Harvesting and Processing Algae

The harvest of algae was carried out on the Algerian West Coast. The algae were identified by specialists in the field using the standard identification keys. Algal specimens were collected by hand and stored in plastic bins, filled with seawater, until processed the same day in the laboratory. After removing debris, small crustaceans, and other algae, the thalli are rinsed with distilled water several times to clean the epiphytes completely. These algae are dried in the open air and away from light for 7 to 10 days until complete dehydration, then ground into a very fine powder, then sifted and stored in a dark and dry place [9].

Extraction

The extraction method adopted was maceration by mixing two organic solvents. 50 g of each powdered algae is added to 400 ml of two solvents: 200 ml of methanol and 200 ml of toluene. The first is polar, and the second is non-polar. The resulting mixture was stirred continuously for 48 hours in the absence of light at room temperature, then filtered with Wattman N^o1 paper. The filtrate obtained was evaporated under reduced pressure at a temperature of 40°C using a “Büchi” type rotary evaporator [10].

Each residue was weighed, and the extract recovered with a few drops of methanol was stored in sterile pill bottles at 4°C until its use for the different dosages [11].

Determination of Yield

The determination of the yield of the dry extract is performed by applying the following formula: R (%) = (P1 – P2/PE) × 100 knowing that: P1: Weight of the balloon after evaporation. P2: Weight of the empty flask before evaporation. PE: Weight of dry algal material.

Dosage of Flavonoids

A volume of 1 ml of the extract is added to 1 ml of 2% aluminum trichloride. The mixture was shaken and incubated for 30 min at room temperature in the dark, then the absorbance was measured at 430 nm on a spectrophotometer, with quercetin used as a reference standard. The results are expressed in micrograms of quercetin equivalent per milligram of dry extract (μg EQ/mg extract) [12].

Dosage of Polyphenols

Add 200 µl of the extract in a tube to the mixture (1 ml of Folin-Ciocalteu reagent diluted 10 times and 0.8 ml of 7.5% sodium carbonate). Shake the tube and store at room temperature for 30 minutes. Use a spectrophotometer to measure the absorbance at 765 nm against a blank. Use gallic acid as a positive control and run the calibration curve in parallel under the same operating conditions. Results are expressed in milligrams (mg) of gallic acid equivalent per gram of dry algae (mg EAG/g DM).

Dosage of Chlorophyll a, b, and Total Carotenoids

The determination of chlorophyll and carotenoids was carried out according to the spectral method [13, 14]. It uses characteristic wavelengths which correspond to the maximum absorbances of chlorophylls and carotenoids. 3 g of crushed algae were added to 30 ml of methanol. After adding 300 mg of magnesium sulfate, the mixture was incubated for 24 hours at 4°C. The mixture obtained was filtered and then centrifuged at 5000 rpm for 10 minutes. The supernatant was collected and the absorbances were read at 666, 653, and 470 nm for chlorophylls a, b, and total carotenoids, respectively. The chlorophyll and carotenoid contents were calculated in μg/ml according to the following formulas:

Ca (μg/ml) = 15.65 A666 – 7.340 A653

Cb (μg/ml) = 27.05 A653 – 11.21 A666

Cc (μg/ml) = 1000 A470 – 2.860 Ca – 129.2 Cb/245

Evaluation of Antioxidant Activity

Evaluation of Antibacterial Activity

The bacterial activity was evaluated on seven bacteria (Pseudomonas sp, Escherichia coli, Bacillus subtilus, Bacillus cereus, Methicillin-resistant Staphylococcus aureus or SARM, Klebsiella sp, Staphylococcus aureus). In order to evaluate the antimicrobial activity of the extracts of the three algae, we used the diffusion method on the Muller Hinton agar medium [14]. Sterile Wattman filter paper discs measuring 6 millimeters in diameter are impregnated with 40 μl of the different extracts. Using sterile forceps, the discs are placed on the surface of a medium seeded with a microbial suspension with an optical density of 0.5 McFarland (10⁸CFU/ml). After diffusion, the plates are incubated for 18 to 24 hours at 37°C. Discs impregnated with different solvents will serve as controls.

