marine drugs

Article
Investigation into the Phytochemical Composition, Antioxidant
Properties, and In-Vitro Anti-Diabetic Efficacy of Ulva
lactuca Extracts
Safae Ouahabi 1 , Nour Elhouda Daoudi 2,3 , El Hassania Loukili 4 , Hbika Asmae 1 , Mohammed Merzouki 1 ,
Mohamed Bnouham 2 , Allal Challioui 1 , Belkheir Hammouti 4 , Marie-Laure Fauconnier 5 , Larbi Rhazi 6, * ,
Alicia Ayerdi Gotor 7 , Flore Depeint 6 and Mohammed Ramdani 1

1 Laboratory of Applied and Environmental Chemistry (LCAE), Faculty of Sciences, Mohammed First
University, B.P. 717, Oujda 60000, Morocco; ouahabi.safae@ump.ac.ma (S.O.);
asmaae.hbika@gmail.com (H.A.); moh.merzouki@gmail.com (M.M.); allal.challioui@gmail.com (A.C.);
moharamdani2000@yahoo.fr (M.R.)
2 Laboratory of Bioresources, Biotechnology, Ethnopharmacology and Health, Faculty of Sciences,
Mohammed First University, B.P. 717, Oujda 60000, Morocco; nourelhoudada95@gmail.com (N.E.D.);
mbnouham@ump.ac.ma (M.B.)
3 Higher Institute of Nursing Professions and Health Techniques, Oujda 60000, Morocco
4 Euromed Research Center, Euromed Polytechnic School, Euromed University of Fes (UEMF),
Fes 30000, Morocco; e.loukili@ump.ac.ma (E.H.L.); hammoutib@gmail.com (B.H.)
5 Laboratory of Chemistry of Natural Molecules, University of Liège, Gembloux Agro-Bio Tech. 2, Passage des
Déportés, B-5030 Gembloux, Belgium; marie-laure.fauconnier@uliege.be
6 Institut Polytechnique UniLaSalle, Université d’Artois, ULR 7519, UniLaSalle, 19 rue Pierre Waguet, BP 30313,
60026 Beauvais, France; flore.depeint@unilasalle.fr
7 Institut Polytechnique UniLaSalle, AGHYLE, UP 2018.C101, UniLaSalle, 19 rue Pierre Waguet, BP 30313,
60026 Beauvais, France; alicia.ayerdi-gotor@unilasalle.fr
* Correspondence: larbi.rhazi@unilasalle.fr
Citation: Ouahabi, S.; Daoudi, N.E.;
Loukili, E.H.; Asmae, H.; Merzouki, Abstract: In this research, the chemical compositions of various extracts obtained from Ulva lactuca,
M.; Bnouham, M.; Challioui, A.; a type of green seaweed collected from the Nador lagoon in the northern region of Morocco, were
Hammouti, B.; Fauconnier, M.-L.; compared. Their antioxidant and anti-diabetic properties were also studied. Using GC–MS tech-
Rhazi, L.; et al. Investigation into the nology, the fatty acid content of the samples was analyzed, revealing that palmitic acid, eicosenoic
Phytochemical Composition,
acid, and linoleic acid were the most abundant unsaturated fatty acids present in all samples. The
Antioxidant Properties, and In-Vitro
HPLC analysis indicated that sinapic acid, naringin, rutin, quercetin, cinnamic acid, salicylic acid,
Anti-Diabetic Efficacy of Ulva lactuca
apigenin, flavone, and flavanone were the most prevalent phenolic compounds. The aqueous ex-
Extracts. Mar. Drugs 2024, 22, 240.
tract obtained by maceration showed high levels of polyphenols and flavonoids, with values of
https://doi.org/10.3390/
md22060240
379.67 ± 0.09 mg GAE/g and 212.11 ± 0.11 mg QE/g, respectively. This extract also exhibited an
impressive ability to scavenge DPPH radicals, as indicated by its IC50 value of 0.095 ± 0.12 mg/mL.
Academic Editor: Marcelo D.
Additionally, the methanolic extract obtained using the Soxhlet method demonstrated antioxidant
Catarino
properties by preventing β-carotene discoloration, with an IC50 of 0.087 ± 0.14 mg/mL. Results from
Received: 2 May 2024 in-vitro studies showed that extracts from U. lactuca were able to significantly inhibit the enzymatic
Revised: 21 May 2024 activity of α-amylase and α-glucosidase. Among the various extracts, methanolic extract (S) has
Accepted: 21 May 2024 been identified as the most potent inhibitor, exhibiting a statistically similar effect to that of acarbose.
Published: 25 May 2024 Furthermore, molecular docking models were used to evaluate the interaction between the primary
phytochemicals found in these extracts and the human pancreatic α-amylase and α-glucosidase
enzymes. These findings suggest that U. lactuca extracts contain bioactive substances that are capable
of reducing enzyme activity more effectively than the commercially available drug, acarbose.
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
Keywords: Ulva lactuca; fatty acids; phenolic compounds; antioxidant activity; enzyme inhibition;
distributed under the terms and anti-diabetic properties; molecular docking
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Mar. Drugs 2024, 22, 240. https://doi.org/10.3390/md22060240 https://www.mdpi.com/journal/marinedrugs

Mar. Drugs 2024, 22, 240 2 of 22

1. Introduction
Ulva lactuca, also known as sea lettuce, forms a very thin blade made up of two layers
of cells, orbicular in shape and transparent, reminiscent in texture of lettuce [1]. The thallus,
which is flat and entire, can measure up to 10 cm in width and is attached by a small,
disc-shaped spike. The cells of the bi-stromatic blade are rectangular and contiguous, 20 to
23 µm long and 20 to 21 µm high, and contain a single parietal chloroplast. The U. lactuca is
an alga found along coastlines worldwide [2–4]. This macroalga is the only one capable of
causing dense blooms [5,6]. Although it can grow at depths of up to 15 m, it is typically
found at a depth of 1 m due to its requirements for light color and intensity [5]. The growth
rate of this alga depends on the amount of light, nitrogen, and sporulation. Additionally,
the presence of epiphytes and grazers can limit its growth [7]. Due to its two-cell-layered
thallus, the surface area per unit volume of Ulva lactuca is highly significant, enabling it
to absorb large quantities of nutrients through its cell wall. This absorption capacity is
particularly high when growth rates are elevated, and under favorable conditions, U. lactuca
can absorb up to between four and six times more nutrients than other algal species [8]. In
response to high nutrient levels, U. lactuca increases its nutrient absorption, grows rapidly,
and stores intracellular nutrients for future growth [9].
Insoluble fibers, such as hemicellulose, cellulose, and lignin account for around a third
of the seaweed’s dry matter, which is significantly lower than in terrestrial plants [10].
Cellulose, the most abundant organic compound on earth, is present in both marine and
terrestrial plants [11]. However, the cellulose of algal species forms a more porous network,
which differs considerably from the cellulose of higher plants [12]. Cellulose contents
vary considerably from one algal species to another: crude cellulose contents of 11%, but
also 0.85% of dry weight have been found [13]. Similarly, cellulose concentrations vary
according to the different parts of the alga (frond or stipe) [14]. In comparison, the cell walls
of terrestrial plants are composed of a high content of cellulose (45% DW), hemicellulose
(18% DW), and lignin (20% DW), which play an essential role in vessel construction,
strengthen the entire plant structure, and prevent the plant collapsing into the air [11,15,16].
Algae, on the other hand, need no support as they grow in an aquatic environment. The
cellulose content of Ulva lactuca is significantly lower than that of terrestrial plants, and
although compounds comparable to lignin have been found in primitive algae, most
seaweeds contain little or no lignin [11,14,16]. The lignin content of Ulva lactuca is only
1.6%, which is extremely low compared with the 17–24% found in the grass and legume
families [17]. Ulva lactuca polysaccharides are easily hydrolyzed due to their low lignin
content, enabling high concentrations of bioethanol to be obtained per unit weight [14].
Seaweed is rich in nutrients such as vitamins and minerals, while being low in fat
and calories compared to foods of animal origin, giving it a high nutritional value [18]. In
particular, Ulva lactuca is a source of high-quality protein containing 17 different amino
acids, including all the essential ones, making it excellent for human consumption [19]. In
addition to its food potential, seaweed can also be used to produce biofuels. Ulva lactuca is
an efficient biomass source for biofuel production, with a high potential yield of 24 tons of
dry biomass per hectare per year, similar to that of sugar beet and three times higher than
brown seaweed [20].
The aim of the present study was to undertake a comprehensive chromatographic
analysis of the fatty acids and polyphenols present in U. lactuca extracts. For this purpose,
two extraction techniques were employed, using solvents with differing polarities for
comparative analysis. Furthermore, the research aimed to evaluate the antioxidant potential
of the extracts and assess their impact on the activities of pancreatic α-amylase and α-
glucosidase. The results of these analyses will provide a better understanding of the
potential health advantages of U. lactuca extracts sourced from the Marchica lagoon, as well
as their potential applications in food and pharmaceutical industries.
Mar. Drugs 2024, 22, 240 3 of 22

2. Results
2.1. Yields, Phenols, and Flavonoid Contents
This research aims to assess the efficacy of different extraction procedures by using
two distinct methods, namely Soxhlet (90 ◦ C) and maceration (25 ◦ C). The evaluation was
conducted using four solvents with increasing polarity, including hexane, ethyl acetate,
methanol, and water. The phenols, flavonoids, and extraction yield of U. lactuca extracts
were studied, and the results are presented in Table 1.

