Variability on the Phytochemical Composition, Antioxidant and Antimicrobial Activities of Ruppia cirrhosa Extract Using Two Different Methods of Extraction

determination of polyphenolic contents and antioxidant activity


Introduction
Natural products have been applied to cure human diseases since the beginning of mankind (1).The aquatic ecosystems are an important resource for a broad range of natural bioactive products (2), and the attention to the discovery of new bioactive substances from marine sources is increasing (3).Polyphenols from marine organisms are far less studied than those from terrestrial sources since their structural diversity and variability require powerful analytical tools.However, both their biological relevance and potential properties make them an attractive group deserving of increasing scientific interest.The marine seagrasses are known as magnoliophytes, and they are flowering plants, namely, angiosperms (4).Nowadays, marine seagrasses give rise to dense formations called "herbariums", which are found in almost all coastal environments around the world (5).These beds characterize the infralittoral stage where they preferentially colonize soft substrates.Five species of marine seagrasses are reported in the Mediterranean, including Cymodocea nodosa, Halophila stipulacea, Posidonia oceanica, Zostera marina, and Nanozostera noltii (6).For Ruppia cirrhosa and Ruppia maritima, these two species are widespread in lagoon environments (7).These two species belong to the widgeon grass family (Ruppiaceae), and they can exist in single populations with no other vascular plants present, and they are barely ever found together (2).
Polyphenols from marine organisms are far less studied than those from terrestrial sources since their structural diversity and variability require powerful analytical tools (8).R. cirrhosa was found to be rich in chicoric acid ( ≤ 30.20 mg/g) (2), has promising pharmacological effects in antiviral, contain anti-inflammatory, antihyperglycemic, antihyperlipidemic, antimicrobial, antioxidant, and antiaging properties, and impacts digestive system illness (9).The findings concerning secondary metabolites of R. cirrhosa, especially polyphenolic contents are limited (2,10).Additionally, the determination of the biological properties of R. cirrhosa is still scarce (2,11).Therefore, this research aimed to characterize the phytochemical composition as well as antioxidant and antimicrobial activities of Tunisian RCE using two different extraction methods.

Seagrass material
Ruppia cirrhosa was manually taken from the North Lake of Tunis and was put in an icebox.It was thoroughly washed using tap water and then sterile distilled water to be air dried at room temperature for one week.Afterward, dried R. cirrhosa was ground with a blender into powder to be analyzed.

Extract Preparation Extraction by Maceration
For this extraction, 2 g of dry R. cirrhosa powder was mixed with 20 mL of polar solvent (80% acetone) for 30 minutes.The mixture was stirred with a magnetic stirrer, standing for 24 hours at 4 °C in the dark and then was filtered through filter Watman paper No. 4 (12).

Extraction by Successive Solvents
This extraction was done using 3 solvents with different polarities, namely, hexane (Polarity: 0), petroleum ether, and acetone (Polarity: 5.4) at 80%.Weigh 5 g of the magnoliophyte R. cirrhosa using a precision balance.Before proceeding with the actual extraction, successive washes with hexane and petroleum ether were considered to remove pigments and lipids.For this, 50 mL of hexane was added to each sample.The mixture was then stirred for 1 hour and then filtered through ashless filter paper (Wattman No. 4); afterward, another 50 mL of petroleum ether was added to each sample.Once a clear and transparent supernatant was obtained, 50 mL of 80% acetone was added to the various algae samples which were stirred for 30 minutes and reserved at rest for 24 hours at 4 °C in the dark.Subsequently, the solution was filtered through ashless filter paper (Whatman No. 4).

Phytochemical Contents
The assay of polyphenols and the total flavonoid assay were done according to Dewanto et al (13), and the assay of condensed tannins was determined following Sun et al (14).

