Exploration of Antimicrobial Potential in Oualidia Algae – Unveiling Cystoseira Humilis as a Promising Source of Bioactive Molecules

The potential of seventeen marine seaweed extracts (five Chlorophyceae, six Pheophyceae, six Rhodophyceae) was investigated to determine their antibacterial activity, aiming to evaluate their viability in pharmaceutical applications. The assessment of their bioactivity involved utilizing crude extracts from dried samples against five Gram-positive bacteria, one Gram-negative bacteria, yeast, and fungus using the disk diffusion technique. These samples were collected from Oualidia, situated along Morocco’s Atlantic coast. Out of the diverse macroalgae studied, 36% of the extracts exhibited activity against at least one of the tested microorganisms. This outcome strongly supported the notion of leveraging algae extracts as a promising source of antibacterial compounds. In particular, extracts from Cystoseira humilis, Bifurcaria bifurcata and Asparagopsis armata showed zones of inhibition greater than 17 mm. Purification of the compounds responsible for the inhibitory activity against several types of C. humilis microorganisms was performed using chromatography and thin layer chromatography


INTRODUCTION
Several natural molecules used by man for pharmacological purposes have a marine origin, demonstrating the potential wealth of molecules with original structures and promising biological activities [Maggio et al.,  The Moroccan beaches and lagoons are a veritable reservoir of plant and animal species because of the immense diversity of marine flora and their unique richness.This presents Morocco with significant economic potential.In recent years, a great deal of research has been carried out into active substances of marine origin in the algae of the Atlantic coast of El Jadida.This work has led to the selection of the most active algal species and the isolation of numerous molecules with significant pharmacological activity [Fayzi et al., 2022;Khaya et al., 2022;Rhimou, 2013].
In this study, algal screening was performed to investigate the antibacterial activity of crude extracts (dichloromethane/ethanol) of seventeen species of marine algae, five green algae, six brown algae, and six red algae collected from the Oualidia lagoon on the Atlantic coast of Morocco.C. humilis is one of the species that showed a high inhibition zone (19mm), and the active molecule was purified.

Seaweed harvesting occurs in March and
April in the Oualidia lagoon.After harvesting, the algae are rinsed several times with distilled water to remove any foreign matter that might interfere with the analysis of biological activities.The algae are then sorted and identified.

ECOLOGICAL ENGINEERING & ENVIRONMENTAL TECHNOLOGY
For each species, the prepared algal powder was extracted in a mixture of organic solvents methanol/ethanol (50:50) at a rate of 5 g algal powder/ml solvent, following the extraction protocol described by Caccamese [Caccamese & Azzolina, 1979].The extracts are then filtered through Whatman paper and evaporated in a rotary evaporator.Extracts dried in this way are stored at 4 °C until they are used for biological testing.

Measuring antimicrobial activity
The antimicrobial activity was quantified using cellulose disks for organic extracts [Bauer et al., 1966].Quantities of 100 µg to 500 µg of the extract to be analyzed were deposited on cellulose paper disks, and after evaporation of the solvent, these disks were applied directly to a Petri dish previously inoculated with the test strain.
After 16 h of incubation at 37 °C, the antibacterial activity was measured as the diameter (in mm) of the zone of inhibition that appeared around the pellets.Similarly, antifungal activity was assessed after 24 h of incubation at 27 °C.Discs impregnated with standard antibiotics such as chloramphenicol, streptomycin, or tetracycline are used (at 50 or 100 µg/ml) as controls in antibacterial activity tests.

Column chromatography
This method relies on the phenomena of adsorption.A column of varying length and crosssection is filled with solid-phase silica.The extract is deposited at the top of the column, and the components separate because the eluent flows continuously through the column either under low pressure or by gravity.Compound displacement is accelerated by progressively increasing the polarity of the eluent employed.
Depending on their solubility in the eluent and affinity for the adsorbent, molecules are transported downhill at varying speeds.A series of colored cylindrical zones that split as they move downhill form the chromatogram.Various fractions were gathered on the basis of their polarity.
Every recovered fraction was examined for its potential to inhibit microorganisms or reduce inflammation.

Thin layer chromatography
Thin-layer chromatography (TLC) is mainly based on adsorption phenomena.The extract migrates through the solvent or solvent mixture.After being deposited on a 0.2 mm thick silica 60F254 plate (MERK), the substances migrate at a speed that depends on their polarity and that of the solvent.All recovered fractions were tested for antibacterial or anti-inflammatory activity.

Screening for the antibacterial activity of algal extracts
The various dichloromethane/methanol extracts prepared from the five species of green algae (Chlorophyceae) harvested in the Oualidia region C. tomentosum, U. lactuca, U. crispa E. linza, and E. intestinalis, were tested against the following microbial strains: S. aureus, B. cereus, B. thuringiensis, B. subtilis, C. sporogenes, E. coli, C. neoformens, and C. tropicalis (Figure 1).Of the extracts tested, 30% showed an inhibitory effect on the strains tested.The extracts of E. linza, E. intestinalis, and U. lactuca showed an inhibition diameter greater than 10 mm for S.  The other extracts showed only weak inhibitory activity against some of the microbial strains tested.With the exception of S. bulbosa, no activity was observed.A similar result was obtained [Elkouri et al., 2004].
The results obtained for F. spiralis and L. ochroleuca extracts showed inhibitory activity against S. aureus and B. cereus.The same result was obtained by [Etahiri, 2002].F. spiralis extract harvested showed significant inhibitory activity against S. aureus and B. subtilis  Several species of brown algae in the Cystoseira genus (Cystoseiraceae, Pheophyta) are widespread in the Atlantic and Mediterranean, and some species have been reported to contain biologically active compounds [Valls et al., 1993].
The antibacterial effect induced by C. humils extract, harvested on the Moroccan coast, has been reported by [Etahiri, 2002;Ibtissam et al., 2009], whereas the methanolic extract of C. humilis, harvested on the Canary Islands, shows no antimicrobial activity [Val et al., 2001].
In the screening performed, C. humilis extract showed an inhibition diameter of 18 mm against B. cereus.
To purify the molecule responsible for this activity, we adopted the protocol shown in Figure 5.
In this assay, 20g of dried powder was extracted in a dichloromethane/methanol (1:1) solvent mixture.After filtration and evaporation of the mixture, 1g of dry extract was obtained.The separation was performed on a normal-phase silica column, elution was performed with a mixture of solvents of increasing polarity, and a 3rd separation was performed on a TLC plate with a mixture of dichloromethane/methanol solvents (90:10).Fractions 4 and 7, 8, and 9 showing interesting antibacterial activity were collected (inhibition diameter greater than 20 mm against S. aureus and B. cereus) for purification by HPLC.

CONCLUSION
This study reveals the existence of algae with significant biological activity, which will undoubtedly become a valuable source of pharmaceutically interesting molecules in the future.In view of these results, we have the opportunity to pursue the purification of C. humilis extracts by HPLC to isolate the molecules responsible for its antimicrobial activity and to extend the purification to other selected antimicrobial active species.