Table 1. Phenols and flavonoid contents of different extracts of U. lactuca.

Extraction Polyphenols Flavonoids

Solvent
Methods (mg GAE/g) (mg QE/g)
M 198.09 ± 0.11 102.10 ± 0.09
Ethyl acetate
S 145.74 ± 0.15 65.27 ± 0.05
M 47.53 ± 0.05 27.18 ± 0.09
Methanol
S 32.46 ± 0.08 20.24 ± 0.04
Water M 379.67 ± 0.09 212.11 ± 0.11
GAE: gallic acid equivalent per gram of dry weight; QE: quercetin equivalent; M: maceration; S: Soxhlet.

In terms of maceration extraction, the aqueous extract demonstrated the highest

extraction yield at 9.45 ± 0.05%, followed by the methanolic extract at 1.18 ± 0.09%.
Furthermore, the methanolic extract obtained through Soxhlet extraction had the highest
polyphenolic yield with 4.21 ± 0.04%, followed by the hexanic extract with 2.23 ± 0.07%.
The ethyl acetate extract had the lowest yield at 1.34 ± 0.06%.
The total phenolic content of the extracts was quantified in terms of gallic acid
equivalent (GAE), as presented in Table 1. The highest total polyphenolic content was
observed in the aqueous extract obtained through maceration, registering a value of
379.67 ± 0.09 mg GAE/g. Following this, the ethyl acetate extract exhibited a total phenolic
content of 198.09 ± 0.11 mg GAE/g and 145.74 ± 0.15 mg GAE/g, using the maceration and
Soxhlet extraction methods, respectively. These findings indicate the efficacy of both water
and ethyl acetate as solvents for phenolic compound extraction from U. lactuca, regardless of
the extraction method employed. In contrast, the methanolic extracts displayed the lowest
total phenolic content, with values of 47.53 ± 0.05 mg GAE/g and 32.46 ± 0.08 mg GAE/g.
The flavonoid content of the different extracts was measured in terms of quercetin equiv-
alent (QE), as detailed in Table 1. Flavonoid concentrations ranged from 20.24 ± 0.04 mg
QE/g to 212.11 ± 0.11 mg QE/g. The highest concentration was observed in the aqueous
extract obtained through the maceration method. Following this, the ethyl acetate extract
exhibited concentrations of 102.1 ± 0.09 mg QE/g and 65.27 ± 0.05 mg QE/g, utilizing the
maceration and Soxhlet extraction techniques, respectively.

2.2. Fatty Acid Analysis

The chemical compositions of Ulva lactuca hexane and ethyl acetate extracts are pre-
sented in Table 2. Analysis by GC–MS revealed the presence of nine saturated and unsatu-
rated fatty acids, the content of which varied according to the solvent used. Compared to
terrestrial plants, marine algae have a unique fatty acid (FA) profile. Fatty acid composition
in samples is expressed in terms of total fatty acids. The percentages of saturated fatty acids
(SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) vary
according to the solvent and extraction method used. The main constituents of Ulva lactuca
extracts were palmitic acid, eicosenoic acid, and linoleic acid.
Mar. Drugs 2024, 22, 240 4 of 22

Table 2. Fatty acids composition of hexane and ethyl acetate extracts from green algae U. lactuca.

RT HE (%) EAcE (%)

Fatty Acids
(min) M S M S
Eicosenoic acid (C20:1) 20.08 35.44 ± 0.02 6.93 ± 0.04 29.62 ± 0.07 nd
7,10-Hexadecadienoic acid (C16:2) 21.17 2.60 ± 0.05 2.93 ± 0.01 13.52 ± 0.04 nd
Palmitoleic acid (C16:1) 23.12 5.01 ± 0.07 6.37 ± 0.03 24.72 ± 0.06 nd
Palmitic acid (C16:0) 23.31 28.05 ± 0.03 46.81 ± 0.10 32.14 ± 0.08 49.92 ± 0.07
Margaric acid (C17:0) 23.87 nd 6.90 ± 0.06 nd nd
Oleic acid (C18:1) 24.55 nd nd nd 28.60 ± 0.04
Linoleic acid (C18:2) 25.04 25.48 ± 0.01 23.50 ± 0.05 nd 21.48 ± 0.05
Linolenic acid (C18:3) 25.09 nd 6.56 ± 0.02 nd nd
Stearic acid (C18:0) 25.26 3.42 ± 0.01 nd nd nd
SFA a 31.47 31.47 53.71 32.14 49.92
UFA b 68.53 68.53 46.29 67.86 50.08
UFA/SFA 2.18 2.18 0.86 2.11 1
RT: retention time; M: maceration; S: Soxhlet, HE: hexane extract; EAcE: ethyl acetate extract; nd: not detected;
a : saturated fatty acids (SFA); b : unsaturated fatty acids (UFA).

In the hexane extract, eicosenoic acid (C20:1) was the most abundant fatty acid ob-
tained via maceration extraction, with 35.44%, followed by palmitic acid (16:0), with a high
amount also at 28.05%, and by linoleic acid, with relatively the same concentration in both
extraction methods at 23–25%. However, the hexane extract (46.81%) and the ethyl acetate
extract (49.92%) obtained via Soxhlet exhibited the highest concentration of palmitic acid.
Specifically, in the ethyl acetate extract obtained through Soxhlet, oleic acid (C18:0) was
exclusively detected at a concentration of 28.60%, while linoleic acid (C18:2) showed a
relatively high concentration of 23.85%. Nevertheless, margaric acid, linolenic acid, and
stearic acid were found in low amounts in only one extract of U. lactuca, ranging from 3.42%
to 6.9%. 7,10-Hexadecadienoic acid and palmitoleic acid accounted for 2.60% to 24.72% of
the total fatty acids.
The hexane extract and the ethyl acetate extract obtained through the maceration
method revealed the highest UFA/SFA ratio on the order of 2. These results draw attention
to the possibility of using these extracts as a natural source of polyunsaturated fatty acids
for nutritional, cosmetic, or pharmaceutical purposes.

2.3. HPLC Analysis of U. lactuca Extracts

High-performance liquid chromatography coupled with a diode array detector (HPLC-
DAD) was utilized to analyze the chemical composition of the ethyl acetate and methanol
extracts derived from U. lactuca. The obtained results were compared with standard com-
pounds based on retention time and ultraviolet spectra; the findings are presented in
Table 3. The investigation identified nine phenolic acids (4-hydroxybenzoic acid, chloro-
genic acid, sinapic acid, cinnamic acid, gallic acid, caffeic acid, syringic acid, p-coumaric
acid, and salicylic acid) along with ten flavonoids (catechin, vanillin, quercetin-3-glucoside,
7,3′ ,4′ -flavon-3-ol, rutin, quercetin, naringin, apigenin, kaempferol, flavone, and flavanone).
Mar. Drugs 2024, 22, 240 5 of 22

Table 3. Chemical composition of ethyl acetate and methanolic extracts from green algae U. lactuca.