All analyses were performed in triplicates and the experimental data were expressed as means±standard deviation using Microsoft Office Excel 2007.

Extraction Yields

Yields of crude extracts were determined relative to 50 g of plant material, and the results are shown in Fig. 1.

The method of extracting bioactive materials from plants plays an important role in providing consumers with a high-quality herbal product, and modern “advanced” extraction methods are superior to conventional methods. Extraction is considered one of the most crucial procedures in the manufacture of herbal products, which affects the active ingredients in the sample both qualitatively and quantitatively [16].

The best yield extraction obtained is that of Cystoseira amentacea var. stricta with a value of 6.4%, followed by that of Ulva (4.50%), and the lowest is recorded by the extract of Corallina elongata, with a rate of 1.2%. Results that are close to ours put forward a yield value of 2.53% for the methanolic extraction of Corallina elongata [17].

Fig. 1.

Extraction yields of the algae.

Calculations of extraction yields are based on several factors, namely the extraction temperature, the initial plant material, and humidity [18]. This difference between species can also be attributed to the polarity of the compounds present in these algae, in addition to the nature of the solvent used and the chemical properties of the molecules to be extracted [19]. Likewise, the extraction method (mace­ ration, decoction, infusion) also plays an important role in determining the yield and the chemical composition of the prepared extract [20].

Dosage of Phenolic Compounds

The contents of polyphenols and total flavonoids in our samples are reported in Table 1. A single dissolvent cannot extract all classes of phenolic compounds simultaneously and at a maximum concentration [21]. Therefore, we have used a mixture of two solvents.

From the results of a dosage of polyphenols we find that the methanol-toluene extract of Corallina elongata is rich in total polyphenols with a value of 219.43 mg/g, followed by the green algae extract Ulva lactuca with 79.7743 mg/g, while the extract of the brown algae Cystoseira amentacea is lower than the other two with a content of 73.92 mg/g. These results are not in agreement with other studies [22], which indicate that brown algae have higher polyphenolic contents compared to green and red algae. On the other hand, the values found are close to those [23] which put forward more or less comparable concentrations in Cystoseires: Cystoseira crinita (56.5 mg/g), C. sedoides (50.3 mg/g), C. compressa (61 mg/g), and which [24] recorded values of 37.5 mg/g of polyphenols in Corallina elongata.

According to the literature, methanolic extracts are the richest in phenolic compounds, therefore methanol remains the best solvent for extracting these compounds. This is due to the ability of methanol to inhibit the action of polyphenol oxidase, which causes the oxidation of polyphenols in plant tissues [25, 26].

A wide range of algae species, including Ulva fasciata, were screened for antimicrobial activity. Researchers stated that drying reduced the bioactive compounds in the chosen algal species, and they found that methanol: toluene was the best solvent for extracting the antimicrobial compounds from fresh algae, and that the antimicrobial activity from chosen algae was due to a lipophilic compound that was stable over a broad range of temperatures (30–60°C) [27].

Many works suggest that maceration time affects the extraction rate [28–30] and the increasing temperature leads to an increasing extraction rate [31].

For flavonoids, we see that the Ulva extract has a higher content of flavonoids with a value of 65.17 mg/g, coming just after the Corallina red algae extract with 61.6 mg/g, the highest content. The lowest was recorded in Cystoseira with a value of 36.2 mg/g.

There exists little work on the flavonoid content in marine algae [32–34] in contrast to the numerous studies on flavonoids from terrestrial plants. A study on the distribution of flavonoids in 27 species of marine algae (6 Chlorophyta, 11 Phaeophyta, and 10 Rhodophyta) showed that they had a completely different flavonoid composition from fruits and vegetables [35].

