Food and Chemical Toxicology 154 (2021) 112348

Contents lists available at ScienceDirect

Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

A comprehensive phytochemical, biological, toxicological and molecular

docking evaluation of Suaeda fruticosa (L.) Forssk.: An edible halophyte
medicinal plant
Hammad Saleem a, b, *, Umair Khurshid c, Muhammad Sarfraz d, Muhammad Imran Tousif e,
Abdulwahab Alamri f, Sirajudheen Anwar f, Abdulhakeem Alamri g, Irshad Ahmad h,
Hassan H. Abdallah i, Fawzi M. Mahomoodally j, Nafees Ahemad b
a
Institute of Pharmaceutical Sciences (IPS), University of Veterinary & Animal Sciences (UVAS), Lahore, Pakistan
b
School of Pharmacy, Monash University, Jalan Lagoon Selatan, 47500 Bandar Sunway Selangor Darul Ehsan, Malaysia
c
Bahawalpur College of Pharmacy, Bahawalpur Medical and Dental College, Bahawalpur, Pakistan
d
College of Pharmacy, Al Ain University, Al Ain, United Arab Emirates
e
Department of Chemistry, Township Campus, University of Education, Lahore, Pakistan
f
Department of Pharmacology & Toxicology, College of Pharmacy, University of Hail, Saudi Arabia
g
Department of Clinical Laboratory Sciences, College of Applied Medical Science, Taif University, P. O. Box 11099, Taif, 21944, Saudi Arabia
h
Department of Pharmacy, The Islamia University of Bahawalpur, Pakistan
i
Chemistry Department, College of Education, Salahaddin University, Erbil, Iraq
j
Department of Health Sciences, Faculty of Medicine and Health Sciences, University of Mauritius, Mauritius

A R T I C L E I N F O A B S T R A C T

Handling Editor: Dr. Jose Luis Domingo Suaeda fruticosa is an edible medicinal halophyte known for its traditional uses. In this study, methanol and
dichloromethane extracts of S. fruticosa were explored for phytochemical, biological and toxicological parame­
Keywords: ters. Total phenolic and flavonoid constituents were determined by using standard aluminum chloride and Folin-
Suaeda fruticose Ciocalteu methods, and UHPLC-MS analysis of methanol extract was performed for tentative identification of
Phytochemicals
secondary metabolites. Different standard methods like DPPH, ABTS, FRAP, CUPRAC, total antioxidant capacity
Antioxidant
(TAC), and metal chelation assays were utilized to find out the antioxidant potential of extracts. Enzyme inhi­
Enzyme inhibition
Cytotoxicity bition studies of extracts against acetylcholinesterase, butyrylcholinesterase, tyrosinase, α-amylase and,
Docking α-glucosidase enzymes were also studied. Likewise, the cytotoxicity was also assessed against MCF-7, MDA-MB-
231, and DU-145 cell lines. The higher phenolic and flavonoids contents were observed in methanol extracts
which can be correlated to its higher radical scavenging potential. Similarly, 11 different secondary metabolites
were tentatively identified by UHPLC profiling. Both the extract showed significant inhibition against all the
enzymes except for α-glucosidase. Moreover, docking studies were also performed against the tested enzymes. In
the case of cytotoxicity, both the samples were found moderately toxic against the tested cell lines. This plant can
be explored further for its potential therapeutic and edible uses.

1. Introduction For example, an edible halophyte plant named Mesembryanthemum edule

Halophytes have the greater ability to survive and bear toxic reactive
oxygen species (ROS) due to a very strong antioxidant systems,
including enzymatic and non-enzymatic components (Ksouri et al.,
2009). The literature data indicates that a number of medicinal halo­
phytes have been reported to possess important bioactive compounds,
including polyphenols which are helpful for different remedial purposes.
is a traditional remedy for bacterial and fungal infections and also used
as a treatment for diarrhoea, sinusitis, infantile eczema, and tuberculosis
(van der Watt and Pretorius, 2001), likewise Tamarix gallica is utilized as
a detergent, diuretic, expectorant, laxative, and astringent (Ksouri et al.,
2009).
S. Fruticosa is also known as shrubby seablite, belongs to the genus
Suaeda of the family Amaranthaceae. This plant is widely distributed