EAcE (%) ME (%)

N◦ Compounds RT (min)
M S M S
1 Gallic acid 15.47 nd nd nd 0.64
2 Catechin 18.68 1.52 8.18 nd nd
3 4-hydroxy benzoïque 18.91 4.11 18.77 nd 3.56
4 Chlorogenic acid 19.15 1.09 4.20 11.66 7.71
5 Caffeic acid 19.45 nd Nd 35.64 24.24
6 Syringic acid 19.74 nd 3.09 3.67 nd
7 Vanilline 23.10 3.45 nd nd nd
8 p-Coumaric acid 23.63 Nd nd nd 5.26
9 Sinapic acid 24.09 9.53 nd nd nd
10 Quercetine 3glucoside 24.52 8.07 nd nd nd
11 7,3′ ,4′ -flavon-3-ol 24.92 8.31 20.68 nd nd
12 Naringin 25.01 8.34 17.75 21.06 12.63
13 Rutin 25.16 8.29 nd nd nd
14 Salicylic acid 25.32 nd 15.61 14.90 11.85
15 Quercetine 25.46 8.67 nd nd nd
16 Cinnamic acid 25.48 8.94 nd nd 9.66
17 Luteolin 25.64 8.91 nd nd 9.38
18 Apigenine 25.87 7.83 nd nd nd
19 Kaempferol 26.1 9.11 11.69 8.10 9.11
20 Flavone 26.92 nd nd 4.95 5.94
21 Flavanone 27.412 nd 3.79 nd nd
RT: retention time; M: maceration; S: Soxhlet; EAcE: ethyl acetate extract; ME: methanolic extract; nd: not detected.

The most abundant phenolic compounds found in Ulva lactuca extracts were sinapic
acid (10.46%) in EAcE (M), (14.17%) in EAcE (S), (14.43%) in EM (M), and (12.69%) in EM
(S); 7,3′ ,4′ -flavon-3-ol (10.94%) in EAcE (S); naringin (12.81%) in EAcE (M) and (13.11%)
in EM (M); and rutin (10.30%) in EAcE (S) and (12.42%) in EM (S). We also found salicylic
acid (11.83%) in ME (M), quercetin (10.10%) in EAcE (M) and (9.89%) in EAcE (S), cinnamic
acid (8.94%) in EAcE (M) and (9.44%) in EAcE (S), apigenin (11.58%) in EAcE (S), flavone
(11.29%) in EAcE (S), and flavanone with a value of (13.62%) in EAcE (M) and (22.72%) in
EAcE (S).
In this study, the results revealed that naringin predominated in EAcE (M), while
sinapic acid was the majority compound in EAcE (S), EM (M), and EM (S). In contrast,
flavanone was found to be the main compound in ME (M) and ME (S). Other compounds
were identified in Ulva lactuca extracts but at low percentages, namely gallic acid, catechin,
4-hydroxy-benzoic acid, chlorogenic acid, caffeic acid, syringic acid, vanillin, p-Coumaric
acid, quercetin 3-glucoside, and kaempferol.

2.4. Antioxidant Activity

Antioxidants have garnered considerable attention in research owing to their capacity
to maintain food quality and their potential in treating diseases linked to oxidative stress.
In this study, we employed the DPPH free radical scavenging assay and the β-carotene
bleaching assay. The IC50 values for U. lactuca ethyl acetate extract (EAcE), methanol extract
(ME), and aqueous extract (AQE) are detailed in Table 4.
The results indicate that the highest antioxidant activity was observed in the case of
the aqueous extract, with a value of 0.09 mg/mL, compared to the reference antioxidant,
ascorbic acid, which exhibited an IC50 value of 0.06 mg/mL. The findings also suggest
that ethyl acetate extracts (0.55–0.62 mg/mL) generally exhibit antioxidant activity similar
to methanolic extracts (0.59–0.65 mg/mL), regardless of the extraction method employed.
This may be explained by the fact that antioxidant compounds present in water are more
effectively solubilized and extracted than those in organic solvents.
Mar. Drugs 2024, 22, 240 6 of 22

Table 4. IC50 values of U. lactuca extracts.

IC50 (mg/mL)
Extracts
DPPH β-Carotene
M 0.55 ± 0.02 0.47 ± 0.04
EAcE
S 0.62 ± 0.01 0.08 ± 0.14
M 0.59 ± 0.13 0.41 ± 0.22
ME
S 0.65 ± 0.21 0.10 ± 0.05
AQE M 0.09 ± 0.12 0.11 ± 0.17
Ascorbic Acid 0.06 -
BHA - 0.02
EAcE: ethyl acetate extract; ME: methanolic extract; AQE: aqueous extract; M: maceration; S: Soxhlet.

The aqueous extract obtained by maceration showed high antioxidant activity by the
DPPH method, compared with ascorbic acid used as a reference. The richness of this extract
in polyphenols and flavonoids could explain this high activity [21].

2.5. In-Vitro α-Amylase Inhibition

Table 5 presents the effect of Ulva lactuca extracts on α-amylase and α-glucosidase
inhibition activity, using acarbose as a positive control. In-vitro studies were conducted
to examine the impact of various doses of the extracts on α-amylase enzymatic activity.
Results showed that all extracts significantly inhibited α-amylase enzymatic activity at all
tested doses. Among the different samples, the concentration of 1.14 mg/mL had the most
active effect, exhibiting inhibitory activities of 57.64 ± 0.47 for EACE (M), 57.61 ± 0.21 for
EACE (S), 57.42 ± 0.10 for ME (M), 67.06 ± 0.06 for ME (S), and 57.67 ± 0.34 for AQE (M).
The results revealed that EACE (M), EACE (S), ME (M), and AQE (M) possess the same
inhibitory effect among them, with lower inhibitory activity than acarbose (p < 0.001 for
EACE (S), ME (M), and AQE (M); and p < 0.01 for EACE (M) compared with acarbose).
However, ME (S) exhibits a higher inhibitory effect compared to the other extracts and has
an effect statistically similar to acarbose.

Table 5. IC50 values of U. lactuca extracts and acarbose in α-amylase and α-glucosidase inhibition.

IC50 (mg/mL)
Inhibitors
α-Amylase α-Glucosidase
Acarbose 0.35 ± 0.08 0.39 ± 0.04
M 0.77 ± 0. 09 ** 0.42± 0.04 ***
EAcE
S 0.78 ± 0. 16 *** 0.37± 0.05 ns
M 0.81 ± 0. 05 *** 0.44± 0.06 **
ME
S 0.63 ± 0. 03 ns 0.27± 0.04 **
AQE M 0.89 ± 0. 21 *** 0.51± 0.06 ***
Data are expressed as mean ± SD. ns: non-significant; significant ** p < 0.01; *** p < 0.001.

As for the effect of Ulva lactuca extracts on pancreatic α-glucosidase inhibitory activity,
using acarbose as a positive control, the results showed that EM (S) was the most effective,
closely followed by EAcE (S), EAcE (M), EM (M), and finally AQE (M).

2.6. Molecular Modeling Studies

The study of molecular docking provides valuable insights into the regulation of en-
zymes involved in carbohydrate digestion, such as α-amylase and α-glucosidase [22]. These
enzymes play crucial roles in breaking down carbohydrates during digestion. Specifically,
α-amylase acts on starch, breaking it down into smaller carbohydrates, while α-glucosidase
further breaks these down into simple sugars like glucose [23]. The absorption of these
Mar. Drugs 2024, 22, 240 7 of 22

sugars into the bloodstream can contribute to elevated blood sugar levels in individuals
with diabetes if not properly regulated [24]. To address this issue and manage blood
sugar levels effectively, some individuals with diabetes may consider the use of α-amylase
or α-glucosidase inhibitors, which work to slow down the digestion and absorption of
carbohydrates [25]. Molecular docking studies can enhance our understanding of the inter-
actions between these inhibitors and the targeted enzymes, providing valuable insights
for potential therapeutic interventions. All molecules identified in Ulva lactuca extracts by
HPLC-DAD exhibited remarkable inhibitory activity against α-amylase and α-glucosidase
compared to acarbose (standard). It was observed that all these compounds remained
stable in the active sites, presenting particularly high energy values, except for cinnamic
acid in both proteins. The presence of negative and low docking score values suggests that
the compounds engaged in robust and advantageous binding interactions (Table 6).

Table 6. The docking scores of the selected docked compounds were identified in Ulva lactuca extracts
with the proteins (1B2Y) and (5NN8) using SP docking.

