Gracilaria

marine drugs
Article
Study of the Phytochemical Composition, Antioxidant
Properties, and In Vitro Anti-Diabetic Efficacy of Gracilaria
bursa-pastoris Extracts
Safae Ouahabi 1 , El Hassania Loukili 1 , Nour Elhouda Daoudi 2 , Mohamed Chebaibi 3 , Mohamed Ramdani 4 ,
Ilyesse Rahhou 5 , Mohamed Bnouham 2 , Marie-Laure Fauconnier 6 , Belkheir Hammouti 1,7,8 , Larbi Rhazi 9, * ,
Alicia Ayerdi Gotor 10 , Flore Dépeint 9 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.); e.loukili@ump.ac.ma (E.H.L.);
hammoutib@gmail.com (B.H.); 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 Biomedical and Translational Research Laboratory, Faculty of Medicine and Pharmacy of the Fez,
University of Sidi Mohamed Ben Abdellah, Fez 30000, Morocco; mohamed.chebaibi@yahoo.fr
4 Biochemistry and Biotechnology Laboratory, Faculty of Sciences, Mohamed First University, B.P. 717,
Oujda 60000, Morocco; ramdanimed@yahoo.fr
5 Higher Institute of Nursing Professions and Health Techniques (ISPITSO), Oujda 63303, Morocco;
ilyesse@hotmail.com
6 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
7 CREHEIO Centre de Recherche de l’Ecole des Hautes Etudes d’Ingénierie, Oujda 60000, Morocco
8 Université Euro-Méditerranéenne de Fès, Fez BP 51, Morocco
9 Institut Polytechnique UniLaSalle, Université d’Artois, ULR 7519, UniLaSalle, 19 Rue Pierre Waguet,
BP 30313, 60026 Beauvais, France; flore.depeint@unilasalle.fr
10 Institut Polytechnique UniLaSalle, AGHYLE, UP 2018.C101, UniLaSalle, 19 Rue Pierre Waguet, BP 30313,
Citation: Ouahabi, S.; Loukili, E.H.;
Daoudi, N.E.; Chebaibi, M.;
60026 Beauvais, France; alicia.ayerdi-gotor@unilasalle.fr
* Correspondence: larbi.rhazi@unilasalle.fr
Ramdani, M.; Rahhou, I.;
Bnouham, M.; Fauconnier, M.-L.;
Hammouti, B.; Rhazi, L.; et al. Study Abstract: In this study, a comparison was made of the chemical makeup of different extracts obtained
of the Phytochemical Composition, from Gracilaria bursa-pastoris, a type of red seaweed that was gathered from the Nador lagoon situated
Antioxidant Properties, and In Vitro in the northern part of Morocco. Additionally, their anti-diabetic and antioxidant properties were
Anti-Diabetic Efficacy of Gracilaria investigated. The application of GC-MS technology to analyze the fatty acid content of the samples
bursa-pastoris Extracts. Mar. Drugs revealed that linoleic acid and eicosenoic acid were the most abundant unsaturated fatty acids across
2023, 21, 372. https://doi.org/ all samples, with palmitic acid and oleic acid following in frequency. The HPLC analysis indicated that
10.3390/md21070372 ascorbic and kojic acids were the most prevalent phenolic compounds, while apigenin was the most
Academic Editor: Patrícia Valentão common flavonoid molecule. The aqueous extract exhibited significant levels of polyphenols and
flavonoids, registering values of 381.31 ± 0.33 mg GAE/g and 201.80 ± 0.21 mg QE/g, respectively.
Received: 7 June 2023
Furthermore, this particular extract demonstrated a remarkable ability to scavenge DPPH radicals, as
Revised: 17 June 2023
evidenced by its IC50 value of 0.17 ± 0.67 mg/mL. In addition, the methanolic extract was found to
Accepted: 23 June 2023
possess antioxidant properties, as evidenced by its ability to prevent β-carotene discoloration, with an
Published: 24 June 2023
IC50 ranging from 0.062 ± 0.02 mg/mL to 0.070 ± 0.06 mg/mL. In vitro study showed that all extracts
significantly inhibited the enzymatic activity of α-amylase and α-glucosidase. Finally, molecular
docking models were applied to assess the interaction between the primary phytochemicals identified
Copyright: © 2023 by the authors. in G. bursa-pastoris extracts and the human pancreatic α-amylase and α-glucosidase enzymes. The
Licensee MDPI, Basel, Switzerland. findings suggest that these extracts contain bioactive substances capable of reducing enzyme activity
This article is an open access article more effectively than the commercially available drug acarbose.
distributed under the terms and
conditions of the Creative Commons
Keywords: Gracilaria bursa-pastoris; phenolic compounds; antioxidant activity; anti-diabetic properties;
Attribution (CC BY) license (https://
enzyme inhibition
creativecommons.org/licenses/by/
4.0/).
Mar. Drugs 2023, 21, 372. https://doi.org/10.3390/md21070372 https://www.mdpi.com/journal/marinedrugs

Mar. Drugs 2023, 21, 372 2 of 25
1. Introduction
As the demand for high-quality food products continues to rise among consumers,
the use of advanced technology and ingredients in the development of functional foods
has become a common response [1]. In accordance with the FFC (Functional Food Center),
foods that offer established clinical benefits for the prevention, management, or treatment
of chronic diseases are known as functional foods, and can either be natural or processed.
These foods contain biologically active compounds in specific amounts that are well-
defined [2]. Seaweeds, also known as marine macroalgae, have gained significant attention
from researchers seeking novel bioactive ingredients for both medication and food since the
year 2000 [3,4]. These organisms are divided into three categories, which are determined by
analyzing their morphology, composition, and other characteristics. These groups are the
chlorophytes, also known as green algae, phaeophytes, or brown algae, and rhodophytes,
which are commonly referred to as red algae [5]. In traditional Japanese cuisine, seaweeds
are frequently used as vegetables, in sauces, and as sushi wrappers [6] and provide a
multitude of advantages to various sectors, such as food, pharmaceuticals, animal nutrition,
skincare, and agrochemical industries. Pharmaceutical and food sectors, specifically, rely
on seaweeds as a source of thickening and emulsifying agents (phycocolloids) [6–10]. Algae
possess a wealth of bioactive components, including polysaccharides, minerals, vitamins,
proteins, lipids, polyphenols (phenolic acid, flavonoids), pigments (fucoxanthins, astax-
anthins, carotenoids), alkaloids, and terpenoids, which play a significant ecological and
economic role and have numerous applications across various fields [11–13]. These bioac-
tive substances exhibit various biological activities such as antimicrobial [13–20], antiviral,
antifungal, insecticidal, cytotoxic, phytotoxic, antiproliferative [17,21,22], and hypolipi-
demic [23] properties. With a global count of between 20,000 and 30,000 species, seaweeds
make up approximately 18% of the plant kingdom [24]. The chemical composition of these
organisms varies depending on genus, habitat, maturity, and environmental conditions.
Algae lack leaves, stems, and roots and consist primarily of a vegetative apparatus known
as the thallus. They are multicellular, macroscopic marine organisms that can reproduce
sexually and/or asexually. Among the diverse types of algae, 6600 species of red algae
have been described [25]. They are referred to as Rhodophyceae and are found in saltwater
environments deeper than 6 m. These red algae require blue light (and some green light)
and contain chlorophyll a and d and pigments such as phycoerythrin and phycocyanin.
They are mostly multicellular and attach to rocks or shells of mollusks. Gracilaria species
are among the red algae and are widely distributed and commonly harvested or farmed for
agar production [26,27]. G. bursa-pastoris, a member of the Gracilariaceae family, is primarily
recognized as a food source rather than an herb. It is a type of edible seaweed widely
consumed in various cultures around the world, particularly in Asian cuisines, and can
be found in Japanese, Korean, and Chinese dishes. This species has a red or discolored
appearance and a fleshy, cartilaginous texture. Its thicker cylindrical axes have a diameter
of between 0.5 and 5 mm and are slightly compressed at the branching point. Branches are
irregular and can reach 35 cm in length. The base of the branches remains thick, and they
end in spikes. G. bursa-pastoris is commonly found in temperate and tropical regions of the
northeast Atlantic, as well as in the Mediterranean.
Diabetes mellitus is a prevalent health condition characterized by an imbalance in
sugar metabolism, leading to chronic hyperglycemia [28]. Current epidemiological studies
indicate that diabetes affects 463 million individuals worldwide, with projections suggest-
ing a rise to 700 million by 2045. This condition is generally classified into two main types:
Type 1 diabetes and Type 2 diabetes, which are the most common forms globally. Over time,
diabetes mellitus results in metabolic disturbances that contribute to the development of
long-term complications, primarily affecting the vascular system. These complications can
be broadly categorized as microvascular complications, also known as microangiopathy,
and macrovascular complications, commonly referred to as macroangiopathy [29]. The
onset and progression of these complications are closely linked to oxidative stress, which
triggers the production of free radicals and activates various metabolic pathways [30].
Mar. Drugs 2023, 21, 372 3 of 25
In diabetic patients, the primary goal of treatment is to maintain blood glucose levels
close to physiological norms to prevent and minimize the onset of diabetic complications.
Treatment for Type 1 diabetes involves insulin injections, while the management of Type 2
diabetes includes lifestyle modifications, dietary changes, and oral anti-diabetic medica-
tions to enhance insulin sensitivity [31]. Despite the beneficial effects of these treatments in
controlling blood sugar levels and maintaining body homeostasis, insulin therapy and anti-
diabetic drugs can have side effects. The specific side effects and their severity may vary
depending on the type of anti-diabetic drug and the individual’s response to it. Common
side effects include gastrointestinal disorders, diarrhea, nausea, the risk of lactic acidosis,
edema, heart failure, weight gain, flatulence, severe hypoglycemia, and vomiting [31–34].
Consequently, the search for natural anti-diabetic products has become a necessity [35].
Seaweeds have gained attention in the field of diabetes research. Several studies have
highlighted the positive effects of seaweeds on various aspects of diabetes, including blood
glucose control, insulin sensitivity, and the prevention of diabetic complications.
The study of the benthic algae along the Moroccan coast dates back to the end of the
19th century, with the work of P. K. Schousboe on the Tangier coast and subsequent revi-
sions by E. Bornet (1892). Further studies emerged early in the 20th century, including the
investigations of P. Hariot (1909–1919) on the Moroccan coast. Over the past few decades,
the Moroccan coast has drawn renewed attention from researchers and authors [36]. Cur-
rently, in collaboration with academics, relevant institutions are working towards creating
comprehensive studies of the algal flora along the national coastlines and preserving this
valuable resource. To the best of our knowledge, the composition of G. bursa-pastoris
seaweeds in the Nador lagoon has not been extensively studied.
This innovative research endeavors to conduct a comprehensive chromatographic
examination of the fatty acids and polyphenols present in G. bursa-pastoris extracts through
the use of advanced analytical techniques, including GC-MS and HPLC-DAD. Moreover,
the study aims to assess the antioxidant capability of the extracts and evaluate their impact
on pancreatic α-amylase and α-glucosidase activities. This is a landmark undertaking
as it represents the first time that the composition of G. bursa-pastoris extracts has been
characterized through GC/MS and HPLC, and the effect on pancreatic digestive enzymes
has been studied.
2. Results
2.1. Yields, Phenols and Flavonoids Contents
The phenols, flavonoids, and extraction yield of G. bursa-pastoris extracts were studied,
and the results are presented in Table 1. The methanolic extract had the highest polyphenolic
yield with 3.42 ± 0.06%, followed by the ethyl acetate extract with 1.80 ± 0.08%, both using
Soxhlet extraction. The hexane extract had the lowest yield, at 1.16 ± 0.32%.
Table 1. Yields, phenols and flavonoids contents of different extracts of G. bursa-pastoris.
Extraction Polyphenols Flavonoids

