Bioactivity of natural compounds extracted from Oedogonium cilitum isolated from Qalachwalan pond

2.1 Algal sample collection

Algal bloom samples were collected from Qalachwalan pond freshwater in Sulaimani district, Kurdistan region of Iraq. Fig. 1 during October 2021, and December 2021. After being carefully chosen from a blooming of algal biomass, samples were cleansed of epiphytes, extraneous matter, and necrotic particles. They were then put in clean plastic containers with their original water and taken to the laboratory. The sample was then dried at 40C in an oven for 2–3 days until the dry weight was constant after being rinsed with distilled water, shade-dried, chopped into small pieces, and powdered. (Elsie and DhanaRajan,2010).

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Fig. 1

Oedogonium cilitum Bloome at Qalachwalan open pond.

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2.2 Morphological identification of algal

The algae sample was identified as Oedogonium cilitum according to the keys of identification (Prescott,1982). The filamentous green alga belonging to the genus Oedogonium featured cylindric cells, some of which were ring-shaped at the end of each cell had pyrenoids and partial chloroplast. Oedogonium species are often found in ponds, lakes, and rivers. Fig. 2.

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Fig. 2

Oedogonium cilitum.

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2.3 Extract preparation

According to (Wong and Shahirah,2019) instructions were followed while making the organic extraction, with some modifications. Soxhlet extraction was performed using 20 g of powdered algae and 200 ml of methanol. The samples were then incubated for 96 h at 56 °C.

1. Gas Chromatography-Mass Spectrometer (GC MS) analysis:

Freshwater microalgae were analyzed using a gas chromatograph using a Carbowax capillary column-equipped quadrupole gas chromatography-mass spectrometer (GC–MS) from Shimadzu (30 m 0.25 mm ID; 0.25im film thickness) (intercut DB5MS. Japan). The capillary column was filled with a sample volume of two microliters. As a carrier gas, helium was used. Temperatures were set at 210 °C for the injector and detector. Split mode was used to inject the fluid (1:30). The column's temperature was programmed to begin at 50 °C for 1 min before rising by 3 °C each minute to 210 °C at the end. NIST Library 2008′s mass spectral database is used to compare the mass spectra, peaks in the separation of bioactive chemicals at constant pressure (100 kPa) were found. The components of the test materials' names, molecular weights, and structures were determined. (Toshihiro Obata.,2013; Saeed et al., 2020; Palaniappan, 2022).

1. Free radical-scavenger activity assessment (DPPH^° assay):

A method for conducting the DPPH assay. Extreme potential for scavenging was calculated according to the recorded technique (Blois, 1958). At different concentrations, 1.5 ml of 0.25 m DPPH mixture and 1.5 ml of extract were diluted in methanol. For a constant state, this combination was vigorously shaken at ambient temperature. After thirty minutes DPPH decolorization was measured by taking a spectrophotometer (UVMini-1240 model, Shimadzu, Columbia, USA). to measure the absorbance at 517 nm. Then, the process of scavenging DPPH radicals was calculated using the following equation. By using a linear regression analysis, the half-maximal inhibitory concentration (IC₅₀) was determined and expressed as the mean of three measurements. A positive control was ascorbic acid. $$\mathrm{S}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{e}\mathrm{q}\mathrm{u}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=[\frac{\mathit{A}0-\mathit{A}1}{\mathit{A}0}\times 100].$$

• A0 denoted the control absorbance (unqualified, with no extraction).

• A1 Denoted the absorbance when the excerpt or regular example is present.

