Active and phenolic compounds in Spirogyra sp. PDNA1 is an antibiotic for some bacteria and fungi

: Green algae are a biological source rich in phenolic compounds and potentially inhibit the growth of microorganisms. Spirogyra sp. PDNA1 is one of the most types of green algae found in freshwater. Because of the increasing resistance of most bacteria and fungi to available antibiotics, a continuous search is required for the most effective, economical


Introduction:
Algae are living, autotrophic organisms, and they represent a wide range of organisms that can carry out the process of photosynthesis, and they are widespread on the surface of the globe as they live in land and water areas Algae produce various compounds such as alkaloids, carbohydrates, fatty acids, vitamins, enzymes, and proteins [3]. It is also a useful source of phenolic compounds. Although these compounds are usually associated with plants, algae are also a rich source of these compounds It is a genus of filamentous green algae consisting of thin, unbranched chains of cylindrical cells whose width ranges between (10-100) micrometers and whose length is greater than its width and may reach several centimeters. The cell wall is made of an inner layer of cellulose and an outer layer of pectin, which is responsible for the sticky texture of algae, so it is called water silk and can produce masses that float near the surface of streams and ponds supported by oxygen bubbles emitted in the process of photosynthesis [9,10].

Spectrometry(GC-MS)
To identify the active compounds using GC-MS, the volume of the methanolic extract was reduced and the solvent was removed by a rotary evaporator device. The analysis of the components of the methanolic extract was carried out in the central chromatography laboratory / College of Agriculture and Forestry / Food Research and Protection Laboratories Consumer -University of Basrah using gas chromatography connected to the mass spectrometer. The GC-MS type (QP 2010) supplied by a Japanese company, Shimadzu, contains a capillary separation column with dimensions (diameter 0.32, thickness of the static phase 0.25 micrometers, length 30 meters) in which high-purity helium gas was used as a directed gas with a flow rate of (1.69 ml/min) and the initial temperature was programmed in it from 50 to 280 ˚m. Samples were injected into it by direct injection at the top of the separation column through a plug The compounds were identified by comparing them with materials with known structure by comparing them with the database of known compounds in the GC-MS library and relying on the evidence of retention for each compound. Whereas the hexane extract was detected previously for the same sample of this alga in the study of [16].

Acidolysis and isolation of phenols from crude hexane and methanolic extracts
Since phenols do not exist in a free form, but are linked by a glycosidic bond with sugar, so

Identification of active phenolic compounds using high-performance liquid chromatography (HPLC)
The separation device used is High-Performance Liquid Chromatography (HPLC), which relies on the capillary and polar property, to separate the phenolic compounds separated from plants, as most of these compounds are characterized by being weakly acidic, that is ionizes under basic conditions and dissolves in polar solvents easily [18,19]. As the diagnostic process for these compounds was carried out in the HPLC Lab/Mosul the decomposition process for them using a high-performance liquid chromatography (HPLC) type. Shimadzu, Japan) where the carrier phase was used: methanol: distilled water (75: 25) and the separation column was

Antibacterial activity of green algae extracts
The sensitivity method (diffusion in agar well) was used according to the method of [21] and to test the inhibitory effectiveness of the extracts used under study. Young colonies of pathogenic bacteria were transferred to the nutrient broth medium and incubated at a temperature of 37 °C for 24 hours. The diluted bacterial suspension was spread on the nutrient agar medium in a homogeneous manner using a sterile cotton swab. The Agar Wells Diffusion Method was adopted to study the effect of the extracts on the bacteria, as holes were made in the nutrient agar using a stainless still pourer with a diameter of 6 mm and the extracts were added at a volume of 50 microliters to each hole.

Antifungal activity of green algae extracts
The Well Diffusion Method [22] was prepared. Potato dextrose agar (PDA) fungus development medium was prepared under sterile conditions, poured into Petri dishes, then inoculated with pathogenic fungi with a diameter of 6 mm, and extracts were added at a volume of 50 microliters to each well.

Results and Discussion
The results of the GC-MS technique for the methanolic extract of Spirogyra alga showed the active compounds, where 30 compounds were identified including alkaloids, phenols, and esters, and the highest percentage was for the compound Oleic acid for a retention time of 21.494 minutes and a concentration of 32.89%, as in Table (1) and Figure (1,2). One of the reasons for inhibition with algae extracts is that they contain steroidal fatty and protein        Then, the effect of the phenolic-methanolic and hexane extracts on the bacteria and fungi used in the study, where the results showed that the highest inhibition rate of the phenolicmethanolic extract was against bacteria (Klebsiella pneumoniae) with a diameter of (21) mm., Salmonella typhi with a diameter of (14) mm, and the least inhibition percentage was against (Pseudomonas aeruginosa) with a diameter of (12) mm. The effect of phenol on the fungi used in the study was recorded by the phenolic methanolic extract, the highest percentage of inhibition against (Candida albicans) was with a diameter of (26) mm, and the least inhibition was against the fungus (Aspergillus niger) with a diameter of (20) mm, and the phenolic hexane extract had the highest inhibition against (Candida albicans) fungus, with a diameter of (26) mm, and the lowest inhibition rate was in (Aspergillus niger), with a diameter of (18) mm. The Inhibition diameters higher than the crude extract of algae indicated that it affected its pure isolated form better than it was mixed with the rest of the materials and components, which reduces its effect and concentration in the concentration used against bacteria, and this result was consistent with a study [25].

Conclusions
The quantitative and qualitative diagnosis of some different phenolic compounds and other biologically active compounds in the extracts of the crude methanolic Spirogyra sp. was carried out using the GC-MS technique. The study also succeeded in isolating phenols from the crude methanolic and hexane extracts of the alga. In addition to the identification of the isolated phenols by HPLC technique. Including fatty acids such as oleic acid in the crude methanolic extract, and phenols, the predominant of which is Rutin in the phenolic extracts.