Docking Score (kcal/mol)

N◦ Compound Name
Alpha-Amylase Alpha-Glucosidase
1 Gallic acid −5.298 −7.334
2 Catechin −5.806 −5.592
3 4-hydroxy-benzoïc acid −5.064 −5.034
4 Chlorogenic acid −4.228 −4.589
5 Caffeic acid −4.629 −5.425
6 Syringic acid −4.946 −4.767
7 Vanilline −5.272 −5.621
8 p-Coumaric acid −4.223 −4.044
9 Sinapic acid −4.304 −3.813
10 Quercetin-O-3-glucoside −5.704 −5.218
11 7,3′ ,4′ -flavon-3-ol −5.923 −6.620
12 Naringin −5.601 −5.055
13 Rutin −6.807 −6.060
14 Salicylic acid −5.336 −4.886
15 Quercetin −5.187 −4.654
16 Cinnamic acid −3.589 −3.522
17 Apigenin −5.811 −4.766
18 Kaempferol −5.539 −4.899
19 Flavone −5.994 −5.319
20 Flavanone −5.433 −5.599
21 Acarbose (standard) −4.877 −4.925

The compounds exhibiting the highest stability in the active site of α-amylase are rutin
and flavone, with docking scores of −6.807 kcal/mol and −5.994 kcal/mol, respectively.
These values surpass the docking score of acarbose, which is −4.877 kcal/mol. Notably,
these compounds form various interactions through the active site, such as conventional
hydrogen bonds, carbon–hydrogen bonds, pi–alkyl, alkyl, pi–pi T-shaped, and van der
Waals interactions with the amino acid residues ALA307, THR163, ASP300, ASP197, TYR62,
LEU165, GLU165, LEU162, GLU233, LYS200, GLU233, ALA198, HIS201, GLY306, and
GLU240 (Figure 1). On the other hand, the compounds displaying the highest stability in the
active site of α-glucosidase are gallic acid and 7,3′ ,4′ -trihydroxyflavone, with docking scores
of −7.334 kcal/mol and −6.620 kcal/mol, respectively. These values surpass the docking
score of acarbose, which is −4.925 kcal/mol. Notably, these compounds form various
interactions within the active site, such as conventional hydrogen bonds, carbon–hydrogen
bonds, pi–alkyl, pi–sigma interactions, attractive charge interactions, sulfur–x bonds, and
van der Waals forces with amino acid residues following ILE823, GLU856, HIS708, ARG725,
GLU748, GLY747, THR711, VAL756, VAL763, LYS760, GLU762, ASP518, ARG600, MET519,
ASP282, ASP616, and PHE525 (Figure 2). While in-silico studies have shown promising
results regarding the inhibitory potential of the identified compounds in Ulva lactuca
Mar. Drugs 2024, 22, x 8 of 22

Mar. Drugs 2024, 22, 240 16 Cinnamic acid −3.589 −3.522 8 of 22

17 Apigenin −5.811 −4.766
18 Kaempferol −5.539 −4.899
on α-amylase
19 and α-glucosidase proteins, further research
Flavone −5.994 is needed to determine
−5.319 their
therapeutic
20 effectiveness in a clinical
Flavanone setting. Clinical trials
−5.433 involving human subjects
−5.599 are
essential21
to assess the safety and pharmacokinetics
Acarbose (standard) of these
−4.877 compounds [26].
−4.925

Figure 1. 2D intermolecular interactions between (A) rutin, (B) flavone, and (C) acarbose (standard)
Figure 1. 2D intermolecular interactions between (A) rutin, (B) flavone, and (C) acarbose (standard)
with the active site of α-Amylase (PDB: 1B2Y) protein.
with the active site of α-Amylase (PDB: 1B2Y) protein.
Mar.
Mar. Drugs 2024, 22,
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x 99 of
of 22

Figure 2. 2D intermolecular interactions between (A) gallic acid, (B) 7,3′,4′-flavon-3-ol, and (C) acar-
Figure 2. 2D intermolecular interactions between (A) gallic acid, (B) 7,3′ ,4′ -flavon-3-ol, and (C) acar-
bose (standard) with the active site of α-glucosidase (PDB: 5NN8) protein.
bose (standard) with the active site of α-glucosidase (PDB: 5NN8) protein.
3. Discussion
3. Discussion
In this
In this study,
study,the
theresults
resultsshow
showthat using
that usingpolar solvents
polar likelike
solvents water and and
water methanol pro-
methanol
duced greater extraction yields compared to less polar solvents like ethyl acetate
produced greater extraction yields compared to less polar solvents like ethyl acetate and and hex-
ane. ThisThis
hexane. suggests thatthat
suggests increasing the polarity
increasing of the
the polarity solvent
of the leads
solvent to an
leads toincrease in the
an increase in
extraction
the yield,
extraction which
yield, is confirmed
which by previous
is confirmed research
by previous [27,28].
research In addition,
[27,28]. polarpolar
In addition, sol-
vents, likelike
solvents, water, can
water, extract
can phenolic
extract phenoliccompounds
compoundsthat thatare
areattached
attachedto tosugars
sugarsor
orproteins,
proteins,
saponins, glycosides, organic
organic acids,
acids, and
and selenium
selenium [29].
[29].
The phenolic content values found in Ulva lactuca are higher than those obtained by
Oucif et al. [30] (4.27–8.69 mg GAE/g DE) as is the flavonoid content (4.66–1.0 mg GAE/g
Mar. Drugs 2024, 22, 240 10 of 22

The phenolic content values found in Ulva lactuca are higher than those obtained by
Oucif et al. [30] (4.27–8.69 mg GAE/g DE) as is the flavonoid content (4.66–1.0 mg GAE/g
DE) in the methanolic and aqueous extract. The observed difference can be explained
by several factors, such as the origin of the algae sample, the extraction method used,
the stage of maturity of the algae, the growth conditions, or the type of solvent used for
extraction [31,32].
The analysis of the chemical composition of EAcE and HE by gas chromatography–
mass spectrometry (GC–MS) highlights the predominance of palmitic acid, eicosenoic acid,
and linoleic acid as major compounds. Palmitic acid exhibits inhibitory effects on prostate
cancer cell proliferation and metastasis by suppressing the PI3K/Akt pathway [33]. Omega-
3 fatty acids, particularly linolenic acid, play a vital role in regulating blood glucose levels
by enhancing insulin secretion from pancreatic beta cells in the islets of Langerhans [34].
Linoleic acid, an essential fatty acid, demonstrates hypocholesterolemic effects [35] and
facilitates glucose uptake by C2C12 muscle cells [36]. Oleic acid has been observed to
stimulate insulin secretion in glucose-sensitive INS-1 cell lines, notably even in the presence
of the inflammatory cytokine TNF-α [37]. Stearic acid promotes glucose uptake into
adipocytes by activating insulin/insulin receptor signaling while inhibiting PTP1B [38].
The expression of GLUT4 transporter and glucose uptake facilitation in 3T3-L1 adipocytes
are significantly influenced by polyunsaturated fatty acids, leading to increased abundance
of both GLUT4 and GLUT1 transporters [39,40]. Furthermore, the impact of eicosenoic
acid, specifically 7,10-hexadecadienoic acid, on diabetes remains relatively understudied
compared to other omega fatty acids such as omega-3 and omega-6 fatty acids.
It is imperative to acknowledge that the composition and ratio of fatty acids in a
diet can profoundly influence its overall impact on health [41,42]. For instance, elevated
levels of saturated fatty acids (SFAs) have been correlated with an increased risk of cardio-
vascular disease, while polyunsaturated fatty acids (PUFAs) have been associated with a
reduced risk. Conversely, monounsaturated fatty acids (MUFAs) are generally regarded
as beneficial for health due to their potential to lower cholesterol levels and mitigate the
risk of heart disease [43]. PUFAs, constituting a significant portion of cell membrane
phospholipids, play a vital role in human metabolism [44,45]. Their metabolic signifi-
cance is underscored by their diverse biological properties, encompassing antibacterial
activity [46–48], anti-inflammatory effects [49,50], antioxidant capacity [51], potential for
preventing cardiovascular disease [52], and inhibition of tumor growth [53,54].
Other studies found significant variations in the FA composition of the same species of
green seaweed, collected from different locations. This poses a challenge in establishing a
direct correlation between a specific FA profile and a particular green seaweed species. For
example, U. lactuca collected from the North California coast in November exhibited 11%
α-linolenic acid, 22% stearidonic acid (18:4 ω3), 1% oleic acid (18:1 ω9), and 24% palmitic
acid [55]. On the other hand, U. lactuca collected from the North Sea in September/October
had 20% α-linolenic acid, 8% stearidonic acid, 20% oleic acid, and 12% palmitic acid [56].
The major phenolic molecules of the different EAcE and ME were determined through
HPLC analysis. The study revealed that U. lactuca extracts are an excellent source of
flavonoids and polyphenols. Flavonoids and their glycosides represent a significant class
of plant secondary metabolites. These compounds are widely distributed in nature and
are of particular interest due to their antioxidant properties and their potential role in
preventing various diseases, including cancer, cardiovascular diseases, neurodegenerative
diseases, diabetes, and osteoporosis [57]. Flavonols, also referred to as hydroxyflavones,
are distinguished from flavones by the presence of a hydroxy group at position 3 in the
chromen-4-one ring [58]. Despite their structural similarity, natural flavonols do not origi-
nate from chalcones through flavones as intermediates but rather through an alternative
biochemical pathway involving different enzymes, via flavanones. Flavanones serve as
common precursors for both flavones and flavonols [59].
Rutin (30,40,5,7-tetrahydroxy-flavone-3-rutinoside) is a flavonol glycoside with docu-
mented clinically relevant properties, potentially advantageous in disease prevention and
Mar. Drugs 2024, 22, 240 11 of 22