Antioxidant Activity
The total antioxidant measurement was based on the reduction of Mo 6 + ions to Mo 5 + by the seagrass extract to form the green-colored phosphate-Mo 5 + complex at an acid pH (15).The antiradical activity was measured by 2,2'-diphenyl-1-picrylhydrazyl (DPPH).A test sample of 1 mL of the extract at different concentrations (10, 50, 75, 100, 500, 1000, and 1500 µg/mL) was placed in the presence of 250 µL of a DPPH solution (0.2 mM in methanol).The mixture remained at rest for 30 minutes and in the dark for incubation (16).
For reducing power assay, samples at different concentrations (from 10 to 1500 μg/mL) were mixed with 1 mL K 3 [Fe(CN) 6 and 1 mL tryptone phosphate water (0.2 M, pH 6.6) (1%), incubated in a water bath at 50 °C for 20 minutes, then 1 mL of tricarboxylic acid (10%) was added to each reaction.Afterward, 1 mL of this solution was added to 1 mL of distilled water and 0.2 mL of 0.1% FeCl 3 solution ( 16).
The iron chelating power assay involved mixing 100 µL of the extract of known concentrations with 50 µL of FeCl 2 6H 2 O solution (2mM: 0.00379g/100mL) to leave them to rest for 5 minutes at room temperature.A dose of 100 µL of a solution of Ferrozine (5 mmol/L) was added afterward to trigger the reaction.Finally to adjust the mixture to 3mL, 2750 µL of distilled water must be added (17).
For β-carotene bleaching activity, a sample of 2 mg of β-carotene was dissolved in 20 mL of chloroform.Then, 2 mL of the mixture was added to 20 mg of linoleic acid and 200 mg of Tween 40.After the evaporation of the chloroform at 40 °C, 100 mL of hydrogen peroxide was added to the residue.After vigorous stirring, 150 µL of the β-carotene/linoleic acid mixture was added to 10 µL of the samples and prepared in the extraction solvent at different concentrations.Then, three repetitions were carried out for each concentration (16).The antibacterial and antifungal activities of seagrass extracts against pathogenic bacteria and fungi were carried out by the solid diffusion method (18).To fix the strains of bacteria and yeasts on the agar medium, an incubation was carried out for 15 minutes at 37 °C.The boxes were then removed and the excess liquid was discarded under sterile conditions.Another incubation of these boxes in the oven at 37 °C for a quarter of an hour allows them to dry, and thus a carpet of bacteria or yeasts is obtained.Sterile Whatman paper discs 6 mm in diameter were placed on the bacteria and yeast mats.Then,10 μL doses of the extracts (0.5 mg/mL) were put on the discs.The effect of RCE on the growth of the strains around the disc was observed after incubation at 37 °C for 24 hours.The formation of a halo around each disk reflects the inhibition of the growth of bacteria and yeasts (19).This activity consisted of measuring the diameter of inhibition, and the positive controls used for these activities were antibiotics (Gentamicin).

Phytochemical Characterization
The analysis of the obtained results concerning the amounts of polyphenols showed significant variability between the two extraction methods (Figure 1).The successive extraction of R. cirrhosa extract relatively indicated higher total phenol (38.1 mg GAE/g) and condensed tannin (18.07 mg CE/g) contents than the extraction of maceration (35.43 mg EAG/g and12.99 mg CE/g, respectively).However, the total flavonoid content of RCE was higher in the maceration extraction (33.09 mg CE/g) than in the successive extraction (21.27 mg CE/g).

Antioxidant Activity
Table 1 presents the antioxidant activities of RCE obtained by maceration and successive extraction using different assays.The total antioxidant capacity of RCE showed a decrease in this activity after fractionation.Indeed, this activity decreased RCE from 47.8 to 37.83 mg GAE/g, and the positive control quercetin had the lowest total antioxidant capacity (28.15 mg GAE/g).Furthermore, the results indicated a decrease in the antiradical activity using DDPH assay after fractionation as well as an increase of 2.5 folds for IC 50 ranging from 6.34 to 16.04 μg/mL.RCE obtained by the two extraction methods showed moderate antioxidant activity using reducing power and β-carotene bleaching assays.Additionally, a decrease in the reducing power and β-carotene bleaching effect of RCE was highlighted after fractionation.For reducing power, IC 50 increased from 380 μg/mL to 490 μg/mL , while for the β-carotene bleaching effect, IC 50 increased from 110 μg/ mL to 310 μg/mL.Values are given as mean ± SD. (n = 3) followed by different letters (a-c) imply the significant differences (P < 0.05) according to the Tukey HSD test.