The dosage of flavonoids is very important because they are a class of secondary plant phenols possessing potent antioxidant activity, thereby scavenging a wide range of reactive oxygen species and inhibiting lipoperoxidation, making them a potential therapeutic agent against a wide variety of diseases [36].

Antimicrobial Activity

A study of 82 different marine macroalgae from the Chlorophyceae, Phaeophyceae, and Rhodophyceae classes were screened for antimicrobial activity [41]. Among the selected taxa, Phaeophyceae were reported to be the most active, with 84% of the algae from this taxa showing antimicrobial activity.

The results obtained for the antibacterial activity, reported in the histogram in Fig. 2, and expressed in terms of the diameter of the inhibition zone, showed that all bacteria are sensitive to one or other of the extracts except Pseudomonas which revealed its resistance for all three algae. The crude extracts of Ulva act on B. cereus, B. subtilus, and SARM with diameters of (8 mm), (10 mm), and (22 mm), respectively. This is consistent with the work [42] that noted the activity of methanolic extracts of U. lactuca on SARM with inhibition zones greater than 10 mm. Unlike those [43], who showed that antimicrobial activity is absent in Ulva algae extracts. A previous study [44] reported that the methanolic and aqueous extracts of 19 marine algal species collected along the western coast of Libya against 4 Gram-positive and 4 Gram-negative bacteria revealed that the observed inhibition zones values with methanolic extract of U. lactuca ranged between 11 mm for S. aureus and Bacillus subtilis and 14 mm for P. aeruginosa and Klebsiella spp. They also reported that Cystoseira crinite (Phaeophyceae) of the 19 examined algae, was the most potent against tested isolates.

The same results are noted for Cystoseira extract. Indeed, the latter acts on B. cereus, B. subtilus, and SARM with inhibition zones of (20 mm), (14 mm), and (21 mm), respectively. Researchers [42] who worked on two brown algae Cystoseira humilis and C. bifurcata showed that they had good activity against SARM with an inhibition zone diameter greater than 15 mm. Cystoseira crude extract has no effect on Klebsiella, Staphylococcus, E. coli, and Pseudomonas, contrary to other researchers [45], who reported that different Cystoseira species exhibited potent antibacterial activity against Gram-negative bacteria: Salmonella typhi, E. coli, P. aeruginosa, and Klebsiella sp.

The extracts of the two algae Ulva and Cystoseira have no effect on E. coli. These results are consistent with others [42, 46] which noted that only a few species of algae presented weak inhibitory activity against E. coli. However, a study [47] reported that the antibacterial activity of methanolic extracts of U. lactuca and Enteromorpha compressa (Chlorophyta), against 4 Gram-negative and 2 Gram-positive isolates showed that U. lactuca extract exhibited the most inhibitory effect for K. pneumonia, S. aureus, P. vulgaris, B. subtilis, and P. aeruginosa, followed by E. compressa. Nonetheless, the methanolic extract of U. lactuca was inactive against E. coli. Reports [48] have been made about the antibacterial activity of methanol/methylene chloride of Ulva lactuca, Codiumto mentosum, and Hypnea musciformis collected from the Suez Canal, Egypt. The latest study showed that U. lactuca has inhibitory activity only against Salmonella typhimurium, K. pneumonia, E. coli, Shigella boydii and S. aureus with inhibitory zones ranging between 6–9 mm.

As for the Corallina extract, its effectiveness is noted against most strains except SARM and Pseudomonas. The inhibition zones vary from (16 mm) against B. subtilus, to 8mm for Kleibseila and Staphylococcus, passing through an inhibition zone of (12 mm) for E. coli.

Our results agree with those [49] which reported that high activity was shown on E. coli (32.00±1.73 mm) and E. faecalis (21.66±0.57 mm) with a methanolic extract of the red alga Corallina officinalis. Works [50] about the antimicrobial activities of some marine algae from the Mediterranean Sea (Algeria) show that the chloroform extracts of Ulva lactuca and Corallina elongata had the highest activity against E. coli and Salmonella sp.