* Corresponding author. Institute of Pharmaceutical Sciences (IPS), University of Veterinary & Animal Sciences (UVAS), Lahore, Pakistan.
E-mail address: hammad.saleem@uvas.edu.pk (H. Saleem).

https://doi.org/10.1016/j.fct.2021.112348
Received 23 May 2021; Received in revised form 7 June 2021; Accepted 12 June 2021
Available online 16 June 2021
0278-6915/© 2021 Elsevier Ltd. All rights reserved.
H. Saleem et al.
over the coasts of northern Africa, the Mediterranean region, Atlantic
coasts of southern Spain, Portugal, France, south-eastern England, Iran,
Afghanistan, and the Indian sub-continent (Jalas and Suominen, 1988).
S. fruticosa is a medicinal and edible halophyte that can be grown under
saline conditions (Ksouri et al., 2009). It commonly grows in flooded
alluvial areas, drier areas, salt marshes, salt flats, and coastal areas. It is
also found over the saline regions in southern Africa (Goodall et al.,
2009). The seeds and young shoots of this plant have been reported to be
consumed (cooked or raw) by humans (Wickens et al., 2012). The seeds
contain a high-quality edible oil that is abundant in unsaturated fatty
acids (Weber et al., 2007). The plant can be utilized to reduce soil
salinity and to remediate hazardous metal-contaminated soils (Hameed
et al., 2012). The plants’ high salt content will most likely limit their
usage as stand-alone fodder crops; instead, they will most likely be
employed as components of a feed mix (Ahmad and Malik, 2013).
The different parts of this plant have hypolipidaemic, hypoglycemic,
and antiophthalmic effects (Bennani-Kabchi et al., 1999). S. fruticosa
cultivation may help in bioremediation and reclamation of the soil that
is contaminated with salinity and toxic metal (Bareen and Tahira, 2011).
S fruticosa contains a large number of compounds like chromo alkaloids
which contain betacyanins and betaxanthine, collectively known as
betalains (Naija et al., 2014). This plant also contains tannins, saponins,
terpenoids, coumarins, fatty acids like palmitic acid, oleic acid, linolenic
acid, and polyphenols (Ashraf et al., 2016). S. fruticosa can be used for
wound healing. Its leaves are used to treat ophthalmia. This plant also
acts as a diuretic and laxative. It is also used for various other disorders
such as pain, fever, skin, respiratory, toothache, digestive, and genitor
urinary disorders (Qasim et al., 2011).
Taking into account the medicinal importance of this halophyte
edible plant, the present work was designed to evaluate the detailed
phytochemical, biological, and toxicological potential of methanol of
dichloromethane (DCM) extracts of S. fruticosa whole plant. The
phytochemical composition was ascertained by determining total
phenolic and total flavonoid contents, and ultra-high performance liquid
chromatography mass-spectrometry (UHPLC-MS) secondary metabolite
analysis. For the biological evaluation, antioxidant and enzyme inhibi­
tion assays were performed. Antioxidant potential was evaluated via
free radical scavenging (2,2-diphenyl-1-picrylhydrazyl -DPPH and 2,2′ -
azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) -ABTS), reducing
power (ferric reducing antioxidant power -FRAP and cupric reducing
antioxidant capacity -CUPRAC), total antioxidant power (phosphomo­
lybdenum), and metal chelation assays. Similarly, the enzyme inhibition
studies against the clinically important enzymes involved in the com­
mon pathologies, including neurological disorders (acetylcholinesterase
-AChE and butyrylcholinesterase -BChE), diabetes (α-amylase and
α-glucosidase), and skin problems (tyrosinase) were performed. The
cytotoxicity of both the extracts was done using MTT assay against the
MCF-7, MDA-MB-231 breast cancer cell lines, and DU-145 prostate cell
line. In addition, in order to highlight the interaction and mechanism of
enzyme inhibition, the major phytochemicals as tentatively identified in
the methanol extract were further studies for in-silico molecular docking.
2. Material and methods
2.1. Plant collection and extraction
The whole plant of S. fruticosa was collected from the peripheral
areas of Bahawalpur, Pakistan. The plant collected was dried under
shade for few days and grounded to powder form. The dried powdered
plant material was extracted using the maceration technique with DCM
and methanol solvents (for 72 h with each solvent). A rotary evaporator
was used to concentrate these resulting extracts.
2
Food and Chemical Toxicology 154 (2021) 112348
2.2. Phytochemical composition
2.2.1. Total phenolic and flavonoid contents
Total phenolic (TPC) and total flavonoid (TFC) contents were
determined via previously described standard Folin-Ciocalteu and
aluminum chloride protocols, respectively (Yadav and Agarwala, 2011;
Zengin et al., 2016). The results of TPC were noted as mg GAE/g of
extract (milligrams of gallic acid equivalent), while the findings of TFC
were recorded as mg QE/g extract (milligrams of quercetin equivalent).
2.2.2. UHPLC-MS analysis
Secondary metabolites of methanol extract were determined UHPLC-
MS analysis as describe previously (Saleem et al., 2019). The details of
UHPLC-MS instrumentation are provided in the supplementary material
section.
2.3. Biological activities
2.3.1. Antioxidant activities