genome stability preservation [60]. The Dietary Supplement Label Database currently lists
over 860 products containing rutin marketed in the U.S. [61]. Rutin supplementation is
particularly recommended for managing various conditions such as varicose veins, internal
bleeding, or hemorrhoids. Common oral dosages range from 500 mg to 2000 mg daily and
can be safely continued for extended periods, up to 6 months [62].
Salicylic acid (SA) is a widely used nonsteroidal anti-inflammatory drug (NSAID)
known for its antibacterial, anti-biofilm-formation, antipyretic, and anticoagulation proper-
ties [63]. Apigenin and nobiletin have been shown to modulate the expression of crucial
inflammatory signaling pathways, including nuclear factor erythroid 2-related factor 2
(Nrf2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The
antioxidant attributes of various natural flavones stem from their capacity to regulate the
expression of Nrf2/heme oxygenase-1 (HO-1), thereby reducing levels of free radicals and
oxidative stress.
Sinapic acid stands out as one of the four most prevalent hydroxycinnamic acids,
distributed widely across the plant kingdom. Numerous researchers have suggested its
potency as an antioxidant [64–67]. Its efficacy is purported to surpass that of ferulic acid [68],
another hydroxycinnamic acid extensively utilized as a natural antioxidant in various food,
beverage, and cosmetic products [69], and is comparable to caffeic acid [64,70,71]. Sinapic
acid exhibits antimicrobial [72–77], anti-inflammatory [78], anticancer [79], and anti-anxiety
properties [80].
The DPPH assay relies on the capacity of the stable free radical DPPH (•) to reduce
its color intensity in the presence of antioxidants. This radical possesses an unpaired
electron, which imparts a deep purple coloration that is attenuated in the presence of
antioxidants [81]. The DPPH assay is commonly utilized as a tool to evaluate the antioxidant
activity of various plant extracts [82,83].
The ability of U. lactuca extracts to arrest or retard lipid peroxidation was assessed
using the β-carotene molecule bleaching method. In this test, linoleic acid oxidation
generates peroxide radicals following the abstraction of hydrogen atoms from linoleic acid
methylene groups. These free radicals then oxidize the highly unsaturated β-carotene,
causing it to lose its red color, which is monitored spectrophotometrically at 470 nm.
However, the presence of an antioxidant could neutralize linoleic acid-derived free radicals
and thus prevent β-carotene oxidation and bleaching.
The findings indicated that extracts derived from the green algae U. lactuca, exhibit-
ing high total phenol content, displayed notable antioxidant activity. This suggests that
polyphenols found in algae may play a pivotal role in the radical scavenging properties
of these extracts. Existing literature has highlighted a strong correlation between antioxi-
dant activity and the presence of polyphenols and flavonoids. Consequently, these algae
represent a valuable reservoir of bioactive compounds and hold considerable potential
for antioxidant activity, as demonstrated by the enhanced efficacy observed with heating
during Soxhlet extraction.
It is important also to note that antioxidant activity determined by various meth-
ods may provide inconsistent findings [84]. The differences in the findings of the two
experiments may be traced to the distinct processes involved.
The α-amylases (1,4-α-D-glucosyl-hydrolase) are enzymes present in plants, animals,
and microorganisms [85]. They constitute a part of both salivary and pancreatic secretions,
playing a crucial role in the breakdown of complex carbohydrates [86]. Pancreatic α-
amylase, functioning as a digestive enzyme, initiates the hydrolysis of starch, converting
it into maltose and eventually glucose. This process involves the hydrolysis of the α-
1,4-glucan bonds present in starch and glycogen, resulting in the production of glucose
and maltose [87]. α-glucosidases, positioned at the brush border of enterocytes, serve
as digestive enzymes catalyzing the hydrolysis of α-(1,4) glucosidic bonds in undigested
dietary disaccharides reaching the jejunum without modification, as well as the residues
generated from starch digestion. This leads to the formation of absorbable glucose within
intestinal cells [88]. Pancreatic α-amylases and intestinal α-glucosidase inhibition activity
Mar. Drugs 2024, 22, 240 12 of 22

become part of a strategic approach to delay the glucose absorption from the digestive tract,
which helps prevent rapid spikes in blood sugar levels after meals, contributing to better
overall glycemic control [89]. Medications such as acarbose, known as α-amylases and
α-glucosidase inhibitors, are commonly employed to achieve this goal [90]. Nevertheless,
despite the positive impact of this treatment on glycemic control and overall body balance,
it is accompanied by side effects, including diarrhea and flatulence [91]. This prompts
individuals to actively seek natural alternatives. Among the natural sources that have
shown an effect on diabetes, marine algae are noteworthy [92]. In our research, we explored
the impact of different extracts from marine algae Ulva lactuca to assess their inhibitory and
therapeutic potential against these two enzymes.
The main findings of the in-vitro study on the inhibitory effect of α-amylase and
α-glucosidase summarize that each Ulva lactuca extract showed significant inhibition of
the enzymatic activity of α-amylase at all tested doses. The IC50 values revealed similar
inhibitory effects among EACE (M), EACE (S), ME (M), and AQE (M), all exhibiting lower
activity compared to acarbose, the reference molecule. However, ME (S) demonstrated a
higher inhibitory activity than the other extracts, displaying a statistically similar effect to
acarbose, which means that the extraction method of this alga does not influence enzyme
inhibition, except for the methanolic extract. It is observed that extraction using Soxhlet
yields better results than maceration. This can be explained by the fact that the Soxhlet
method extracted a much higher amount of active principles responsible for the beneficial
effect compared to maceration. Our results are in agreement with [93], which suggests that
the Soxhlet methanolic extract of Galium aparine L. has demonstrated a more pronounced
α-amylase inhibition effect and radical scavenging activity than other extracts (maceration
and ultrasonic). This effect is attributed to the high concentration of phenolic compounds,
flavonoids, and tannins. In fact, our results align with a previous study, which reported
that the IC50 values for α-amylase inhibition by aqueous extracts of seaweed Ulva lactuca,
Sargassum polycystum, G. Gracilaria edulis, and Gracilaria corticate are, respectively,
67 µg/mL, 60 µg/mL, 83 µg/mL, and 82 µg/m [94]. Furthermore, the study added that the
extraction using ethyl acetate of the green seaweed Ulva fasciata exhibited lower inhibitory
activity against α-amylase (IC50 = 69.12 g/mL) compared to the positive control, acarbose
(IC50 = 49.34 µg/mL) [95].
Ulva lactuca is rich in minerals, vitamins, and polysaccharides that exhibit several bioac-
tive properties, including anticoagulant, antiviral, anti-inflammatory, immunomodulatory,
and antioxidant activities [96]. Phenolic compounds, notably tannins and flavonoids such
as quercetin, kaempferol, and apigenin, play a crucial role as antioxidant and anti-diabetic
agents, modulating oxidative stress and inflammation [97]. According to Sok Yen et al. [98],
flavonoids exhibit both hypoglycemic and antioxidant effects in diabetic animals. Moreover,
they have been found to inhibit both α-amylase and α-glucosidase activities with IC50
values of 770 µg/mL, 32 µg/mL for quercetin and 287.53, 231.13 µg/mL for apigenin,
respectively [90,99]. Fatty acids, including linoleic acid, gamma-linolenic acid, oleic acid,
linolelaidic acid, and α-linoleic acid, are also reported as a main component of Ulva lactuca.
Oleic acid demonstrates the most potent anti-α-glucosidase activity, followed by linoleic
acid. Their activities proved more potent than acarbose [100]. In addition, Ulva lactuca
contains pigments such as chlorophylls that play a role in photosynthesis and exhibit
antioxidant properties. Also, it contains carotenoids such as beta-carotene, lutein, and
zeaxanthin that have hypoglycemic activity and a strong antioxidant effect. It may also
play an essential role in diabetic complications [101]. All these biomolecules are collectively
contributing to the nutritional and therapeutic potential of Ulva lactuca.