Solvent Yield (%)
Methods (mg GAE/g) (mg QE/g)
M 0.11 ± 0.09 - -
Hexane
S 1.16 ± 0.34 - -
M 0.21 ± 0.04 165.42 ± 0.21 84.53 ± 0.07
Ethyl acetate
S 1.80 ± 0.08 150.24 ± 0.11 75.47 ± 0.02
M 0.81 ± 0.26 37.69 ± 1.02 15.29 ± 0.18
Methanol
S 3.42 ± 0.07 28.68 ± 0.07 13.70 ± 0.03
Water M 1.25 ± 0.06 381.31 ± 0.33 201.80 ± 0.21
GAE: gallic acid equivalent; QE: quercetin equivalent; M: maceration extraction; S: Soxhlet extraction.
Mar. Drugs 2023, 21, 372 4 of 25
In terms of maceration extraction, the aqueous extract had the highest yield with
1.25 ± 0.06%, followed by the methanolic extract with 0.81 ± 0.26%. The yields of the ethyl
acetate and hexane extracts were 0.21 ± 0.04% and 0.11 ± 0.09%, respectively.
The total phenolic content of the extracts was determined based on its equivalent to
gallic acid (GAE), as seen in Table 1. The highest total polyphenolic content was found
in the aqueous extract using the maceration, with a value of 381.31 ± 0.33 mg GAE/g.
This was followed by the ethyl acetate extract, with a value of 165.42 ± 0.21 mg GAE/g or
150.24 ± 0.11 mg GAE/g, using the maceration or Soxhlet method, respectively. These re-
sults suggest that both water and ethyl acetate are effective solvents for extracting phenolic
compounds in G. bursa-pastoris using either maceration or Soxhlet methods. The methanolic
extracts showed the lowest values of 37.69 ± 1.02 mg GAE/g and 28.68 ± 0.07 mg GAE/g.
The flavonoid content of the various extracts of G. bursa-pastoris was quantified as the
quercetin equivalent (QE), as presented in Table 1. The concentrations of flavonoids were
found to range from 13.70 ± 0.03 mg QE/g to 201.80 ± 0.21 mg QE/g. The highest concen-
tration was observed in the aqueous extract and was obtained via the maceration method,
followed by the ethyl acetate extract with 84.53 ± 0.07 mg EQ/g and 75.47 ± 0.02 mg EQ/g,
using the maceration and Soxhlet methods, respectively.
2.2. Fatty Acid Analysis

The results of the GC-MS analysis presented in Table 2 showed that both the Soxhlet
and maceration extraction techniques of G. bursa-pastoris resulted in a significant concen-
tration of saturated fatty acids (SFAs) and unsaturated fatty acids (UFAs). Compared to
terrestrial plants, seaweeds have a unique fatty acid (FA) profile.
Table 2. Fatty acids composition of hexane and ethyl acetate extracts from red algae G. bursa-pastoris.
RT HE (%) EAcE (%)