Antioxidant properties of S. fruticosa extracts were estimated by
using previously described standard protocols, including free radical
scavenging (DPPH and ABTS), reducing antioxidant power (FRAP and
CUPRAC), total antioxidant capacity (phosphomolybdenum), and metal
chelating power assays (Grochowski et al., 2017). The results of all the
antioxidant assays were reported as mg TE/g extract, except for the
metal chelation assay for which the results were presented as mg
EDTAE/g extract. The detailed methodology of antioxidant assays is
provided in the supplementary material section.
2.3.2. Enzyme inhibition studies
The studied plant extracts were tested against different enzymes,
including acetylcholinesterase (AChE), butyrylcholinesterase (BChE),
tyrosinase, α-glucosidase, and α-amylase by using in vitro standard
methods as detailed earlier (Abirami et al., 2014; Mollica et al., 2017).
The standard used for AChE and BChE was galantamine, and their
inhibitory activity was measured as mg GALAE/g extract (milligrams of
galantamine equivalent per gram of extract). Likewise, acarbose was
used as a standard for α-glucosidase and α-amylase inhibition, and the
results were expressed in millimoles of acarbose equivalent per gram of
extract (mmol ACAE/g extract). For tyrosinase inhibition, kojic acid was
employed as standard, and results were recorded as milligrams of kojic
acid equivalents per gram of extract (mg KAE/g extract). The detailed
methodology of enzyme assays is provided in the supplementary mate­
rial section.
2.3.3. Cytotoxicity assay
The cytotoxicity activity of the tested samples was tested against two
breast cancer cell lines, i.e., MDA-MB 231 and MCF-7 cells employing
the previously described method, with slight modifications (Nem­
udzivhadi and Masoko, 2014). The cell viability percentage (%) after 72
h at the concentration of 200 μg/mL was determined as follows:
Percentage cell viability = ABSs – ABSc × 100
Where ABSs: absorbance of the sample; ABSc: absorbance of control.
2.3.4. In-Silico studies/Docking calculations
Three dominant compounds were selected for docking studies,
namely, 3,4-dihydroxybenzoic acid, 3-O-acetylhamayne, and uplandi­
cine. The starting structures of the three compounds were found online
from the zinc database (Irwin et al., 2012). AM1 semi-emperical
method, which is implemented in gaussian09 software, was used to
optimize the 3D structure to the ground state energy. The optimized
structures were docked at the active site of five enzymes, namely, AChE,
BChE, tyrosinase, α-amylase, and α-glucosidase enzymes. The co­
ordinates of these enzymes were extracted from the crystal structures of
H. Saleem et al.
these enzymes in Protein Databank RCSB PDB. PDB structure with the
code: 4EY6 was used as the AChE receptor in which the enzyme was
crystallized in complex with galantamine. PDB code:1P0P is represent­
ing the crystal structure of the BChE in complex with the inhibitor
butyrylthiocholine at the active site of the enzyme. Similarly, the PDB
code: 5I38 was used as the crystal structure of the tyrosinase enzyme as a
complex with kojic acid. PDB codes 7TAA and 3W37 were used to
represent the enzymes α-amylase and α-glucosidase, respectively. Both
enzymes were crystallized as a complex with the acarbose inhibitor at
the active site. Prior to docking, inhibitors, co-crystallized molecules,
and all water molecules were removed from the retrieved structures.
Autodock4 software (Molinspiration Database) was used to add polar
hydrogens, neutralize the protein structures using Kollman united atom
charges and to perform the docking calculations. Docking the three in­
hibitors was performed in a 60 × 60 x 60 grid box with 0.375 Å distance
between points using Lamarckian genetic algorithm (LGA) for the pre­
diction of binding free energies. Two hundred fifty conformations were
produced for each enzyme-inhibitor complex and the control ligand. The
docked conformations were ranked into clusters based on the binding
free energy (ΔG). Discovery studio 5.0 visualizer was used to visualize to
docked inhibitors at the active site and identify the intermolecular in­
teractions with the active site.
2.4. Statistical analysis
All experiments were done in triplicates, and their mean values were
calculated. The results obtained were expressed as mean values with
their standard deviation (SD). For the analysis of data, SPSS software
was used. One-way analysis of variance (ANOVA) and Tukey’s test were
employed to analyze the results. A statistically significant value of p <
0.05 was obtained.
3. Results and discussion
3.1. Total phenolic and flavonoid contents
Plants are responsible for the production of a large number of organic
compounds called primary and secondary metabolites (Zengin et al.,
2019). Both primary and secondary metabolites are of great importance,
as the primary metabolites are directly involved in respiration, photo­
synthesis, development, and growth. In contrast, the secondary metab­
olites play an important role in defensive mechanism against herbivores
in plants, provide protection against UV light, also used as signal mol­
ecules, dyes, fibers, glues, oils, waxes, drugs, perfumes, flavouring
agents, insecticide, herbicides, and antibiotics (Crozier et al., 2008). In