4. Materials and Methods

4.1. Chemicals and Reagents
Analytical-grade chemicals and reagents were procured from Merck (Darmstadt,
Germany) and Carl Roth GmbH (Karlsruhe, Germany) to determine the total phenolic
and flavonoid components. Methanol, ethyl acetate, and N-hexane were acquired from
 4. Materials and Methods
4.1. Chemicals and Reagents
Analytical-grade chemicals and reagents were procured from Merck (Darmstadt,
Germany) and Carl Roth GmbH (Karlsruhe, Germany) to determine the total phenolic
Mar. Drugs 2024, 22, 240 13 of 22
and flavonoid components. Methanol, ethyl acetate, and N-hexane were acquired from
Merck (Darmstadt, Germany). α-amylase, α-glucosidase, β-carotene, 1,1-Diphenyl-2-pic-
rylhydrazyl (DPPH•), acarbose, and 3,5-Dinitrosalicylic acid (DNSA) were obtained from
Merck (Darmstadt, Germany). α-amylase, α-glucosidase, β-carotene, 1,1-Diphenyl-2-
Merck (Sigma–Aldrich, St. Louis, MA, USA). Phenolic standards including ascorbic acid,
picrylhydrazyl (DPPH•), acarbose, and 3,5-Dinitrosalicylic acid (DNSA) were obtained
kojic acid, gallic acid, apigenin, succinic acid, cholesterol, and tannic acid were sourced
from Merck (Sigma–Aldrich, St. Louis, MO, USA). Phenolic standards including ascorbic
from Merck and Carl Roth GmbH (Karlsruhe, Germany).
acid, kojic acid, gallic acid, apigenin, succinic acid, cholesterol, and tannic acid were sourced
from Merck and Carl Roth GmbH (Karlsruhe, Germany).
4.2. Plant Material and Extraction
In April
4.2. Plant 2021,and
Material theExtraction
green algae species U. lactuca was harvested from the Nador lagoon
located at 35°08′26.9″
In April 2021, the greenN 2°29′09.6″ W in northern
algae species U. lactucaMorocco. The classification
was harvested from the Nadorof this algae
lagoon
species was ◦carried
′ out
′′ by
◦ Dr.
′ M.′′ Ramdani, who is affiliated with the
located at 35 08 26.9 N 2 29 09.6 W in northern Morocco. The classification of this algae Faculty of Sciences
at the University
species was carriedMohammed
out by Dr. M. I in Oujda, Morocco.
Ramdani, who is affiliated with the Faculty of Sciences at
the University Mohammed I in Oujda,was
The collected seaweed sample transported to the laboratory for further pro-
Morocco.
cessing. U. lactucaseaweed
The collected was carefully
samplecleaned and rinsed
was transported to with distilled water
the laboratory before
for further being ex-
processing.
posed
U. to light
lactuca was for 48 h. The
carefully dried and
cleaned sample waswith
rinsed then placed
distilledinwater
an oven at 35 being
before °C for exposed
24 h. The
seaweed was then lyophilized and ground into a fine powder for
to light for 48 h. The dried sample was then placed in an oven at 35 C for 24 h. The extraction
◦ purposes, as
illustrated in Figure 3. To extract the valuable components, we utilized
seaweed was then lyophilized and ground into a fine powder for extraction purposes, as maceration and
Soxhlet techniques
illustrated in Figurewith 3. Tofour solvents:
extract hexane,components,
the valuable ethyl acetate,we methanol,
utilized and distilledand
maceration wa-
ter. Thetechniques
Soxhlet resulting extracts
with four were then filtered
solvents: hexane,using
ethyl aacetate,
glass filter crucible
methanol, and(50 mL, with
distilled po-
water.
rosity
The 4, Isolab,
resulting Wertheim,
extracts Germany)
were then filtered and
usingplaced
a glassinfilter
flasks. The extracts
crucible (50 mL,werewith then dehy-
porosity 4,
dratedWertheim,
Isolab, using a rotary evaporator
Germany) (Laborota
and placed 4000,The
in flasks. Heidolph
extractsInstruments, Schwabach,
were then dehydrated Ger-
using
amany).
rotary This thorough
evaporator extraction
(Laborota process
4000, ensures
Heidolph that the resulting
Instruments, extracts
Schwabach, are of the high-
Germany). This
est quality, making them ideal for further studies, and the extraction
thorough extraction process ensures that the resulting extracts are of the highest quality,yield was calculated
using the
making following
them ideal forequation:
further studies, and the extraction yield was calculated using the
following equation: 𝑚𝑎𝑠𝑠 𝑑𝑟𝑖𝑒𝑑 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 (g)
Yield (%) = × 100
𝑚𝑎𝑠𝑠
mass 𝑑𝑟𝑖𝑒𝑑
dried 𝑚𝑎𝑡𝑟𝑖𝑥
extract (g)(g)
Yield (%) = × 100
mass dried matrix (g)
The recorded datum is the median of three extraction replicates.

Figure3.3.(a)
Figure (a)U.
U.lactuca
lactucabefore
beforedrying,
drying,(b)
(b)powder
powderofofdried
driedU.
U.lactuca.
lactuca.

4.2.1.The
Maceration
recorded Extraction
datum is the median of three extraction replicates.
To extract soluble compounds from a solid substance, maceration is the most com-
4.2.1. Maceration Extraction
mon method, involving immersion in a cold liquid. In this case, extracts were prepared
To extractby
successively soluble compounds
stirring from a solid
100 g of macroalgae substance,
powder withmaceration is the most
200 mL of solvent common
of increasing
method, involving immersion in a cold liquid. In this case, extracts were prepared
polarity (99% n-hexane for 2 h, ethyl acetate for 24 h, methanol for 24 h, and distilled succes-
water
sively by stirring 100 g of macroalgae powder with 200 mL of solvent
for 24 h) at room temperature. This process is illustrated in Figure 4. of increasing polarity
(99% n-hexane for 2 h, ethyl acetate for 24 h, methanol for 24 h, and distilled water for 24 h)
at room temperature. This process is illustrated in Figure 4.
Mar.
Mar. Drugs 2024, 22,
Drugs 2024, 22, 240
x 14
14 of 22
of 22

Figure 4. Extraction process of U. lactuca with various solvents.