Fatty Acids
(min) M S M S
Eicosenoic acid (C20:1) 20.08 53.06 ± 0.05 33.25 ± 0.41 5.21 ± 0.01 nd
7,10-Hexadecadienoic acid (C16:2) 21.17 2.88 ± 0.23 5.40 ± 0.01 5.67 ± 0.03 3.84 ± 0.06
Palmitic acid (C16:0) 23.30 22.88 ± 0.01 40.72 ± 0.31 50.55 ± 0.05 38.24 ± 0.02
Margaric acid (C17:0) 23.87 nd nd nd 16.19 ± 0.07
Oleic acid (C18:1) 24.54 3.32 ± 0.07 nd 10.48 ± 0.02 13.01 ± 0.09
Linoleic acid (C18:2) 25.03 9.51 ± 0.31 10.08 ± 0.11 15.57 ± 0.23 23.85 ± 0.11
Linolenic acid (C18:3) 25.09 3.58 ± 0.04 4.49 ± 0.17 6.09 ± 0.06 nd
Stearic acid (C18:0) 25.26 4.77 ± 0.43 6.06 ± 0.08 6.43 ± 0.07 4.87 ± 0.04
SFA a 27.65 46.78 56.98 59.3
UFA b 72.35 53.22 43.02 40.7
UFA/SFA c 2.61 1.13 0.75 0.68
RT: retention time; M: maceration; S: Soxhlet extraction, HE: hexane extract; EAcE: ethyl acetate extract; nd: not
detected; a : saturated fatty acids (SFA); b : unsaturated fatty acids (UFA); c : unsaturation ratio = UFA/SFA.
The composition of fatty acids in the samples is expressed in terms of total fatty acids.
The percentages of total saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs),
and polyunsaturated fatty acids (PUFAs) varied in function of the solvent and the extraction
method used, ranging from 13.01% to 59.3%.
In the hexane extract, eicosenoic acid (C20:1) was the most abundant fatty acid ob-
tained via maceration extraction, with 53.06%, followed by palmitic acid (16:0), with also
a high amount at 40.72%, and by linoleic acid, with relatively the same concentration in
both extraction methods at 10.08%. However, the highest concentration of palmitic acid
(50.55%) was found in the ethyl acetate extract obtained through maceration, followed by
linoleic acid (C18:2), with a relatively high concentration (23.85%) obtained through Soxhlet
Oleic acid (C18:1) 24.54 3.32 ± 0.07 nd 10.48 ± 0.02 13.01
Linoleic acid (C18:2) 25.03 9.51 ± 0.31 10.08 ± 0.11 15.57 ± 0.23 23.85
Linolenic acid (C18:3) 25.09 3.58 ± 0.04 4.49 ± 0.17 6.09 ± 0.06 n
Stearic
Mar. Drugs 2023, 21, 372 acid (C18:0) 25.26 4.77 ± 0.43 6.06 ± 0.08 6.43 ± 0.075 of 25 4.87 ±
SFA a 27.65 46.78 56.98 59
UFA b 72.35 53.22 43.02 40
extraction. Margaric acid (C17:0) was detected in only one extract, the ethyl acetate extract
UFA/SFAobtained through Soxhlet extraction, at2.61
c
a value of 16.19%.1.13
In this extract, oleic0.75
acid (C18:1) 0.
RT: more
was retention time;than
dominant M: in
maceration; S: Soxhlet
the hexane extract, withextraction, HE:ofhexane
concentrations 13.01%extract; EAcE: ethy
and 10.48%
extract;
in Soxhletnd:
andnot
maceration a: saturated
detected;extraction, fatty acids
respectively. (SFA);
Stearic b: unsaturated
acid fattyfor
(C18:0) accounted acids (UFA); c: u
4.77%
to 6.43%
tion of=the
ratio total fatty acids. Figure 1 shows the distribution of SFA, MUFA and PUFA
UFA/SFA.
in each type of solvent and extraction process.
100
SFA
MUFA
90
PUFA
80
70
% Fatty Acids
60
50
40
30
20
10
0
HE (M) HE (S) EAcE (M) EAcE (S)
Figure 1. Fatty acid (FA) content in different extracts of G. bursa-pastoris (SFAs: saturated fatty acids;
Figure 1. Fatty acid (FA) content in different extracts of G. bursa-pastoris (SFAs: saturated fa
MUFAs: monounsaturated fatty acids; PUFAs: polyunsaturated fatty acids; HE: hexane extract; EAcE:
MUFAs: monounsaturated fatty acids; PUFAs: polyunsaturated fatty acids; HE: hexane
ethyl acetate extract; M: maceration; S: Soxhlet extraction).
EAcE: ethyl acetate extract; M: maceration; S: Soxhlet extraction).
In addition, the hexane extract obtained through the maceration method revealed the
2.3. HPLC
highest Analysis
unsaturated of followed
ratio, G. bursa-pastoris Extracts
by the hexane extract under Soxhlet extraction. These re-
sults indicate that the hexane extract was rich in unsaturated fatty acids, notably eicosenoic
High-Performance
acid. G. bursa-pastoris possessesLiquid Chromatography
great nutritional combined
value in terms of humanwith a Diode Array
consumption. D
(HPLC-DAD) was implemented to examine the chemical composition of the ethy
2.3.
andHPLC Analysisextracts
methanol of G. bursa-pastoris Extracts
of G. bursa-pastoris. The results were compared with thos
High-Performance Liquid Chromatography
standards based on the retention time and ultraviolet combined with aspectra
Diode Array
and Detector
are displayed
(HPLC-DAD) was implemented to examine the chemical composition of the ethyl acetate
3. The study identified nine phenolic acids (4-hydroxy benzoic acid, chlorogenic a
and methanol extracts of G. bursa-pastoris. The results were compared with those of
ulicstandards
the acid, cinnamic
based onacid, gallic acid,
the retention caffeic
time and acid, syringic
ultraviolet spectra andacid,
are p-coumaric
displayed in acid, a
cylic 3.acid)
Table and ten
The study flavonoids
identified (catechin,
nine phenolic acids quercetin-3-glucoside, 7,3′,4′-flavon-3-o
(4-hydroxy benzoic acid, chlorogenic
acid, ferulic acid, cinnamic acid, gallic acid, caffeic acid, syringic
quercetin, luteolin, apigenin, kaempferol, flavone, and flavanone). acid, p-coumaric acid, and acid
Caffeic
salicylic acid) and ten flavonoids (catechin, quercetin-3-glucoside, 7,30 ,40 -flavon-3-ol, rutin,
most abundant in both ME (M) (35.64%) and ME (S) (24.24%), while sinapic acid
quercetin, luteolin, apigenin, kaempferol, flavone, and flavanone). Caffeic acid was the
most abundant in both ME (M) (35.64%) and ME (S) (24.24%), while sinapic acid was the
most prevalent in EAcE (M) (9.53%). EAcE (S) had a predominant amount of flavonoid,
specifically 7,30 ,40 -flavon-3-ol (20.68%) (Figure 2).
Mar. Drugs 2023, 21, 372 6 of 25
Table 3. Chemical composition of ethyl acetate and methanolic extracts from red algae G. bursa-pastoris.
EAcE (%) ME (%)

N◦ Compounds RT (min)
M S M S
1 Gallic acid 15.47 nd nd nd 0.637
2 Catechin 18.68 1.516 8.188 nd nd
3 4-hydroxy benzoïc acid 18.91 4.105 18.778 nd 3.561
4 Chlorogenic acid 19.15 1.096 4.202 11.666 7.714
5 Caffeic acid 19.45 nd nd 35.642 24.239
6 Syringic acid 19.74 nd 3.092 3.673 nd
7 Vanilline 23.1 3.457 nd nd nd
8 p-Coumaric acid 23.63 nd nd nd 5.269
9 Sinapic acid 24.09 9.534 nd nd nd
11 Quercetin-O-3-glucoside 24.52 8.074 nd nd nd
12 7,30 ,40 -flavon-3-ol 24.92 8.316 20.682 nd nd
13 Naringin 25.01 8.344 17.754 21.069 12.626
14 Rutin 25.16 8.291 nd nd nd
15 Salicylic acid 25.32 nd 15.613 14.901 11.854
16 Quercetin 25.46 8.669 nd nd nd
17 Cinnamic acid 25.48 8.941 nd nd 9.662
18 Luteolin 25.64 8.912 nd nd 9.387
19 Apigenin 25.87 7.839 nd nd nd
20 Kaempferol 26.1 9.108 11.690 8.100 9.109
21 Flavone 26.92 nd nd 4.948 5.940
Mar. Drugs 2023, 21, 372 22 Flavanone 27.412 nd 3.795 nd nd7 of 26
RT: retention time; M: maceration; S: Soxhlet extraction; EAcE: ethyl acetate extract; ME: methanolic extract; nd:
not detected.
Figure 2. Structures
Figure 2. of the
Structures of the main
main compounds
compounds present
present in
in the
the red
red algae
algae G.
G. bursa-pastoris.
bursa-pastoris.
2.4. Antioxidant
In all four Activity
extracts, several other compounds were discovered, including naringin
chlorogenic
Antioxidantsand
acid, kaempferol.
are the Naringin
focus of research due was present
to their roleininmoderate levels,
preserving foodwith values
quality and
ranging from 8% to 21%, and chlorogenic acid was found at varying percentages,
their potential to treat diseases associated with oxidative stress. In this study, the DPPH from 7% to
11% in ME and
free radical at low levels
scavenging assayinand
EAcE,thewhile kaempferol
β-carotene was assay
bleaching detected in moderate
were used. The amounts
IC50 val-
in each
ues extract.
for G. Additionally,
bursa-pastoris the extracts
EAcE, ME, and AQEcontained
extracts aremoderate
presented quantities
in Table 4.of The
4-hydroxy
results
benzoic acid, quercetin-3-glucoside, salicylic acid, quercetin, cinnamic acid,
indicate that the best antioxidant activity was observed in the case of Soxhlet-extracted luteolin, and
apigenin. While gallic acid, catechin, syringic acid, vanillin, p-coumaric
ME as well as for maceration-extracted ME, with IC50 values of 0.06 and 0.07 mg/mL, re- acid, flavone,
and flavanone
spectively, were present
in comparison in the
with the reference
extracts in low quantities.
antioxidant The presence
BHA, which presented of certain
an IC50
compounds in maceration extracts and their absence in Soxhlet
value of 0.02 mg/mL. Additionally, AQE constituted a modest antioxidant with an extracts (and vice versa)
IC50
can be attributed to the extraction conditions, including solvent, temperature,
value of 0.21 mg/mL; however, the EAC extracts revealed the lowest antioxidant activity. time, and the
stabilityacid
Caffeic of natural
is among products under certain conditions.
the hydroxycinnamic acids used to enhance the stability of dietary
products [37,38]. It constituted the most abundant compound in both maceration and
Soxhlet-extracted methanol extracts (ME). The presence of this compound at a high con-
centration in these extracts (35.64% and 24.24%) could explain the high antioxidant poten-
tial observed in ME extracts.
Table 4. IC50 values of G. bursa-pastoris extracts.

Mar. Drugs 2023, 21, 372 7 of 25
2.4. Antioxidant Activity

Antioxidants are the focus of research due to their role in preserving food quality and
their potential to treat diseases associated with oxidative stress. In this study, the DPPH free
radical scavenging assay and the β-carotene bleaching assay were used. The IC50 values
for G. bursa-pastoris EAcE, ME, and AQE extracts are presented in Table 4. The results
indicate that the best antioxidant activity was observed in the case of Soxhlet-extracted
ME as well as for maceration-extracted ME, with IC50 values of 0.06 and 0.07 mg/mL,
respectively, in comparison with the reference antioxidant BHA, which presented an
IC50 value of 0.02 mg/mL. Additionally, AQE constituted a modest antioxidant with
an IC50 value of 0.21 mg/mL; however, the EAC extracts revealed the lowest antioxidant
activity. Caffeic acid is among the hydroxycinnamic acids used to enhance the stability of
dietary products [37,38]. It constituted the most abundant compound in both maceration
and Soxhlet-extracted methanol extracts (ME). The presence of this compound at a high
concentration in these extracts (35.64% and 24.24%) could explain the high antioxidant
potential observed in ME extracts.
Table 4. IC50 values of G. bursa-pastoris extracts.
IC50 (mg/mL)
Extracts
DPPH β-Carotene
M 0.40 ± 0.51 0.83 ± 0.32
EAcE
S 0.30 ± 0.31 0.65 ± 0.04
M 0.55 ± 0.25 0.07 ± 0.06
ME
S 0.46 ± 0.36 0.06 ± 0.64
AQE M 0.17 ± 0.07 0.21 ± 0.14
Ascorbic Acid 0.06 -
BHA - 0.02
EAcE: ethyl Acetate extract; ME: methanolic extract; AQE: aqueous extract; M: maceration; S: Soxhlet.
2.5. In Vitro α-Amylase Inhibition