the current research work, the methanol and DCM extracts of S. fruticosa
were tested for the determination of total phenolic and total flavonoid
contents, and the results are presented in Table 1. The higher amount of
phenolic (11.09 mg GAE/g extract) and flavonoid (20.50 mg QE/g
extract) contents were observed in the methanol extracts, while low
phenolic (9.25 mg GAE/g extract) and flavonoid (4.98 mg QE/g extract)
contents were observed in the case of DCM extracts (Table 1). A similar
type of results was reported in a previous study in which the higher
flavonoid contents were noted in the methanol extract (29.8 mg CE/g) of
Reaumuria vermiculata as compared to DCM extracts (25.12 mg CE/g)
(Karker et al., 2016).
Table 1
Total phenolic and flavonoid contents in Suaeda fruticosa extracts.
Extracts Total phenolic content (mg GAE/g) Total flavonoid content (mg QE/g)
SF-M 11.09 ± 0.08 20.50 ± 0.68
SF-D 9.25 ± 0.07 4.98 ± 0.16
SF-M: Suaeda fruticosa methanol extract; SF-D: Suaeda fruticosa DCM extract.
Data from three repetitions, with mean ± standard deviation; GAE: gallic acid
equivalent; QE: quercetin equivalent.
3
Food and Chemical Toxicology 154 (2021) 112348
3.2. UHPLC-MS phytochemical analysis
Ultra-high-pressure liquid chromatography (UHPLC) is a system
working at ultra-high pressure and has decreased the time of analysis for
different compounds. This technique is widely used for profiling sec­
ondary metabolites (Grata et al., 2009). In the present study, for the
tentative identification of different phytocompounds in methanol
extract of S. fruticose, negative ionization mode of UHPLC-MS analysis
was employed. The total ion chromatogram (TIC) with different peaks of
the tentatively identified secondary metabolites as in methanol extract is
represented in Fig. 1. A total of 11 different secondary metabolites
belonging to different classes of phytochemicals were tentatively iden­
tified, and the list of these compounds is given in Table 2. Two phenolic
compounds were found, namely 3, 4-dihydrobenzoic acid, and gingerol.
Three compounds, namely pitheduloside B, 5, 8, 12-trihydroxy-9-
octadecenoic acid, and kamahine C were also tentatively identified as
saponin, fatty acid, and spiroketal, respectively. Similarly, D-threonic
acid (carbohydrate) and allantoin (urea derivative) were also identified.
Likewise, two alkaloidal compounds, i.e., uplandicine and 3-O-acetylha­
mayne were also tentatively identified.
3.3. Antioxidant activities
Reactive oxygen species (ROS) are radicals with unpaired electrons
that can damage other biomolecules. They are also known as pro-
oxidants. ROS are responsible for causing damage to biomolecules
including as proteins, lipids, carbohydrates, fatty acids, and nucleic
acids. They are also capable of causing DNA damage, which is the major
cause of mutations (Halliwell, 1990) and may also cause different
common diseases (Alho and Leinonen, 1999; Duh, 1998). Antioxidants
are becoming increasingly important because they block the oxidation
of other molecules, hence preventing the formation of free radicals
(Halliwell, 1995).
In the present work, the antioxidant potential of methanol and DCM
extracts S. fruticose were determined by using different six different
antioxidant assays, including free radical scavenging (DPPH and ABTS),
reducing power (FRAP and CUPRAC), total antioxidant capacity
(phosphomolybdenum), and metal chelation assays and the results are
assembled in Table 3. The higher radical scavenging antioxidant values
were observed in the case of methanol extract (DPPH: 9.06 mg TE/g
extract; ABTS:16.46 mg TE/g extract). Similarly, the methanol extract
was also found most active for the CUPRAC assay, while the DCM extract
showed the highest FRAP value. Likewise, the phosphomolybdenum
assay was carried out to find out the total antioxidant capacity, and the
higher value of antioxidant capacity was exhibited by the DCM extract
(0.55 mg TE/g extract) as compared to methanol extract (0.24 mg TE/g
extract). Furthermore, the DCM extract also presented superior metal
chelating power with a value of 18.15 mg EDTAE/g extract.
3.4. Enzyme inhibition studies
Alzheimer’s disease (AD) falls in the category of neurodegenerative
disease, which is the main reason of gradual and irreversible damage to
the brain (Parihar and Hemnani, 2004). AD is linked to aging and is
accountable for damaging different parts of brain. It affects the cholin­
ergic neurons, and as result neurotransmitters transmission is stopped
(Querfurth and LaFerla, 2010; Ritter, 2012). AChE is in charge of con­
verting acetylcholine into choline and acetic acid. As a result, the
enzyme AChE plays an important role in the development of AD. By
limiting acetylcholine breakdown, inhibitors stimulate neurotransmis­
sion (García-Ayllón et al., 2011). Butyrylcholinesterase is the second
member of the cholinesterase family, but its functions have not been
thoroughly investigated, and is found to be associated with the AD
(Karlsson, 2013). In the present study, the methanol and DCM extracts of
S. fruticosa were tested for the AChE and BChE inhibition, and as pre­
sented in Table 4, a considerable inhibition potential was observed for
H. Saleem et al. Food and Chemical Toxicology 154 (2021) 112348