2.4 Antimicrobial bioassays

Dioxane, Chloroform, and Dichloromethane were used to extract Oedogonium cilitum from three different organic solvents. These solvents were utilized to treat one Gram-positive bacteria, Staphylococcus aureus, as well as three Gram-negative bacteria, Escherichia coli, Acinetobacter species, and Enterobacter species. Muller Hinton agar was used for the bacterial culture's incubation (24 hr. at 37^οC). With the help of a cork borer, four wells measuring 6 mm each were created into the medium to assess the extracts' antimicrobial activity using the Agar well diffusion method. After allowing bacteria to acclimate to medium on Petri dishes for 15 min, extracts (50 l) were added to the wells. After 24 h, the agar plates were incubated at 37 °C for the negative control well (which contained only the necessary solvent) and the positive control well (15 g) of erythromycin, and the inhibition zones were measured using a ruler. Tests were carried out in duplicate. Following incubation, the inhibition zones surrounding the wells were measured underneath and represented in millimeters as proof of antibacterial activity (Abdo et al.,2012).

3.1 Chemical composition of algal extracts

By using GC/MS analysis, a total of fifteen bioactive chemicals were discovered in the peaks of methanolic extracts of fresh (Oedogonium cilitum). Table 1. Contains information on the bioactive chemicals in tabular form. The chromatogram of the chemicals found by GC/MS is shown in Fig. 3. The compounds' mass spectra were described and identified by comparison with the databases from the standard library. There were obviously several types of chemical compounds in (Oedogonium cilitum) such as fatty acids, esters, terpenes, hydrocarbons, and aldehydes.

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Fig. 3

Chromatograms of GC/MS of (Oedogonium cilitum).

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Fatty acids like (n-Hexadecenoic acid %12.71,9-Octadecenoic acid, (E) % 9.59,9-Octadecenoic acid, (Z) 5.67 %), another class of aroma compounds like esters was abundant in this algae as (Acetic acid propyl ester %24.93, Acetic acid, 2-methyl propyl ester %13.64, Acetic acid butyl ester %8.75, Thiosulphuric acids-(2-aminoethyl) ester %2.15). By peak percentage in all three fractions, the total quantity of ester compounds in Oedogonium cilitum was about (47.32 %). For algae to have order and flavor, volatile chemicals are essential. Consequently, it is not surprising that researchers have looked at the volatile chemicals in algae. volatile compounds classed as alkanes, alkene, aldehyde, sulfur compounds, alcohol, and ester were identified in the freshwater macroalgae (Table 1).

Hydrocarbons, including alkane, and feasible. euro −2-methyl cyclopentene 2.71 %, Neophytadiene 2.06 %,2-pentene,4,4-dimethyl 2.32 %. The relative concentration of saturated aldehydes like hexanal has been observed to follow an opposite trend 5.58 %, Nonanal 1.61 %, the first identification of 2-Decenal (E), 3.23 %,2-Undecenal 2.71 %. Unsaturated aldehyde in Oedogonium cilitum extracts shows that parallel-focused and untargeted investigation of previously unidentified chemicals is feasible.

3.2 Antioxidant activity

In recent years, many samples have been investigated using a variety of approaches due to the existence of various bioactive components with antioxidant potential. The DPPH radical scavenging method was chosen in the current investigation to assess the antioxidant potential of the algal extracts since it provided an easy, quick, and practical way to test the antioxidants and radical scavengers (Nickavar et al.,2007). Antioxidants were considered to have a scavenging impact on DPPH radicals because of their capacity to donate hydrogen (Ilhami et al.,2004). The ability of the DPPH radical to reduce was evaluated using the reduction caused by antioxidants. Results are shown in (Table 2), and (Figs. 4 and 6). Antioxidant activity is increased when the IC50 value is reduced. In this study, the standard antioxidant ascorbic acid's IC50 was calculated to be 0.021 mg/ml, which was much lower than the IC50 of the algal extract. Ascorbic acid's scavenging power ranged from 11.44 % at a concentration of 8.25 μg/ml to 80.54 % at a concentration of 66 μg/ml as shown in (Table 3), (Figs. 5 and 7). Extract of Oedogonium cilitum exhibited high antioxidant activity with a relatively low IC₅₀ (0.538 mg/ml). The scavenging effect of the tested extracts at 4.88 μg/ml to 1250 μg/ml increased with increasing concentration as shown in (Fig. 4) which showed higher scavenging activity (56.68 %)on DPPH free radical.