Figure 3 illustrates the effect of G. bursa-pastoris extracts on α-amylase inhibitory
activity in comparison to 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. The
results showed that all extracts significantly inhibited the α-amylase enzymatic activity
at all tested doses. Among the different samples, the concentration of 2.27 mg/mL had
the most active effect, exhibiting inhibitory activities of 51.83% for EAcE (M), 67.64% for
EAcE (S), 78.54% for ME (M), 73.94% for ME (S), and 72.08% for AQE. IC50 values for each
sample were determined (Table 5), revealing that the ME (M), AQE, and ME (S) extracts
exhibited similar inhibitory effects (p < 0.05) and higher inhibitory activities than the other
extracts. EAcE (S) (p < 0.05) and EAcE (M) (p < 0.05) followed. In addition, the results
of the in vitro study demonstrated that all extracts significantly reduced the pancreatic α-
glucosidase activity compared to acarbose (Figure 4). Among the extracts, EM (M) had the
most significant effect (98.32%), followed by AQE (M) (96.68%), ME (S) (94.32%), EAcE (M)
(88.32%), and EAcE (S) (83.32%) at a concentration of 2.27 mg/mL. The observed inhibitory
effects may be attributed to the presence of active chemicals in the extracts that act as
inhibitors of the enzymes responsible for hydrolyzing polysaccharide and disaccharide
chains. These substances may be unique to α-amylase and α-glucosidase.
Mar. Drugs 2023, 21, 372 9 of 26
Mar. Drugs 2023, 21, 372 8 of 25
B Control
Acarbose
EAC (M)
100
EAC (S)
EM (M)
*** EM (S)
Inhibition percentage (%)
80
*** AQE (M)
***
*** ***
*** ***
60 *** ***
*** ***
***
***
40
***
***
***
20
0.0 0.5 1.0 1.5 2.0 2.5

Concentrations (mg/ml)
Inhibition
Figure3.3.Inhibition
Figure percentage
percentage ofofG.G.bursa-pastoris
bursa-pastoris extracts
extracts and
and acarbose
acarbose against
against α-glucosidase
α-glucosidase (A)
(A)
and
andα-amylase
α-amylase (B) atat
(B) different
differentdoses
dosesofofG.G.bursa-pastoris
bursa-pastorisextracts
extractsEAc:
EAc:Ethyl
EthylAcetate; ME:
Acetate; Methanolic
ME: Methanolic
Extract;
Extract;AQE:
AQE:Aqueous
Aqueous Extract; M: Maceration;
Extract; M: Maceration;S: Soxhlet. Data are
S: Soxhlet. expressed
Data as mean
are expressed as± mean
SD. ***±p SD.
<
0.001.
*** p < 0.001.
2.6. Molecular Modeling Studies

Table 5. IC values of G. bursa-pastoris extracts and acarbose in α-amylase and α-glucosidase inhibition.
50
Alpha-amylase and alpha-glucosidase are enzymes that play a role in the digestion
of carbohydrates. IC50 (mg/mL)
Inhibitors α-amylase breaks down starch into smaller carbohydrates, while α-glu-
α-Amylase α-Glucosidase
cosidase breaks down these smaller carbohydrates into simple sugars, such as glucose,
which can Acarbose
be absorbed into the bloodstream. In individuals 0.35 ± 0.08 0.39 ±enzymes
with diabetes, these 0.04
can contribute M 1.86 ± 0.06 *** 0.44 ± 0.02 ns
EAcEto high blood sugar levels if glucose intake or storage are not adequately
S 1.11 ± 0.02 *** 0.57 ± 0.03 **
regulated. To manage blood sugar levels, some people with diabetes may take α-amylase
M 0.72 ± 0.04 ** 0.25 ± 0.09 *
ME inhibitors to slow down carbohydrate digestion and absorption. All mol-
or α-glucosidase S 0.76 ± 0.05 ** 0.37 ± 0.06 ns
ecules identified
AQE in the G. bursa-pastoris M extracts using 0.85 HPLC
± 0.01 ***showed remarkable
0.32 ± 0.07inhibi-
ns
tory activity
EAcE: against
ethyl acetate α-amylase
extract; and α-glucosidase
ME: methanolic extract; AQE: aqueous compared
extract; with acarbose
M: maceration; and metfor-
S: Soxhlet, Data are
expressed
min as mean
(positive ± SD. ns:
controls), asnon-significant * p < 0.05;
shown in Figures ** p <5.0.01;
4 and *** pshowed
Rutin < 0.001. the highest glide gscore
values against α-amylase of −8.109 kcal/mol, followed by naringenin, with a glide gscore
Mar. Drugs 2023, 21, 372 9 of 25
Mar. Drugs 2023, 21, 372 11 of 26
Figure 4. Cont.
Mar. Drugs 2023, 21, 372 10 of 25
Mar. Drugs 2023, 21, 372 12 of 26
Figure 4. Two-dimensional view of ligands interactions. (A): Rutin interactions in the active sites of
α-amylase; (B): naringenin interactions in the active sites of α-amylase; (C): quercetin interactions
in the active(B):
α-amylase; sitenaringenin interactions
of α-glucosidase; (D): in the activeinteractions
kaempferol sites of α-amylase; (C): quercetin
in the active interactions
site of α-glucosidase;
in the active site of α-glucosidase; (D): kaempferol interactions in the active site of α-glucosidase;
(E,F): acarbose interactions in the active site of α-amylase and α-glucosidase; (G,H) metformin in-
(E,F): acarbose
teractions in theinteractions
active site ofinα-amylase
the activeand
siteα-glucosidase.
of α-amylase and α-glucosidase; (G,H) metformin
interactions in the active site of α-amylase and α-glucosidase.
Upon comparing the various extracts to the positive control, it was discovered that all
of the extracts demonstrated a lower inhibitory effect compared to acarbose. The statistical
analysis revealed a significant difference, with a p-value of less than 0.001 for EAcE (M),
EM (S), EAcE (S), and a p-value of less than 0.01 for ME (M), ME (S), and AQE.
2.6. Molecular Modeling Studies

Alpha-amylase and alpha-glucosidase are enzymes that play a role in the digestion
of carbohydrates. α-amylase breaks down starch into smaller carbohydrates, while α-
glucosidase breaks down these smaller carbohydrates into simple sugars, such as glucose,
which can be absorbed into the bloodstream. In individuals with diabetes, these enzymes
can contribute to high blood sugar levels if glucose intake or storage are not adequately
regulated. To manage blood sugar levels, some people with diabetes may take α-amylase
or α-glucosidase inhibitors to slow down carbohydrate digestion and absorption. All
molecules identified in the G. bursa-pastoris extracts using HPLC showed remarkable
inhibitory activity against α-amylase and α-glucosidase compared with acarbose and
metformin (positive controls), as shown in Figures 4 and 5. Rutin showed the highest
glide gscore values against α-amylase of −8.109 kcal/mol, followed by naringenin, with
a glide gscore of −7.198 kcal/mol. Moreover, quercetin and kaempferol were the most
active molecules in the active site of α-glucosidase, with a glide gscore of −7.035 and
−5.698 kcal/mol, respectively (Table 6).
Mar. Drugs 2023, 21, 372 in the active site of α-glucosidase; (D): kaempferol interactions in the active site of α-glucosidase;
11 of 25
(E,F): acarbose interactions in the active site of α-amylase and α-glucosidase; (G,H) metformin in-
teractions in the active site of α-amylase and α-glucosidase.
Mar. Drugs 2023, 21, 372 13 of 26
Figure 5. Three-dimensional view of ligand interactions. (A): rutin interactions in the active sites of
Figure 5. Three-dimensional view of ligand interactions. (A): rutin interactions in the active sites of
in the active site of α-glucosidase; (D): kaempferol interactions in the active site of α-glucosidase;
in theacarbose
(E,F): active site of α-glucosidase;
interactions (D): kaempferol
in the active interactions
site of α-amylase in the active site
and α-glucosidase; of α-glucosidase;
(G,H): metformin in-
(E,F): acarbose interactions in the active site of α-amylase
teractions in the active site of α-amylase and α-glucosidase. and α-glucosidase; (G,H): metformin
interactions in the active site of α-amylase and α-glucosidase.
3. Discussion
In this research, extracts were prepared from extracts of G. bursa-pastoris. The results
showed that polar solvents (methanol and water) produced higher yields than less polar
solvents (hexane). This is supported by published research [39,40]. Additionally, polar
solvents such as water are capable of extracting phenolic compounds that are bound to
sugars or proteins, saponins, glycosides, organic acids, selenium, and mucilage [41]. Fur-
thermore, the study by Bandar et al. [42] concluded that Soxhlet extraction was the most
suitable technique for extracting active molecules of G. bursa-pastoris based on yield.
For EAcE and ME of G. bursa-pastoris, the mean quantity of total polyphenols and
total flavonoids was determined. The polyphenolic content of the methanolic extract of G.
Mar. Drugs 2023, 21, 372 12 of 25
Table 6. Docking results with identified compounds in active sites of α-amylase and α-glucosidase.
α-Amylase (PDB: 1B2Y) α-Gluosidase (PDB: 5NN8)