Fig. 1. Total ion chromatogram (TIC) of Suaeda fruticosa methanol extract.

Table 2
UHPLC-MS of S. fruticosa methanol extract.
S.no RT (min) B. peak (m/z) Tentative compound identification Comp. class MFG formula Mol. mass

1 2.485 332.92 Costatol Alcohol derivative C10H14BrCl3O 333.92

2 2.729 135.03 D-threonic acid Carbohydrate C4H8O5 136.03
3 2.799 157.04 Allantoin Urea derivative C4H6N4O3 158.04
4 12.27 153.02 3,4-Dihydroxybenzoic acid Phenol C7H6O4 154.02
5 12.783 328.12 3-O-Acetylhamayne Alkaloid C18H19NO5 329.12
6 15.153 329.24 5,8,12-trihydroxy-9-octadecenoic acid Fatty acid C18H34O5 330.24
7 15.97 881.49 Pitheduloside B Saponin C46H74O16 882.49
8 16.67 267.13 Kamahine C Spiroketal C14H20O5 268.13
9 17.711 293.18 Gingerol Phenol C17H26O4 294.18
10 17.713 356.17 Uplandicine Alkaloid C17H27NO7 357.17
11 21.065 233.16 Curcumenol Guaianes C15H22O2 234.16

RT: retention time; B. peak: base peak.