4.2.2.
4.2.2. Soxhlet
Soxhlet Extraction
Extraction
In order to extract active compounds from marine macroalgae, we utilized the alterna-
In order to extract active compounds from marine macroalgae, we utilized the alter-
tive technique, namely the Soxhlet extraction method. This method involves employing a
native technique, namely the Soxhlet extraction method. This method involves employing
Soxhlet chamber, an extraction flask, and a condenser [102]. The process begins by placing
a Soxhlet chamber, an extraction flask, and a condenser [102]. The process begins by plac-
35 g of marine macroalgae powder into an extraction thimble, which is then combined with
ing 35 g of marine macroalgae powder into an extraction thimble, which is then combined
300 mL of a selected solvent (such as hexane, ethyl acetate, or methanol) in the extraction
with 300 mL of a selected solvent (such as hexane, ethyl acetate, or methanol) in the ex-
flask. The duration of the Soxhlet extraction process is determined by the time it takes to
traction flask. The duration of the Soxhlet extraction process is determined by the time it
extract all the soluble compounds with affinity for the solvent at a given temperature.
takes to extract all the soluble compounds with affinity for the solvent at a given temper-
ature.
4.3. Phytochemicals Compounds
4.3.1. Quantification of Total Phenolic Constituents
4.3. Phytochemicals Compounds
The study aimed to determine the total amount of polyphenols present in U. lactuca
4.3.1. Quantification
extracts. of Total
To achieve this, PhenolicFolin–Ciocalteu
a modified Constituents method was used. In this method,
200 µLTheof study aimed
the extract to determine
solution the total amount
with a concentration of 1of polyphenols
mg/mL was mixedpresent
within1000
U. lactuca
µL of
extracts. To achieve
Folin–Ciocalteu this,dissolved
reagent a modified Folin–Ciocalteu
in distilled methodby
water, followed was
theused. In this
addition method,
of 800 µL of
200 µL of
sodium the extract
carbonate (75 solution
g/L). Thewith a concentration
resulting mixture was ofthen
1 mg/mL was at
incubated mixed
roomwith 1000 µL
temperature
of Folin–Ciocalteu
for one hour in thereagent dissolved
dark. After in distilled
incubation, water, followed
absorbance readings bywere
the addition
taken atof765800nmµL
using a spectrophotometer,
of sodium carbonate (75 g/L).withThe ethanol
resultingserving
mixtureaswas a control. To generate
then incubated calibration
at room temper-
curves,
ature fortheoneconcentration of gallic
hour in the dark. Afteracid was altered
incubation, (ranging readings
absorbance from 0 towere0.1 mg/mL). All
taken at 765
experiments were conducted in with
nm using a spectrophotometer, triplicate to obtain
ethanol mean
serving as a values
control.and
To their corresponding
generate calibration
standard
curves, thedeviations. The total
concentration amount
of gallic of phenolic
acid compounds
was altered (rangingwas fromexpressed
0 to 0.1inmg/mL).
milligrams
All
of gallic acid equivalents per gram of dry extract (mg GAE/g) [103].
experiments were conducted in triplicate to obtain mean values and their corresponding
standard deviations. The total amount of phenolic compounds was expressed in milli-
4.3.2.
gramsMeasurement
of gallic acid of Total Flavonoid
equivalents per gram Contents
of dry extract (mg GAE/g) [103].
We combined 200 µL of each U. lactuca extract, 1000 µL of distilled water, and 50 µL
of NaNO
4.3.2. 2 (5%). Subsequently,
Measurement of Total Flavonoidafter aContents
6 min incubation period, 120 µL of AlCl3 (10%)
was introduced, followed by a further
We combined 200 µL of each U. lactuca 5 min incubation
extract, 1000 µL period at roomwater,
of distilled temperature
and 50 µLin
darkness. This was followed by the addition of 400 µL of 1 M NaOH and
of NaNO2 (5%). Subsequently, after a 6 min incubation period, 120 µL of AlCl3 (10%) was 230 µL of distilled
water. The absorbance was then measured at 510 nm. To construct the calibration curve,
introduced, followed by a further 5 min incubation period at room temperature in dark-
various concentrations of quercetin solution (ranging from 0 to 0.1 mg/mL) were utilized as
ness. This was followed by the addition of 400 µL of 1 M NaOH and 230 µL of distilled
standards. Each measurement was conducted in triplicate to ensure result reproducibility.
water. The absorbance was then measured at 510 nm. To construct the calibration curve,
various concentrations of quercetin solution (ranging from 0 to 0.1 mg/mL) were utilized
as standards. Each measurement was conducted in triplicate to ensure result reproduci-
bility.
Mar. Drugs 2024, 22, 240 15 of 22

4.4. Fatty Acid GC–MS Analysis of U. lactuca Extracts

In line with the methodology outlined in Loukili et al. [42], methyl esters of hexane
fatty acids and ethyl acetate were extracted from U. lactuca with certain adaptations. The
Shimadzu GC system (Kyoto, Japan) equipped with a BPX25 capillary column featuring a
5% diphenyl and 95% dimethylpolysiloxane phase (30 m × 0.25 mm × 0.25 µm) (Kyoto,
Japan) coupled with a QP2010 MS was utilized for the characterization and identification of
fatty acids. Helium gas (99.99%) served as the mobile phase with a flow rate set at 3 L/min.
The temperature settings for the injection, ion source, and interface were maintained at
250 ◦ C. The column oven temperature program began at 50 ◦ C for 1 min, followed by a
gradual increase to 250 ◦ C at a rate of 10 ◦ C/min, and held for an additional minute. Sample
components underwent ionization in electron ionization (EI) mode at 70 eV, scanning a
mass range of 40 to 300 m/z. Each extract, in a volume of 1 µL, was injected in split mode,
and triplicate analyses were conducted for each sample. Compound characterization relied
on comparisons of retention times, mass spectra fragmentation patterns, and databases,
including the National Institute of Standards and Technology’s database (NIST). Data
processing was conducted using LabSolutions (version 2.5, Shimadzu, Kyoto, Japan).

4.5. HPLC Analyses of U. lactuca Extracts

Identification of phenolic compounds within the ethyl acetate and methanolic extracts
was accomplished utilizing high-performance liquid chromatography (HPLC) employing
an Agilent 1100 system (Agilent Technologies, Palo Alto, CA, USA) coupled with a diode
array UV detector (Bruker, Berlin, Germany). Each extract (10 µL) underwent separation
through a Zorbax XDB-C18 column (5 µm, 250 × 4.6 mm) connected to the Agilent 1100 sys-
tem, preceded by a 4 × 3 mm C18 cartridge precolumn (Agilent Technologies). The elution
gradient protocol employed was as follows: 0 to 5 min with 95% solvent A and 5% solvent
B, 25 to 30 min with 65% solvent A and 35% solvent B, 35 to 40 min with 30% solvent A
and 70% solvent B, and finally 40 to 45 min with 95% solvent A and 5% solvent B. Solvent
A comprised water/methanol (9/1) with 0.1% phosphoric acid, while solvent B consisted
of methanol with 0.1% phosphoric acid. The elution was carried out at a constant flow
rate of 1 mL/min under a temperature of 40 ◦ C. Spectrophotometric data were collected at
wavelengths of 254 nm, 280 nm, 320 nm, 370 nm, and 510 nm. Compound identification
was conducted by comparing their retention times and UV spectra with those of standard
compounds.

4.6. Antioxidant Activity

4.6.1. Scavenging 2,2-Diphenyl-1-Picrylhydrazyl Radical Test
The antioxidant activity of different extracts of U. lactuca was assessed using the
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical bleaching method, following the protocol
outlined by Brand-Williams et al. [104] with minor adjustments. The initial concentration
of both the extracts and ascorbic acid was maintained at 1 mg/mL. A solution comprising
0.8 mL of samples or standard (ascorbic acid) at varying concentrations (ranging from 0.02
to 0.32 mg/mL) and 2 mL of DPPH• solution (prepared by dissolving 2 mg of DPPH• in
200 mL of MeOH) was prepared and manually agitated. Following a 30 min incubation
period in darkness at room temperature, the absorbance of the samples was measured
using a UV–visible spectrophotometer at a wavelength of 517 nm, relative to the blank.
Each analysis was conducted in triplicate.
The inhibitory activity of the DPPH radical by a sample was determined using the
following formula:
(Ab − As)
 
Inhibition Percent = × 100
Ab
where Ab is absorbance of the blank and As is absorbance of a sample (or positive control).
The graph plotting inhibition percentage against extract concentration was used to
calculate the IC50 .
Mar. Drugs 2024, 22, 240 16 of 22

4.6.2. β-Carotene Bleaching Assay

The antioxidant potential of U. lactuca extracts was assessed using the method de-
scribed by [105]. This evaluation relied on the extracts’ capacity to mitigate oxidative
damage to β-carotene in an emulsion, employing the carotene bleaching assay. To prepare
the emulsion, 2 mg of β-carotene was dissolved in 10 mL of chloroform, supplemented
with 20 mg of linoleic acid and 200 mg of Tween 80 as an emulsifier. The chloroform
was evaporated under vacuum at 40 ◦ C, followed by the addition of 100 mL of distilled
water while vigorously stirring the solution. Subsequently, 0.2 mL of the emulsion was
dispensed into individual test tubes, along with either the extract or a reference antioxidant
solution (BHA) at a concentration of 1 mg/mL. The absorbance was recorded at 470 nm
using a 96-well microplate reader at t0 (immediately after emulsion addition) and after a
2 h incubation period. All measurements were conducted in triplicate.”
The inhibition of the linoleate/β-carotene radical was determined using the following
equation:

initial (β − carotene)(t0) − (β − carotene) a f ter 2 h

Bleaching inhibition (%) = 100 − × 100
initial (β − carotene)(t0)

4.7. In-Vitro α-Amylase Inhibition

The inhibitory activity of various extracts of U. lactuca against α-amylase was assessed
following the protocol outlined by Daoudi et al. [106]. A reaction mixture was prepared,
comprising 0.2 mL of the sample or acarbose (utilized as a positive control at concentrations
ranging from 2.27 to 0.14 mg/mL), 0.2 mL of phosphate buffer (pH 6.9), and 0.2 mL of
enzyme solution (13 IU). This mixture was pre-incubated at 37 ◦ C for 10 min before the
addition of 0.2 mL of enzyme-substrate solution (1% starch dissolved in phosphate buffer).
The reaction proceeded at 37 ◦ C for 30 min. The enzymatic reaction was halted by adding
0.6 mL of DNSA reagent, followed by an incubation step at 100 ◦ C for 8 min, followed by
cooling in a cold-water bath. Absorbance was measured at 540 nm.
The inhibition percentage was calculated using the following formula:

(ODcontrol 540 nm − ODcontrol blank 540 nm) − (OD sample 540 nm − OD sample blank 540 nm)
Inhibition activity (%) = × 10
OD control 540 nm − OD control blank 540 nm
The IC50 of the various tests was done graphically using the function:

Inhibition percentage (%) = f(log(sample concentration))

4.8. In-Vitro α-Glucosidase Inhibition Assay

The effect of U. lactuca extracts on intestinal α-glucosidase activity was evaluated
using a modified version of the protocol outlined by Hbika et al. [107]. A mixture was
prepared by combining 100 mL of sucrose (50 mM), 1000 mL of phosphate buffer (50 mM,
pH 7.5), and 100 mL of intestinal α-glucosidase enzyme solution (10 IU). This mixture was
then supplemented with 10 mL of distilled water (as control), acarbose (as the positive
control), or U. lactuca extract solution at a concentration of 22 mg/mL. Subsequently, the
mixture was incubated for 25 min at 37 ◦ C in a water bath. To terminate the enzymatic
reaction and quantify the release of glucose, the mixture was heated at 100 ◦ C for 5 min:

(ODcontrol 540 nm − ODcontrol blank 540 nm) − (OD sample 540 nm − OD sample blank 540 nm)
Inhibition activity (%) = × 10
OD control 540 nm − OD control blank 540 nm

4.9. Theoretical Study

4.9.1. Ligand Preparation
To optimize and minimize the energy of the ligands (compounds) identified in Ulva lactuca
extracts by HPLC-DAD, data were retrieved from the PubChem database (https://pubchem.ncbi.nlm.
nih.gov, accessed on 21 April 2024). The LigPrep module within Maestro 12.8 (Schrodinger 2021-2)
was utilized for ligand preparation. In this study, acarbose inhibitor served as the standard [108],
 (https://pubchem.ncbi.nlm.nih.gov, accessed on 21 April 2024). The LigPrep module
within Maestro 12.8 (Schrodinger 2021-2) was utilized for ligand preparation. In this
study, acarbose inhibitor served as the standard [108], and the energy minimization and
optimization procedures adhered to the OPLS_2005 force field. Hydrogen atoms were in-
Mar. Drugs 2024, 22, 240 cluded, and adjustments were implemented to eliminate salt and ionization effects17 at a
of 22
pH of 7 ± 2 [109].

4.9.2.
and theMolecular Docking and
energy minimization Preparation
and ofprocedures
optimization Proteins adhered to the OPLS_2005 force field.
Hydrogen atoms docking
Molecular were included, and adjustments
was conducted usingwere implemented
X-ray to eliminate
crystal structures salt and from
obtained ionization
the
effects atData
Protein of 7 ±(PDB)
a pHBank 2 [109].[103]. Specifically, the structures utilized were “Structure of Hu-
man Pancreatic Alpha-Amylase in Complex with the Carbohydrate Inhibitor Acarbose”
4.9.2. Molecular Docking and Preparation of Proteins
(PDBID: 1B2Y) with an X-ray diffraction resolution of 3.20 Å and “Crystal Structure of
Molecular docking was conducted using X-ray crystal structures obtained from the Protein
Human Lysosomal Acid-Alpha-Glucosidase, GAA, in Complex with Acarbose” (PDBID:
Data Bank (PDB) [103]. Specifically, the structures utilized were “Structure of Human Pancreatic
5NN8) with an X-ray diffraction resolution of 2.45 Å (Figure 5). The preparation of these
Alpha-Amylase in Complex with the Carbohydrate Inhibitor Acarbose” (PDBID: 1B2Y) with an
protein structures
X-ray diffraction involvedofthe
resolution utilization
3.20 of the Protein
Å and “Crystal StructurePreparation Wizard (Schrodinger
of Human Lysosomal Acid-Alpha-
2021-2) [110].GAA,
Glucosidase, During this process,
in Complex ligand and
with Acarbose” water5NN8)
(PDBID: atomswith
were anremoved, and non-polar
X-ray diffraction resolution
hydrogens were5).
of 2.45 Å (Figure consolidated.
The preparation Theofactive site was
these protein definedinvolved
structures as the target center. To
the utilization establish
of the Protein
an optimal docking
Preparation environment,
Wizard (Schrodinger the central
2021-2) grid box
[110]. During thisdimensions
process, ligandwere
andconfigured
water atoms towere
en-
removed, and
compass non-polar
all atoms hydrogens
of the ligandwere
set, consolidated.
with 20 pointsThe allocated
active site was
for defined
each axisas the
(x,target
y, and center.
z).
To establish an optimal docking environment, the central grid box dimensions
Energy minimization was performed with default settings, constraining the root-mean- were configured to
encompass all atoms of the ligand set, with 20 points allocated for each axis (x, y, and z). Energy
square deviation (RMSD) to 0.3 Å. The standard precision (SP) glide score was employed
minimization was performed with default settings, constraining the root-mean-square deviation
for predicting binding and selecting anchored poses. The output docking scores were ex-
(RMSD) to 0.3 Å. The standard precision (SP) glide score was employed for predicting binding and
pressed
selecting as affinityposes.
anchored binding in kcal/mol.
The output dockingSubsequently, the protein
scores were expressed structure
as affinity bindingunderwent
in kcal/mol.
further minimization using the OPLS_2005 force field. Biovia Discovery
Subsequently, the protein structure underwent further minimization using the OPLS_2005 Studio 2021 force[111]
field.
was utilized
Biovia for visualizing
Discovery the protein–ligand
Studio 2021 [111] complexes.the protein–ligand complexes.
was utilized for visualizing

Figure
Figure 5.
5. The
The3D
3Dcrystal
crystalstructure
structureof
ofthe
the proteins
proteins (A)
(A) α-amylase
α-amylase (PDB:
(PDB: 1B2Y)
1B2Y) and
and (B)
(B) α-glucosidase
α-glucosidase
(PDB: 5NN8).
(PDB: 5NN8).

5. Conclusions
Seaweed has many qualities as an organic product, being rich in vitamins and minerals yet
very low in calories. This study compares the chemical composition and bioactive properties of
extracts from Ulva lactuca, a green seaweed collected from the Nador lagoon in Morocco. Using
GC–MS and HPLC analyses, the fatty acid and phenolic compound content of the extracts were
identified. The aqueous extract showed high levels of polyphenols and flavonoids and exhibited
strong antioxidant activity against DPPH radicals. The methanolic extract demonstrated antioxidant
properties by preventing β-carotene discoloration. In-vitro studies revealed that the extracts inhibited
the enzymatic activity of α-amylase and α-glucosidase. Molecular docking models suggested that the
extracts’ phytochemicals interacted with these enzymes more effectively than acarbose, a commercial
drug. These findings highlight the potential of U. lactuca extracts as natural sources of bioactive
compounds with antioxidant and anti-diabetic properties. Seaweed is widely perceived as natural
and beneficial to health by consumers. The rise of seaweed in cosmetics products is undeniable, and
its growing presence in food products is evidence of a new niche for the agri-food and pharmaceutical
Mar. Drugs 2024, 22, 240 18 of 22

industries. The nutritional and therapeutic properties of algae promise to propel them towards greater
valorization in the years to come, extending their influence to other sectors. This trend suggests a
promising potential for future applications of algae in various economic fields.

Author Contributions: Conceptualization, S.O., H.A., M.M., M.B. and B.H.; Data curation, N.E.D.,
E.H.L., M.M., A.C., L.R., A.A.G., F.D. and M.R.; Methodology, S.O., N.E.D., A.C. and M.-L.F.; Supervi-
sion, M.B., B.H. and M.R.; Validation, S.O., E.H.L., H.A., M.B., M.-L.F., L.R. and A.A.G.; Writing—
original draft, S.O., N.E.D., E.H.L., M.M. and A.C.; Writing—review and editing, H.A., M.B., B.H.,
M.-L.F., L.R., A.A.G., F.D. and M.R. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: Data are contained within the article.
Acknowledgments: The authors thank the Faculty of Science Oujda of the Chemical Department
for its help with local physical measurements. Great thanks are addressed to Danny and Thomas
for their help. Support was also provided by the Laboratory of Chemistry of Natural Molecules,
Gembloux Agro-Bio Tech, University of Liege, Belgium.
Conflicts of Interest: The authors declare no conflicts of interest.

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