Glide Gscore Glide Emodel Glide Energy Glide Gscore Glide Emodel Glide Energy
4-Hydroxybenzoic acid −6.079 −27.887 −18.583 −4.366 −20.091 −13.882
7,30 ,40 -flavon-3-ol −4.801 −48.996 −38.542 −3.695 −36.338 −30.331
Apigenin −7.105 −60.626 −41.637 −5.2 −44.196 −33.441
Caffeic Acid −5.893 −39.405 −29.08 −4.237 −26.17 −18.858
Chlorogenic acid −6.801 −63.522 −45.48 −3.738 −43.188 −37.071
Cinnamic acid −3.632 −22.468 −18.689 −3.353 −19.589 −14.942
Flavanone −5.798 −41.242 −30.237 −4.287 −35.284 −27.649
Flavone −6.121 −43.458 −31.445 −4.326 −35.845 −28.673
Kaempferol −7.2 −63.076 −43.51 −5.698 −47.8 −35.946
Luteolin −6.366 −52.124 −38.738 −5.425 −51.621 −37.184
Naringenin −7.198 −60.107 −40.882 −5.246 −38.138 −29.883
p-coumaric acid −5.395 −35.602 −26.441 −3.558 −21.069 −15.352
Quercetin −7.146 −67.907 −45.267 −7.035 −60.46 −42.363
Quercetin 3-glucoside −6.402 −67.452 −49.322 −5.242 −60.877 −49.104
Rutin −8.109 −108.772 −71.453 −5.237 −73.009 −57.522
Salicylic Acid −4.06 −23.996 −18.556 −3.911 −15.12 −12.243
Sinapic acid −3.894 −30.39 −24.001 −3.543 −27.834 −22.072
Syringic acid −5.104 −32.584 −24.351 −3.472 −23.213 −19.151
Vanillin −5.498 −24.397 −17.121 −4.651 −30.76 −23.024
Acarbose −7.067 −108.733 −73.113 −6.989 −111.624 −70.347
Metformin −3.989 −35.344 −22.492 −4.494 −36.414 −21.184
In comparison to the effect of acarbose and metformin in the active site of α-amylase,
apigenin, kaempferol, naringenin, quercetin, and rutin showed higher binding energy than
acarbose (−7.067 kcal/mol) (Table 6).
However, the effect of acarbose and metformin in the active site of α-glucosidase, quercetin
showed higher binding energy than acarbose (−6.989 kcal/mol), while 4-hydroxybenzoic acid,
apigenin, kaempferol, luteolin, naringenin, quercetin, quercetin 3-glucoside, rutin, and
vanillin exhibited higher binding energy than metformin.
3. Discussion
In this research, extracts were prepared from extracts of G. bursa-pastoris. The results
showed that polar solvents (methanol and water) produced higher yields than less polar
solvents (hexane). This is supported by published research [39,40]. Additionally, polar
solvents such as water are capable of extracting phenolic compounds that are bound
to sugars or proteins, saponins, glycosides, organic acids, selenium, and mucilage [41].
Furthermore, the study by Bandar et al. [42] concluded that Soxhlet extraction was the most
suitable technique for extracting active molecules of G. bursa-pastoris based on yield.
For EAcE and ME of G. bursa-pastoris, the mean quantity of total polyphenols and
total flavonoids was determined. The polyphenolic content of the methanolic extract of
G. bursa-pastoris was investigated by Ramdani et al. in Morocco [43] and by Yildiz et al.
in Turkey [44]. Ramdani et al. reported high phenolic content using maceration, with a
value of 99.62 mg GAE/g, and using extraction at 40 ◦ C, with a value of 142.26 mg GAE/g.
In contrast, Yildiz et al. found a relatively low phenolic content of 0.35 mg GAE/g. This
disparity in results could be attributed to differences in the origin of the seaweed sample,
the extraction procedure used, the maturity of the algae, the growing conditions or the
extraction solvent used [45,46].
The chemical composition of EAcE and HE was determined via gas chromatography
coupled with mass spectrometry (GC-MS). It highlights the presence of palmitic acid,
linoleic acid and eicosenoic acid as major compounds. Linoleic acid is an essential fatty
acid and has hypocholesterolemic effects [47]. Palmitic acid inhibits prostate cancer cell
proliferation and metastasis by suppressing the PI3K/Akt pathway [48]. Omega-3 fatty
Mar. Drugs 2023, 21, 372 13 of 25
acids, particularly linolenic acid, play a crucial role in the regulation of blood glucose
levels by enhancing insulin secretion from the beta cells within the pancreatic islets of
Langerhans [49]. Linoleic acid enhances glucose uptake by C2C12 muscle cells [50]. Oleic
acid has been found to promote insulin secretion in the glucose-sensitive INS-1 cell line. Of
particular significance is its ability to enhance insulin secretion even when the inflammatory
cytokine TNF-α is present [51]. Stearic acid enhances glucose uptake into adipocytes by
activating insulin/insulin receptor signaling while inhibiting PTP1B [52]. The expression
of the GLUT4 transporter and the facilitation of glucose uptake by 3T3-L1 adipocytes are
significantly impacted by polyunsaturated fatty acids, resulting in an increased abundance
of both GLUT4 and GLUT1 transporters [53,54]. In addition, the effect of eicosenoic acid,
7,10-hexadecadienoic acid, on diabetes is not well-studied compared to other omega fatty
acids, such as omega-3 and omega-6 fatty acids.
It is important to consider that the type and proportion of fatty acids in a food can
have a significant impact on its overall health effects. For instance, high levels of SFAs
have been associated with an increased risk of cardiovascular disease, while PUFAs have
been linked to a reduced risk. MUFAs, on the other hand, are commonly considered to
be beneficial for health as they may help to lower cholesterol levels and decrease the risk
of heart disease [55]. Polyunsaturated fatty acids, or PUFAs, play a crucial role in human
metabolism [56], as they constitute a major part of cell membrane phospholipids [57]. Their
metabolic significance is reflected in their diverse biological properties, such as antibacterial
activity [58–60], anti-inflammatory effects [61,62], antioxidant capacity [63], potential for
preventing cardiovascular disease [64], and inhibition of tumor growth [65,66].
The major phenolic molecules of the different EAcE and ME extracts were determined
through HPLC analysis. The study revealed that G. bursa-pastoris extracts are an excellent
source of flavonoids and polyphenols. The investigation of natural bioactive compounds
possessing health-promoting properties has been the subject of substantial scrutiny. Among
these compounds, caffeic acid is a hydroxycinnamic acid that is synthesized as a sec-
ondary metabolite by several plant species [67]. It is known for its antioxidant [68,69],
anticancer [70] and anti-diabetic properties [71]. Caffeic acid has also been found to protect
neurons against oxidative damage and neurotoxicity by modulating various signaling
pathways and reducing inflammation in the brain [70].
7,30 ,40 -flavon-3-ol, also known as fisetin, is a natural flavonoid that has been exten-
sively studied in the scientific community due to its various health-promoting properties.
This compound acts as an anti-inflammatory and anticancer agent [72–74] and has also
been shown to have anti-diabetic properties that can improve insulin sensitivity and de-
crease blood sugar levels, making it potentially useful in managing Type 2 diabetes [75].
Furthermore, fisetin has demonstrated beneficial effects in preclinical models of several neu-
rodegenerative diseases, including Alzheimer’s disease, vascular dementia, schizophrenia,
Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease, stroke, depression,
and traumatic brain injury [76].
The DPPH assay is predicated on the ability of the stable free radical DPPH (•) to
diminish coloration in the presence of antioxidants. This radical features an unpaired
electron that confers a deep purple coloration, which is diminished by the presence of
antioxidants [77]. DPPH assay is widely applied as an indicator to assess the antioxidant
activity of diverse plant extracts [78,79]. The β-carotene/linoleic acid bleaching test mea-
sures the impact of antioxidants on the discoloration of a solution caused by the oxidation
of linoleic acid, which generates hydroperoxides that attack β-carotene molecules. An-
tioxidants, on the other hand, inhibit β-carotene bleaching [80]. The results showed that
extracts of the red algae G. bursa-pastoris with high total phenol content demonstrated
robust antioxidant activity, suggesting that algal polyphenols may be the key contributors
to the radical scavenging properties of these extracts. There is a strong correlation between
antioxidant activity and the content of polyphenols and flavonoids, as noted in the litera-
ture. These algae are, therefore, a rich source of bioactive compounds and have significant
potential for antioxidant activity, as evidenced by increased efficacy resulting from heating
Mar. Drugs 2023, 21, 372 14 of 25
during Soxhlet extraction. Ramdani et al., [43] reported a high antioxidant activity, with an
IC50 of 0.09 to 0.1% for the ethanolic extract and 0.17 to 0.18% for the aqueous extract, the
differences of which could be attributed to the extraction method, time, and temperature.
The in vitro anti-diabetic activities of G. bursa-pastoris extracts were investigated
through their potential to inhibit pancreatic enzyme activity. Type 2 diabetes is widely
recognized as the most prevalent metabolic disorder affecting people of all ages across the
globe. This condition is known to cause numerous long-term health complications, such
as retinopathy, nephropathy, neuropathy, cardiopathy, and, unfortunately, even death. In
fact, the mortality rate associated with this condition has seen a concerning increase of
3% between the years 2000 and 2019 [30]. Therefore, it is crucial to have proper medical
supervision, which involves the use of oral anti-diabetic medications such as sulfonamides,
biguanides, and metformin [81]. As the disease progresses, subcutaneous insulin injections
become a common method of treatment. While these injections can be helpful in regulating
blood sugar levels and promoting bodily balance, they also carry a number of adverse
effects, including hypoglycemia, coma, gastrointestinal issues, diarrhea, and even heart
failure, nausea, and vomiting [33]. As such, it has become imperative to conduct research
on natural products with anti-diabetic properties. When it comes to human nutrition,
starch serves as the primary source of carbohydrates that can be digested, and it undergoes
a breakdown process into glucose as a result of digestive enzymes such as α-amylase.
This enzymatic reaction is considered to be the initial stage of starch digestion [82]. This
particular enzyme is capable of being secreted by both the salivary glands and the pancreas
and then released into the intestine. Its primary function is to hydrolyze the 1,4-glucan
bonds of polysaccharides into oligosaccharides and disaccharides [83]. As a means of con-
trolling postprandial hyperglycemia, natural α-amylase inhibitors are considered to be one
of the best options available [84]. In the present work, we examined the inhibitory effects
of different G. bursa-pastoris extracts on pancreatic α-amylase and α-glucosidase in vitro.
Our results showed that the various extracts possessed considerable in vitro inhibitory
α-amylase activity, particularly the methanolic extract, which yielded an IC50 value that
was comparable to that of the positive control, acarbose. This was followed by the aqueous
extract and ethyl acetate extract. G. bursa-pastoris was found to contain a total of 0.35 mg
of gallic acid per gram of phenolic compounds [44]. In other studies, this chemical has
been shown to have significant anti-diabetic activity and has the ability to block digestive
enzymes linked to diabetes, such as pancreatic α-amylase [85,86]. Our samples were found
to contain significant levels of gallic acid, apigenin, and tannic acid, which have all been
shown to exhibit intense α-amylase activity by many researchers. Tannic acid has an IC50
value of 0.75 mg/mL, compared to acarbose’s 0.01 mg/mL. Gallic acid has an IC50 value
of 287.53 mg/mL, compared to acarbose’s 678.43 mg/mL. Apigenin has an IC50 value
of 3.46 mg/mL, compared to acarbose [87–89]. In addition, it was found that gallic acid
can enhance insulin secretion by promoting peripheral glucose uptake by adipocytes and
skeletal muscle [90]. Apigenin, on the other hand, plays a role in decreasing hepatic glucose-
6-phosphatase activity that catalyzes the final reaction of glycogenolysis and glucose release
into the bloodstream [91]. In diabetic mice, kaempferol was observed to contribute to the
improvement of hyperglycemia and reduce hepatic glucose production by decreasing the
activity of pyruvate carboxylase [92]. Quercetin exhibits anti-diabetic properties, includ-
ing reducing postprandial glycemia, achieved by inhibiting glucose uptake by skeletal
muscle [93]. Chlorogenic acid possesses potential effects on diabetes. It has been found to
regulate blood sugar levels and enhance insulin sensitivity [94]. Syringic acid, flavanone,
and naringin have been reported to have antihyperglycemic properties, which can lead to
reduced glucose absorption and improved glycemic control [95–97]. One of the organic
acids identified in our algae extracts is succinic acid. It is present in an amount of 3.01% for
ME (M) and has a high antihyperglycemic effect (IC50 = 6.74 µM/mL, compared to acar-
bose’s 0.21 µM/mL). According to Gamze Yildiz et al., [44], G. bursa-pastoris contains a high
amount of ascorbic acid, with a value of 21.6 mg/100 g, which has been confirmed in our
work. This compound has been shown to have the ability to reduce blood glucose, enhance
Mar. Drugs 2023, 21, 372 15 of 25
insulin secretion, improve insulin resistance, and reduce diabetic complications [98]. The
same study also showed that G. bursa-pastoris [EHL1] contains vitamin E, with an amount
of 57 mg α-tocopherol/100 g, which has a potential anti-diabetic effect. Tocopherols play a
key role in improving insulin resistance and suppressing oxidative stress, both of which
are implicated in diabetes management [99].
On the other hand, molecular docking simulations of the metabolites identified in
G. bursa-pastoris extracts were performed. Several scientific studies have indicated that rutin
plays a significant role in terms of anti-diabetic activity by enhancing glucose homeostasis in
diabetic rats. It achieves this by lowering fasting blood glucose levels and increasing insulin
levels, as well as by increasing glycogen content in the liver and muscle while reducing it
in the kidneys. Rutin also boosts hexokinase activity and reduces glucose-6-phosphatase
and fructose1,6-bisphosphatase activities in the tissues [100,101].
Another study showed that a fraction rich in rutin, naringenin, and quercetin from
Globularia alypum exhibited significant anti-diabetic activity with an inhibitory effect on α-
amylase (IC50 = 0.57 mg/mL) and α-glucosidase (IC50 = 0.52 mg/mL) [102]. In comparison,
another in silico study showed significant affinity of rutin and quercetin against alpha-
amylase and alpha-glucosidase [103]. G. bursa-pastoris is rich in polyphenols, such as
flavonoids (quercetin, kaempferol, catechins) and phenolic acids (chlorogenic acid, caffeic
acid) that have been reported to possess alpha-amylase and alpha-glucosidase inhibitory
effects [104]. Furthermore, polyunsaturated fatty acids have also been studied for their
potential effects on alpha-amylase and alpha-glucosidase inhibitory activities [105]. Some
studies have suggested that omega-3 fatty acids such as linolenic acid and omega-6 fatty
acids such as linoleic acid, present in G. bursa-pastoris, may have inhibitory effects on these
enzymes, potentially contributing to their anti-diabetic properties [106].
Molecular docking of rutin in the active sites of α-amylase showed that rutin formed
eight hydrogen bonds with amino acid residues TRP 59, THR 163, GLN 63, ASP 197, GLU
233, LYS 200, and HIS 201, while naringenin established in this active site of alpha-amylase
two hydrogen bonds with residues ASP 197 and GLN 63 and 2 Pi-Pi stacking bond with
amino residues TYR 62 and TRP 59. Furthermore, quercetin established in the active site of
alpha-glucosidase four hydrogen bonds with residues ASP 518, SER 676 ASP 616, and two
Pi-Pi staking bonds with residue PHE 649, whereas kaempferol established in this active
site of alpha-glucosidase three hydrogen bonds with residues ASP 518, ASP 616, and SER
676 and one Pi-Pi stacking with residue PHE 649.
4. Materials and Methods