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Fig. 4

The percentage of Radical Scavenging Activity of Oedogonium cilitum algae on DPPH radicals.

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Fig. 5

The percentage of Radical Scavenging Activity of Ascorbic acid.

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Fig. 6

The DPPH radical scavenging activities of Oedogonium cilitum extract.

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Fig. 7

The DPPH radical scavenging of Ascorbic acid.

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3.3 Antimicrobial activity

As a potential source of bioactive chemicals, freshwater Oedogonium cilitum is one of the primary topics for the development of many medicinal drugs. In this work, three agal extracts made from three organic solvents and tested against four microorganisms were found to have antibacterial properties. (Table 4). The results showed that algal extracts included many antibacterial substances. The finding demonstrated that the Dioxane extract provided stronger antimicrobial activity than the Dichloromethane and chloroform extract. The present study investigated that microalgae exhibited the strongest antibacterial activity against Staphylococcus aureus (12.4 mm) microalgae extract, and reported the lowest antibacterial activity against Enterobacteria’s(4.4 mm), The results of this study showed that the macroalgae Dioxane extracts had the greatest level and widest range of antibacterial activity against the tested microorganisms. Results show Dioxane extract exhibited strong inhibition against Staphylococcus aureus (12.44 mm) followed by E.coli bacteria (10.6 mm) Acinetobacter sp. (9.9 mm) and Enterobacter sp.(8.4 mm).In contrast, Dichloromethane extract showed a strong inhibition zone against Staphylococcus aureus (10.2), followed by Acinetobacter sp. (6.9 mm)and E.coli (4.8 mm), from the result seen that Dichloromethane extract showed no inhibition zone against Enterobacter sp., chloroform extract showed high inhibition zone (7.2 mm)against Acinetobacter species, followed by E.coli (5.6 mm) and showed lowest inhibition zone against Enterobacter species(4.4 mm).chloroform extract showed no inhibition zone against Staphylococcus aureus, also, investigated that chloroform extract showed least efficiency antimicrobial activity compared to two the extracts. These variations could be related to the different solubility behavior of bioactive compounds. The efficiency of extraction, the solvent used, and the resistance of the tested bacteria are only a few of the variables that may have affected the antibacterial potency of freshwater macroalgae and caused this variation in the results (Seenivasan et al.,2010). Gram-negative bacteria were resistant to the mild antibacterial activity displayed by all three macroalgae extracts, Dioxane extract, and Dichloromethane extract showed high activity against Gram-positive bacteria. While chloroform extract shows no activity against Gram-positive bacteria. The role of bacteria and algae as antibacterial agents, as well as their structure were dependent upon the processes by which the freshwater algae, including both macro and microalgae, were used as antimicrobial agents (Pina-Pérez et al., 2017) and (Breijyeh et al.,2020). Antibiotics were typically less successful against Gram-negative bacteria because of their stiff cell walls, which make them more complex than Gram-positive bacteria. Because of this, it was challenging for active compounds like -lactams, quinolones, and other antibiotics to penetrate bacteria and exert their antibacterial effects. (Breijyeh et al.,2020) The results demonstrated that Gram-positive bacteria were much more resistant to the antibacterial effect than Gram-negative bacteria due to their cell wall structure and components as well as the presence of short-chain fatty acids (Santoyo et al.,2009). Although it is still unclear how the fatty acids were able to block the entry of the active chemicals from algal extracts, numerous studies have found that fatty acids and lipids were the reason for cellular membrane breakdown (Leflaive 2009) and (Al-Saif at el.,2014). The study concluded by stressing the importance of naturally occurring freshwater algae as a possible antibiotic against a variety of harmful and disease-causing bacteria. However, more research is necessary to fully understand the mechanism by which they fight germs and if they could replace traditional antibiotics.