Table 3
Antioxidant properties of Suaeda fruticosa extracts.
Extracts Radical Scavenging activity Reducing power Total antioxidant capacity (TAC) Ferrous chelating

DPPH (mgTE/g ABTS (mgTE/g FRAP (mgTE/g CUPRAC (mgTE/g Phosphomolybdenum (mgTE/g Metal Chelating
extract) extract) extract)) extract) extract) (mgEDTA/g)

SF-M 9.06 ± 0.63 16.46 ± 1.59 25.01 ± 0.49 48.89 ± 1.63 0.24 ± 0.02 1.77 ± 0.19
SF-D 5.69 ± 0.15 10.52 ± 0.20 23.84 ± 0.77 52.52 ± 1.23 0.55 ± 0.09 18.15 ± 1.18

TE: trolox equivalent; EDTAE: EDTA equivalent. Values expressed are means ± S.D. of three parallel measurements.

in the treatment of hyperpigmentation in humans, such as nevus, len­

Table 4 tigo, and post-inflammatory phases (Parvez et al., 2006). The methanol
Enzyme inhibition effects of Suaeda fruticosa extracts.
and DCM extracts of S. fruticosa were screened for tyrosinase inhibition,
Extracts AChE BChE Tyrosinase Glucosidase Amylase and the results are presented in Table 4. Both the extracts revealed
inhibition inhibition (mg KAE/g (mmol (mmol
comparatively same levels of inhibition with values of 125.38 mg KAE/g
(mg (mg extract) ACAE/g ACAE/g
GALAE/g GALAE/g extract) extract) extract for methanol extract and 123.12 mg KAE/g extract for the DCM
extract) extract) extract.
SF-M 4.70 ± 4.64 ± 125.38 ± 0.54 ± 0.02 1.94 ±
Glucosidases (including α-glucosidase and α-amylase enzymes) are
0.22 0.26 1.25 0.01 found in the gastrointestinal (GI) tract, where these enzymes are
SF-D 4.23 ± 4.73 ± 123.12 ± 0.61 ± 0.02 1.96 ± involved in catalyzing the final step of the digestion of carbohydrates.
0.05 0.09 0.63 0.01 Thus, in order to develop the compounds having inhibition potential
GALAE: galatamine equivalent; KAE: kojic acid equivalent; ACAE: acarbose against these enzymes is a promising approach (Mollica et al., 2019).
equivalent; all values expressed are means ± S.D. of three parallel Higher sugar levels in the blood can develop to diabetes, which is the
measurements. major cause of kidney, eye, and cardiovascular diseases. As a result, in
order to cure diabetes, blood sugar levels must be reduced. The search
both the methanol (AChE: 4.70 mg GALAE/g extract; BChE: 4.64 ± 0.26 for α-amylase and α-glucosidase inhibitors is of great significance in the
mg GALAE/g) and DCM (AChE: 4.23 mg GALAE/g extract; BChE: 4.73 treatment of diabetes (Bhandurge et al., 2010). In this research work, the
mg GALAE/g extract) extracts. methanol and DCM extracts of S. fruticosa were examined for α-gluco­
Tyrosinase is a copper-containing enzyme that is important for sidase, and α-amylase inhibition, and the results are depicted in Table 4.
catalyzing oxidation during the early stages of melanin production For α-glucosidase inhibition, a stronger level of inhibition potential was
(Parvez et al., 2006). Because of the prevalence of skin problems, the observed in DCM extract (0.61 mmol ACAE/g extract) as compared to
quest for tyrosinase inhibitors is critical. Tyrosinase inhibitors are useful methanol extract (0.54 mmol ACAE/g extract). Likewise, for the

4
H. Saleem et al. Food and Chemical Toxicology 154 (2021) 112348

α-amylase inhibition activity, almost similar values of inhibition were Table 5

noted for both the methanol (1.94 mmol ACAE/g extract) and DCM Cytotoxicity of Suaeda fruticosa methanol and DCM extracts against tested cell
(1.96 mmol ACAE/g extract) extracts. lines.
Samples % Viability (200 μg/mL)
3.5. Cytotoxicity MCF-7 MDA-MB-231 DU-145