4.1. Chemicals and Reagents
For the purposes of determining the total phenolic and flavonoid components, analytical-
grade chemicals and reagents were procured from Merck (Darmstadt, Germany) and
Carl Roth GmbH (Karlsruhe, Germany). N-hexane, ethyl acetate, and ethanol were ob-
tained from Merck (Darmstadt, Germany). The required acarbose, 3,5-Dinitrosalicylic
acid (DNSA), 1,1-Diphenyl-2-picrylhydrazyl (DPPH•), β-carotene, α-glucosidase, and α-
amylase were purchased from Merck (Sigma-Aldrich, St. Louis, MA, USA). Ascorbic acid,
kojic acid, gallic acid, apigenin, succinic acid, cholesterol, and tannic acid, which served as
phenolic standards, were obtained from Merck and Carl Roth GmbH (Karlsruhe, Germany).
4.2. Plant Material and Extraction

The red algae G. bursa-pastoris was harvested from the Nador lagoon (35◦ 080 26.900 N
2◦ 290 09.600
W) in northern Morocco in April 2021, as shown in Figure 6. The algae species
was classified by Dr. M. RAMDANI, who is affiliated with the Faculty of Sciences at
University Mohammed I in Oujda, Morocco.
with Porosity 4, Isolab, Wertheim, Germany). The extracts were then dehydrated using a
rotary evaporator (Laborota 4000, Heidolph Instruments, Schwabach, Germany). The ex-
traction yield was calculated using the following equation:
( )
Yield (%) = × 100
( )
Mar. Drugs 2023, 21, 372 16 of 25
The data were recorded as the median of three extraction replicates.
Mar. Drugs 2023, 21, 372 17 of 26
with Porosity 4, Isolab, Wertheim, Germany). The extracts were then dehydrated using a
rotary evaporator (Laborota 4000, Heidolph Instruments, Schwabach, Germany). The ex-
traction yield was calculated using the following equation:
( )
Yield (%) = × 100
( )
Figure6.6.Location
Figure Locationofofthe
theG.G.bursa-pastoris
bursa-pastorissampling
samplingsite
siteininthe
theNador
Nadorlagoon
lagoonininthe
theMediterranean
MediterraneanSea.
Sea.
The collected algae sample was brought to the laboratory for processing. G. bursa-
pastoris was thoroughly cleaned and repeatedly rinsed with distilled water before being
dried and left to be exposed to light for 48 h. The dried sample was then placed in an oven
at 35 ◦ C for 24 h (as shown in Figure 7). The seaweed was lyophilized and ground into a
powder for extraction purposes. The extraction process utilized maceration and Soxhlet
techniques, using four solvents: hexane, ethyl acetate, methanol, and distilled water. The
resulting extracts were placed in flasks and filtered using a glass filter crucible (50 mL, with
Porosity 4, Isolab, Wertheim, Germany). The extracts were then dehydrated using a rotary
evaporator (Laborota 4000, Heidolph Instruments, Schwabach, Germany). The extraction
yield was calculated using the following equation:
mass dried
Figure 6. Location of the G. bursa-pastoris sampling extract
site in (g) lagoon in the Mediterranean
the Nador
Sea. Yield (%) = × 100
mass dried matrix (g)
Figure 7. (a) G. bursa-pastoris, (b) powder of dried G. bursa-pastoris.
Figure
Figure7.7.(a)
(a)G.G.bursa-pastoris, (b)(b)
bursa-pastoris, powder of dried
powder G. bursa-pastoris.
of dried G. bursa-pastoris.
1, 372 Mar. Drugs 2023, 21, 372
18 of 26
17 of 25

