mixture of at least 6 compounds (Fig. 5) and their effects(Table 5). However, there were only seven major components which accounted for 86.74% of the total mass. From mass spectral analysis seven compounds were identified and they together accounted for at least 86.74% of the total mass, composition of the remaining 13.26 % could not be ascertained due to their low abundance.

 


 

Figure 5: The chromatogram of GC-Mass spectrophotometery showed that hot ethanolic and extract of Cladophoraglomerata


The major compounds were found in hot methanolic and ethanolic extracts of Cladophoraglomerata hexadecanethis compound return to the acyclic diterpenes. These are diterpenes (compounds made of four consecutive isoprene units) that do not contain a cycle, this compound had antimicrobial activity, also Octadecane which belonged to hydrocarbons class which had several bioactivity such as Lubricant, Transformer oil ,Anti-corrosion agent and Pheromones, also were found in  hotalcoholic crud extracts of algae Pentadecane compound that representas alkane hydrocarbon the generic name for this group of aliphatic hydrocarbons Cn-H2n+2 ,that signified reactive groups. Outcomes are similarity with the investigations reported[27,28] straight chain paraffins (n-alkanes), branched chain paraffins (alkyl-alkanes) and unsaturated hydrocarbons (alkenes) were described in numerous marine algae[29].Alike group of hydrocarbons  Tetradecane, Octadecane and hexadecane have been described as communal main volatile constituents in  the all extracts of algae and this results agreed with other studied [30].


 

Table 4: The compounds where identified in methanol hot extract of Cladophoraglomerata by using GC-Mass spectrophotometer.

No.

Rt.

Area%

Name of compound

Biological Activity

Reference

1

2.153

2.16

Tridecyne

antibacterial activity

[31]

2

15.571

10.51

Hexadecanoic acid

Anti-inflammatory, Antioxidant, hypocholesterolemicnematicide, pesticide, anti androgenic flavor, hemolytic, 5-Alpha reductase inhibitor , potent mosquito larvicide

[27]

[28]

[32]

3

16.821

1.06

Octadecadienoic acid

Anti-cancer, Antiinflammatory, antiandrogenic, cancer preventive,dermatitigenic, irritant, antileukotrieneD4

[30]

4

17.436

47.59

Octadecynoic acid

antioxidant, and antibacterial activities

[33]

5

18.693

4.46

Hexadecanoic acid

Antibacterial and Antifungal

[34]

6

20.299

25.59

Pentadecadien-1-ol

Antimicrobial Activity

[29]

7

22.058

4.16

Tetradecenal

antibacterial activity

[31]

 

Table 5: The compounds where identified inethanol hot extract of Cladophoraglomerata by using GC-Mass spectrophotometer.

No.

Rt.

Area%

Name of compound

Biological Activity

Reference

1

2.955

4.83

Glycerin

antibacterial effect

[35]

2

12.018

6.47

Hexadecanoic acid

Anti-inflammatory, Antioxidant, hypocholesterolemicnematicide, pesticide, antiandrogenic flavor, hemolytic, 5-Alpha reductase inhibitor , potent mosquito larvicide

[27]

[28]

[32]

3

13.905

5.82

Oleic Acid

Antibacterial

[36]

4

14.185

24.10

Hexadecanoicacid

Anti-inflammatory, Antioxidant, hypocholesterolemicnematicide, pesticide, antiandrogenic flavor, hemolytic, 5-Alpha reductase inhibitor , potent mosquito larvicide

[27]

[28]

[32]

5

16.855

43.09

Octadecynoicacid

antioxidant, and antibacterial activities

[33]

6

24.742

2.43

Tetradecenal

antibacterial activity

[31]

 


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 antifungal products, our finding agreed with [37] how reported the most similarly compound in algae where isolated  from green algae.

 

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Received on 01.08.2018          Modified on 20.08.2018

Accepted on 30.08.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(12): 5449-5454.

DOI: 10.5958/0974-360X.2018.00993.9