SF-M 63.44 77.75 62.83

The cytotoxicity of both the methanol and DCM extracts of
SF-D 45.01
S. fruticosa was also evaluated against MCF-7 and MDA-MB-231 breast
cancer cell lines, and the results of the toxicity assay are presented in SF-M: Suaeda fruticosa methanol extract; SF-D: Suaeda fruticosa DCM extract;
Table 5. From the results, it is clear that all the tested extracts presented all values expressed are means ± S.D. of three parallel measurements.
low to moderate toxicity against the tested cell lines. The methanol
extract was noted to be the most cytotoxic against the tested MCF-7,
Table 6
MDA-MB-231, and DU-145 cell lines with percentage viability of Binding energy (kcal/mol), Inhibition constant Ki, interaction sites of the studied
63.44, 77.75, and 62.83%, respectively. Likewise, the DCM extract was compounds with AChE, BChE, tyrosinase, α-amylase and α-glucosidase enzymes.
noted to be most active for the MDA-MB-231 cell line. This is just a
Binding energy/
preliminary toxicity testing of the studied plant extract; the detailed in- inhibition constant Ki
vivo toxicity studies are recommended.
Uplandicine
AChE − 5.58 (81.63 μM)
3.6. Docking results
Currently, docking is one of the efficient and reliable tools to predict BChE − 4.63 (405.60 μM)
binding free energy and to correlate the experimental biological activity.
Tyrosinase − 2.80 (8.94 mM)
In this study, three selected compounds as the dominant compounds α-amylase − 2.88 (7.75 mM)
were docked against AChE, BChE, tyrosinase, α-amylase, and α-gluco­
sidase enzymes. The active site of these enzymes was detected and α-glucosidase − 4.34 (654.70 μM)
confirmed by docking the control ligands. The docking results are shown
3,4-Dihydroxybenzoic acid
in Table 6 and Fig. 2. In Table 6, the calculated binding affinities of the
AChE − 4.54 (470.14 μM)
studied compounds against the five enzymes are listed with the esti­
mated inhibition constants. In addition, the residual amino acids BChE − 4.38 (616.55 μM)
involved in the interactions with the inhibitor at the active site are
shown in Table 6. Uplandicine has shown the highest binding energy Tyrosinase − 4.36 (635.81 μM)
with the AChE enzyme (− 5.58 kcal/mol) and the lowest inhibition α-amylase − 4.41 (585.94 μM)
constant (81.63 μM) in comparison with the rest of the studied enzymes.
This inhibition is mainly attributed to the high number of Hydrogen α-glucosidase − 4.93 (242.02 μM)
bonds formed with Ser 125, Asn 87, Tyr 337, and Gly 122 at the active 3-O-Acetylhamayne
AChE − 9.38 (133.91 nM)
site of the enzyme, as shown in Fig. 2. In contrast, 3,4-dihydroxybenzoic
acid has shown similar affinity with the five enzymes as shown in
Table 6; however, the highest binding free energy is with α-glucosidase BChE − 8.84 (329.84 nM)
enzyme (− 4.93 kcal/mol) and inhibition constant (242.02 μM). Tyrosinase − 7.13 (5.96 μM)
Hydrogen bonds and pi-pi interactions are the dominant interactions
between this inhibitor and the active site of the studied enzymes. Similar α-amylase − 6.37 (21.52 μM)
to uplandicine, 3-O-acetylhamayne has shown higher binding energy
with AChE enzyme with binding free energy equal to (− 9.38 kcal/mol) α-glucosidase − 6.36 (21.87 μM)
and inhibition constant (133.91 nM). The interactions with the active
site involve hydrogen bonds with Tyr 124 and Tyr 133 residues. (HB)* = Hydrogen bond.
67.22 25.88
Interaction site
Ser 125(HB), Asn 87(HB), Tyr 337(HB),
Asp 74, Tyr 124, Phe 297, Phe 338, Gly
122(HB), Ser 203
Tyr 332, Pro 85(HB), Gly 117(HB), His
438, Trp 82
His 208, Val 218, Met 61, His 204, His 60
Trp 83(HB), Arg 344(HB), Tyr 82, Leu 166,
His 122, Asp206
Ala 234, Trp 329, Phe 601, Ala 602, Asp
568(HB), Arg 552(HB) Asp 469(HB)
His 447(HB), Tyr 337, Trp 86, Glu 202
(HB)
Thr 120(HB), Trp 82(HB), Tyr 128(HB),
Glu 197(HB)
His 208, His 69(HB), His 231(HB), Ala
221, Val 218
Asp 340(HB), Asp 206(HB), Arg 204(HB),
Tyr 82
Asp 357(HB), Asp 469, Asp 568(HB)
Tyr 124(HB), Tyr 341, Tyr 337, His 447,
Glu 202, Trp 86, Gly 120, Leu 130, Tyr 133
(HB)
Thr 122(HB), His 438, Ala 328, Trp 82
Asn 205 (HB), Phe 197, Arg 209, Gly 216,
Val 217, Val 218, His 208, His 60, His 204,
Ala 221
Asp 206(HB), Leu 173, Leu 166, Glu 230,
Leu 232, Asp 297
Asp 568(HB), Trp 432, Phe 476, Trp 329,
Phe 601