4.2.1. Maceration Extraction
This extraction
4.2.1.method entails
Maceration immersing a solid substance in a cold liquid to draw
Extraction
out soluble compounds. This The extracts
extraction were
method prepared
entails by mixing
immersing 100 g ofinmacroalgae
a solid substance a cold liquidpow-
to draw
der with 200 mL of solvent (99% n-hexane (2 h), ethyl acetate (24 h), methanol (24 h),powder
out soluble compounds. The extracts were prepared by mixing 100 g of macroalgae and
with 200 mL of solvent (99% n-hexane (2 h), ethyl acetate (24 h), methanol (24 h), and
distilled water (24 h)) at ambient temperature, through stirring (as shown in Figure 8).
distilled water (24 h)) at ambient temperature, through stirring (as shown in Figure 8).
Figure 8. Extraction process

Figure of G. Bursa-pastoris
8. Extraction with various
process of G. Bursa-pastoris solvents.
with various solvents.
4.2.2. Soxhlet Extraction

4.2.2. Soxhlet Extraction
The Soxhlet extraction method requires the use of a condenser, a Soxhlet chamber, and
The Soxhletanextraction method
extraction flask. Then,requires the use
35 g of powder fromofmarine
a condenser,
macroalgaeaspecies
Soxhletwaschamber,
placed in an
extraction thimble and combined with 300 mL of the selected solvent
and an extraction flask. Then, 35 g of powder from marine macroalgae species was placed (hexane, ethyl acetate,
or methanol) in the extraction flask. The duration of the Soxhlet extraction process was
in an extraction thimble and combined with 300 mL of the selected solvent (hexane, ethyl
determined based on the time required for the descending solvent to become colorless.
acetate, or methanol) in the extraction flask. The duration of the Soxhlet extraction process
was determined 4.3.
basedPhytochemicals
on the time Compounds
required for the descending solvent to become color-
less. 4.3.1. Quantification of Total Phenolic Constituents
The total polyphenol content in G. bursa-pastoris extracts was determined using a
modified Folin–Ciocalteu method. Indeed, 200 µL of extract solution with a concentration
4.3. Phytochemicals Compounds
of 1 mg/mL was mixed with 1000 µL of Folin–Ciocalteu reagent solubilized in distilled
4.3.1. Quantification
water,ofand
Total Phenolic
finally, 800 µL ofConstituents
sodium carbonate was added (75 g/L); the resulting mixture
was incubated at room temperature for 1 h in the dark. Then, the absorbance was measured
The total polyphenol content in G. bursa-pastoris extracts was determined using a
at 765 nm using a spectrophotometer against ethanol as a control. The calibration curve is
modified Folin–Ciocalteu method.
created by varying theIndeed, 200 µL
concentration of extract
of gallic solution
acid (0–0.1 withAll
mg/mL). a concentration
experiments were
of 1 mg/mL wasperformed
mixed with 1000 µL
in triplicate of Folin–Ciocalteu
to take reagent solubilized
the mean of the experiments in distilled
with the standard deviation.
water, and finally, 800 µL of sodium carbonate was added. (75 g/L); the resulting mixtureper
The amount of total phenolic compounds was expressed in mg gallic acid equivalents
gram of dry extract (mg GAE/g) [107].
was incubated at room temperature for 1 h in the dark. Then, the absorbance was meas-
ured at 765 nm using a spectrophotometer against ethanol as a control. The calibration
curve is created by varying the concentration of gallic acid (0–0.1 mg/mL). All experiments
were performed in triplicate to take the mean of the experiments with the standard devi-
ation. The amount of total phenolic compounds was expressed in mg gallic acid equiva-
lents per gram of dry extract (mg GAE/g) [107].
Mar. Drugs 2023, 21, 372 18 of 25
4.3.2. Measurement of Total Flavonoids Contents

A modified version of the method described by Kim et al. [108] was used. A quantity
of 200 µL of each extract of G. bursa-pastoris, 1000 µL of distilled water and 50 µL of NaNO2
(5%) were combined. Following a 6 min incubation period, 120 µL of AlCl3 (10%) was
introduced, followed by a 5 min incubation period at room temperature in the dark. Then,
400 µL of 1 M NaOH and 230 µL of distilled water were mixed together. At 510 nm,
the absorbance was calculated. The calibration curve was created by using different
concentrations of quercetin solution (0–0.1 mg/mL) as a standard. All measurements were
determined three times to verify the reproducibility of the results.
4.4. Fatty Acid GC-MS Analysis of G. bursa-pastoris Extracts

In accordance with the protocol described in Loukili et al. [109] with some modi-
fications, methyl esters of hexane fatty acids and ethyl acetate were extracted from G.
bursa-pastoris. The characterization and identification of fatty acids were analyzed using the
Shimadzu GC system (Kyoto, Japan), which was equipped with a BPX25 capillary column
with a 5% diphenyl and 95% dimethylpolysiloxane phase (30 m × 0.25 mm × 0.25 µm)
and connected to a QP2010 MS. Helium gas (99.99%) was used as the mobile phase with a
flow rate of 3 L/min. The temperature of the injection, ion source, and interface was set
at 250 ◦ C, while the temperature program for the column oven was set to 50 ◦ C for 1 min
and then heated to 250 ◦ C at a rate of 10 ◦ C/min and maintained for an additional minute.
The ionization of the sample components was performed in Electron Ionization (EI) mode
at 70 eV, with a scanned mass range of 40 to 300 m/z. A 1 µL volume of each extract was
injected in split mode, and each sample was analyzed in triplicate. The compounds were
characterized by comparing their retention times, mass spectra fragmentation patterns, and
databases, including the National Institute of Standards and Technology’s database (NIST).
Data processing was carried out using LabSolutions (version 2.5, Shimadzu, Kyoto, Japan).
4.5. HPLC Analyses of G. bursa-pastoris Extracts

The phenolic compounds in the ethyl acetate and methanolic extracts were identified
using High-Performance Liquid Chromatography (HPLC) with an Agilent 1100 system
(Agilent Technologies, Palo Alto, CA, USA) connected to a diode array UV detector (Bruker,
Germany). Each extract (10 µL) was passed through a Zorbax XDB-C18 column (5 m,
250 × 4.6 mm) attached to the Agilent 1100 system, preceded by a 4 × 3 mm C18 cartridge
precolumn (Agilent Technologies). The elution gradient used was 0 to 5 min: 95% A and
5% B, 25 to 30 min: 65% A and 35% B, 35 to 40 min: 30% A and 70% B, and 40 to 45 min:
95% A and 5% B. The elution system comprised A (water/MeOH (9/1) + 0.1% phosphoric
acid) and B (methanol + 0.1% phosphoric acid), with a constant flow rate of 1 mL/min at a
temperature of 40 ◦ C. Spectrophotometric measurements were taken at wavelengths of 254,
280, 320, 370, and 510 nm. Compounds were identified by comparing their retention times
and UV spectra to those of standards.
4.6. Antioxidant Activity

4.6.1. Scavenging 2, 2-Diphenyl-1-Picrylhydrazyl Radical Test
The antioxidant activity of various extracts of G. bursa-pastoris was evaluated using
the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical bleaching technique, as described by
Brand-Williams et al. with slight modifications [110]. The initial concentration of extracts
and Ascorbic acid employed is 1 mg/mL. A mixture of 0.8 mL of samples or standard
(ascorbic acid) of varying concentrations (ranging from 0.02 to 0.32 mg/mL) and 2 mL of
DPPH• solution (2 mg of DPPH• in 200 mL MeOH) was prepared and shaken by hand.
After 30 min of incubation in the dark at ambient temperature, the absorbance of the
samples was measured using a UV/visible spectrophotometer at a wavelength of 517 nm,
with respect to the blank. Each determination was performed in triplicate.
Mar. Drugs 2023, 21, 372 19 of 25
The inhibitory activity of the DPPH radical by a sample was calculated using the
following equation:
( Ab − As )
Inhibition Percent = [ ] × 100
Ab
where Ab : Absorbance of the blank, As : Absorbance of sample (or positive control).