Discussion
This study investigated the variability of the phytochemical, antioxidant, and antimicrobial activities using two different extraction methods.Indeed, the amounts of total phenols and condensed tannins increased using successive extraction, but total flavonoid content decreased by 1.5 times.The presence of phenols, flavonoids, and tannins in seagrasses may indicate that their extracts have antioxidant activity.This activity was believed to help prevent several diseases through free-radical scavenging activity (20).
In this study, a decrease was observed in reducing power and β-carotene bleaching activities after fractionation.This study was the first account of findings on reducing power and β-carotene bleaching activities of RCE.In our study, there was also a decrease in the antiradical activity using DDPH assay after fractionation with an increase of 2.5 folds for IC 50 ranging from 6.34 to 16.04 μg/mL.However, Hasle Enerstvedt et al (2) noted that RCE exhibits an IC 50 = 152.9-175.7 µg/mL, showing a low radical scavenging activity.However, after partition with ethyl acetate, the hydraulic R. cirrhosa phase had a very strong antiradical scavenging activity (IC50 = 31.8μg/ mL).The differences recorded between our results and those of these authors can be explained by the fact that the extraction method was different, the solvents were not of the same nature (polarity), and the extraction process (continuous extraction, magnetic stirring, supercritical fluid extraction, and the like) and the extraction conditions (extraction time, temperature) were varied.That is why there was no uniform solvent or extraction method to determine the biological capacity of terrestrial plants let alone aquatic macrophytes.This shows the importance of screening and fractionation to look for interesting and recoverable species at an industrial scale.From RCE, Hasle Enerstvedt et al (2) isolated one phenolic acid (chicoric acid), and 8 flavonoids (four quercetin derivatives and four isorhamnetin derivatives) had a strong antiradical DPPH activity (12.1-88.4μg/mL).Several studies have reported that polyphenols of seagrass extracts are mainly responsible for antioxidant activities (21)(22)(23)(24)(25).
Regarding the antimicrobial activity, the two extraction methods of RCE showed modest to extremely appreciable inhibitory activities of bacterial and fungal strains with IZ varying from 1.33 to 9.33 mm.These inhibitory effects could be due to the presence of active molecules (alkaloids, glycosides, phenolic acids, tannins, flavonoids, saponins, and Terpenes) of RCE.Moreover, Hasle Enerstvedt et al (2) reported that RCE is rich in phenolic chicoric acid ( ≤ 30.20 mg/g) which has a potent antimicrobial activity (26).Abd El-Hady et al (11) found that RCE is not active against the fungus Aspergillus flavus and A. fumigates, but it is only active against Alternaria alternata (IZ = 10 mm).This ethanol RCE also had active inhibitory action against B. subtilis (IZ = 10.50 mm), S. aureus (IZ = 10 mm), E. coli (IZ = 19.50mm), and P. aeruginosa (IZ = 11 mm).In fact, the ethanol extract effect was significant against both tested Gram-positive and negative bacteria.This can be explicated by the presence of a cell wall with a single layer in Gram-positive bacteria but with a multi-layered structure bordered by an outer cell membrane in Gramnegative bacteria (27).

Conclusion
Since RCE is rich in bioactive molecules and has highly promising biological potential, other investigations can be carried out on this seagrass to identify and characterize these bioactive molecules by advanced chromatographic techniques for possible valorization in various fields such as cosmetics, foods, and pharmaceuticals.

Figure 1 .
Figure 1.Total Contents of Total Phenolics, Total Flavonoids, and Condensed Tannins of Ruppia cirrhosa Extracts Obtained by Maceration and Successive Extraction.Note.* Total phenolic contents were expressed as milligrams of gallic acid equivalents per gram of dry extract (mg GAE/g DE); Total flavonoid contents were expressed as milligrams of catechin equivalents per gram of dry extract (mg CE/g DE); Condensed tannin contents were expressed as milligrams catechin equivalents per gram of dry extract (mg CE/g DE).Values are given as mean ± S.D (n = 3).Means followed by the same small letter (a, b) shared significant differences at P < 0.05(Duncan test) 4.67 ± 0.65 b 6 ± 0.00 a -Note.Activity is absent (-) if the inhibition zone (IZ) was < 1 mm, low if IZ = 1 mm, slight if IZ between 2-3 mm, moderate if IZ between 4-5 mm, high if IZ between 6-9 mm, strong if IZ > 9 mm.Values are given as mean ± S.D. (n = 3) followed by different letters (a-c) imply the significant differences (P < 0.05) according to the Tukey HSD test.
Table 2 illustrates the antimicrobial activities of RCE obtained by maceration and successive extraction.RCE obtained by maceration had the greatest antibacterial activity against all tested strains (IZ = 3.33-9.33mm) except for S. typhimurium (IZ = 2 mm), E. faecalis (IZ = 6 mm), and S. aureus (3.67 mm) as compared to those obtained by successive extraction.