The graph plotting inhibition percentage against extract concentration was used to
calculate the IC50 .
4.6.2. β-Carotene Bleaching Assay

The antioxidant activity of G. bursa-pastoris extracts was determined using the method
described by Bekkouch et al. [111]. This evaluation was based on the extracts’ ability to
reduce oxidative damage to β-carotene in an emulsion using the carotene bleaching test. To
prepare the emulsion, 2 mg of β-carotene was dissolved in 10 mL of chloroform, along with
20 mg of linoleic acid and 200 mg of the emulsifier Tween 80. The chloroform was then
evaporated at 40 ◦ C using a vacuum evaporator, and 100 mL of distilled water was added
to the solution while stirring vigorously. 0.2 mL of the emulsion was placed in separate
test tubes, along with either the extract or a reference antioxidant solution (BHA) at a
concentration of 1 mg/mL. Absorbance was measured at 470 nm using a 96-well microplate
reader at t0, immediately after adding the emulsion and after 2 h. All measurements were
performed in triplicate. The inhibition of the linoleate/β-carotene radical was calculated
using the following formula:
initial(β − carotene)(t0) − (β − carotene) after 2 h

Bleaching inhibition (%) = 100 −
initial(β − carotene)(t0)
4.7. In Vitro α-Amylase Inhibition

The inhibitory effect of the different extracts of G. bursa-pastoris on α-amylase was
determined according to the method described by Daoudi et al. [84]. A reaction mixture
was prepared containing 0.2 mL of the sample or acarbose (used as a positive control at
varying 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). The mixture was pre-incubated at 37 ◦ C for
10 min before the addition of 0.2 mL of enzyme–substrate (1% starch dissolved in phosphate
buffer). The reaction was allowed to continue at 37 ◦ C for 30 min. The enzymatic reaction
was then stopped by adding 0.6 mL of DNSA reagent, followed by incubation at 100 ◦ C for
8 min and placement in a cold-water bath. The absorbance was measured at 540 nm.
The following formula was employed to calculate the inhibition percentage:
(OD control 540 nm − OD control 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 test was presented graphically using the function:
Inhibition percentage(%) = f(log (sample concentration))
4.8. In Vitro α-Glucosidase Inhibition Assay

The impact of G. bursa-pastoris extracts on intestinal α-glucosidase activity was de-
termined through a modified version of the protocol described by Hbika et al. [112]. 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 added to 10 mL of distilled water (as control), acarbose (as positive
control), or G. bursa-pastoris extract solution at a concentration of 2.2 g/mL. The mixture
was then incubated for 25 min at 37 ◦ C in a water bath. To inhibit the enzymatic reaction
and measure the release of glucose, the mixture was heated at 100 ◦ C for 5 min:
Mar. Drugs 2023, 21, 372 20 of 25
(OD control 540 nm − OD control 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. Molecular Docking Study

The ligands (compounds) identified via HPLC analysis in Gracilaria bursa-pastoris
were obtained from PUBCHEM.SDF format, then processed for docking using the LigPrep
tool in Schrödinger Software Maestro 11.5 using the OPLS3 force field. The ionization
states at pH 7.0 ± 2.0 were selected, resulting in a maximum of 32 stereoisomers for each
ligand. The protein preparation process involved downloading the 3D crystal structure of
α-amylase (PDB: 1B2Y) and α-glucosidase (PDB: 5NN8) in PDB format from the protein
data bank [113]. The structure was then refined using the Schrödinger-Maestro v11.5’s
Protein Preparation Wizard. This included assigning charges and bond orders, adding
hydrogens to heavy atoms, converting selenomethionines to methionines, and removing
all water molecules. Minimization was performed using the OPLS3 force field with a
maximum heavy atom RMSD limit of 0.30 Å [114].
The generation process is initiated by selecting a ligand atom, leading to the prepa-
ration of a default grid box. The grid box has a 20 × 20 × 20 volumetric spacing, with
its coordinates being x: 18.938, y: 5.742, and z: 46.879 for α-amylase and the following
coordinates x: −14.02, y: −38.177, and z: 95.206 for α-glucosidase. The ligand was then
linked to the protein-generated grid box through “Extra Precision” (XP). The outcome
was assessed based on the XP GScore. The Glide Standard Precision (SP) Ligand Docking
was performed using Schrödinger-Maestro v11.5. In this process, penalties were imposed
on non-cis/trans amide bonds, and the van der Waals scaling factor was set to 0.80, with
a partial charge cutoff of 0.15 for the ligand atoms. The final scoring was based on the
energy-minimized poses displayed as Glide scores. The ligand with the lowest Glide score
was recorded as the best-docked pose.
5. Conclusions
The abundance of G. bursa-pastoris in the Marchica Nador lagoon sparked our curiosity
about potential uses for these natural products. Upon conducting GC-MS analysis, we
discovered that both hexane and ethyl acetate extracts from G. bursa-pastoris contained a
high proportion of unsaturated fatty acids, with palmitic acid being the primary compound
detected in three extracts (HE (M), EAcE (M), and EAcE (S)) at levels ranging from 38% to
50%. The hexane extract obtained through maceration had the highest amount of eicosenoic
acid, while apigenin was the main compound in the ethyl acetate and methanolic extracts.
Although the aqueous extract exhibited moderate antioxidant activity using the DPPH
radical scavenging method, it was lower than that of ascorbic acid. However, these extracts,
containing numerous compounds, could be an alternative to synthetic additives, and it
is likely that once purified, they could exhibit comparable activity to ascorbic acid. The
methanolic extract was particularly noteworthy, displaying excellent antioxidant activity
in the β-carotene bleaching test, with an IC50 (0.062 ± 0.64 mg/mL) closely resembling
BHA when it was used as a reference. Moreover, our study found that G. bursa-pastoris
extracts exhibited a significant anti-diabetic effect in both in vitro and in silico assays,
suggesting the potential as a phytomedicine to regulate blood sugar levels and prevent
health complications in diabetic patients, as well as in healthy subjects.
Author Contributions: Conceptualization, S.O. and E.H.L.; Data curation, E.H.L., N.E.D., M.R.
(Mohamed Ramdani), M.-L.F. and A.A.G.; Methodology, N.E.D., M.C., M.R. (Mohamed Ramdani),
I.R., and M.B.; Supervision, I.R., M.B., B.H. and M.R. (Mohammed Ramdani); Validation, N.E.D.,
M.C., M.R. (Mohamed Ramdani), I.R., M.B., M.-L.F., B.H., L.R. and F.D.; Writing, original draft, S.O.,
E.H.L., M.C., L.R., A.A.G., F.D. and M.R. (Mohammed Ramdani); Writing, review and editing, S.O.,
E.H.L., M.-L.F., B.H., L.R., A.A.G., F.D. and M.R. (Mohammed Ramdani). All authors have read and
agreed to the published version of the manuscript.
Mar. Drugs 2023, 21, 372 21 of 25
Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors thank Faculty of Science Oujda of the Chemical Department for his
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
AgroBio Tech, University of Liege, Belgium.
Conflicts of Interest: The authors declare no conflict of interest.
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Mar. Drugs 2023, 21, 372. https://doi.org/10.3390/md21070372 https://www.mdpi.com/journal/marinedrugs

Table 1. Yields, phenols and flavonoids contents of different extracts of G. bursa-pastoris.

Extraction Polyphenols Flavonoids

2.2. Fatty Acid Analysis

RT HE (%) EAcE (%)

EAcE (%) ME (%)

Table 4. IC50 values of G. bursa-pastoris extracts.

2.4. Antioxidant Activity

Table 4. IC50 values of G. bursa-pastoris extracts.

2.5. In Vitro α-Amylase Inhibition

0.0 0.5 1.0 1.5 2.0 2.5

2.6. Molecular Modeling Studies

2.6. Molecular Modeling Studies

Mar. Drugs 2023, 21, 372 13 of 26

α-Amylase (PDB: 1B2Y) α-Gluosidase (PDB: 5NN8)

4. Materials and Methods

4.2. Plant Material and Extraction

The data were recorded as the median of three extraction replicates.

Mar. Drugs 2023, 21, 372 17 of 26

The data were recorded as the median of three extraction replicates.

Figure 7. (a) G. bursa-pastoris, (b) powder of dried G. bursa-pastoris.

The data were recorded as the median of three extraction replicates.

Figure 8. Extraction process

4.2.2. Soxhlet Extraction

4.3.2. Measurement of Total Flavonoids Contents

4.4. Fatty Acid GC-MS Analysis of G. bursa-pastoris Extracts

4.5. HPLC Analyses of G. bursa-pastoris Extracts

4.6. Antioxidant Activity

where Ab : Absorbance of the blank, As : Absorbance of sample (or positive control).

4.6.2. β-Carotene Bleaching Assay

initial(β − carotene)(t0) − (β − carotene) after 2 h

4.7. In Vitro α-Amylase Inhibition

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

4.8. In Vitro α-Glucosidase Inhibition Assay

4.9. Molecular Docking Study

Funding: This research received no external funding.

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