4. Conclusion Funding

In this study, the phytochemical, biological, and toxicological pa­
rameters of methanol and DCM extracts S. fruticosa were explored. The
methanol extract contains a considerable amount of total phenolic and
flavonoid contents and also presented considerable antioxidant poten­
tial. All the extracts presented strong to moderate enzyme inhibition
potential against the tested enzymes. The UHPLC-MS secondary
metabolite profiling of methanol extract revealed the tentative identi­
fication of several important secondary metabolites. Both the tested
extracts were found to be moderately cytotoxic against breast cancer cell
lines and prostate cancer cells. Theoretically, docking study has
confirmed the inhibition ability of the three selected compounds with
the AChE, BChE, tyrosinase, α-amylase, and α-glucosidase enzymes and
their interactions with the active site are elucidated. Further in­
vestigations can be done for the isolation of new potentially bioactive
compounds.
This research work is partly supported by the project number
(TURSP-2020/288), Taif University, Taif, Saudi Arabia.
CRediT authorship contribution statement
Hammad Saleem: Conceptualization, Methodology, Formal anal­
ysis, Investigation, Writing – original draft. Umair Khurshid: Writing –
original draft. Muhammad Sarfraz: Writing – review & editing.
Muhammad Imran Tousif: Writing – original draft. Abdulwahab
Alamri: Funding acquisition. Sirajudheen Anwar: Funding acquisition.
Abdulhakeem Alamri: Funding acquisition. Irshad Ahmad: Funding
acquisition. Hassan H. Abdallah: Formal analysis, Investigation. Fawzi
M. Mahomoodally: Formal analysis. Nafees Ahemad: Conceptualiza­
tion, Supervision, Funding acquisition.

5
H. Saleem et al.
Fig. 2. The docked compounds at the active site of the enzyme and their interactions.
Declaration of competing interest
I Hammad Saleem is submitting a manuscript entitled for possible
publication in Food and Chemical Toxicology. It is stated that there is no
Conflict of Interest for the submitted paper.
References
Abirami, A., Nagarani, G., Siddhuraju, P., 2014. In vitro antioxidant, anti-diabetic,
cholinesterase and tyrosinase inhibitory potential of fresh juice from Citrus hystrix
and C. maxima fruits. Food Science and Human Wellness 3, 16–25.
Ahmad, R., Malik, K.A., 2013. Prospects for Saline Agriculture. Springer Science &
Business Media.
Alho, H., Leinonen, J., 1999. Total antioxidant activity measured by chemiluminescence
methods. Methods in Enzymology. Elsevier, pp. 3–15.
Ashraf, M.T., Fang, C., Bochenski, T., Cybulska, I., Alassali, A., Sowunmi, A.,
Farzanah, R., Brudecki, G.P., Chaturvedi, T., Haris, S., 2016. Estimation of bioenergy
potential for local biomass in the United Arab Emirates. Emir. J. Food Agric. 99–106.
Bareen, F.-e., Tahira, S.A., 2011. Metal accumulation potential of wild plants in tannery
effluent contaminated soil of Kasur, Pakistan: field trials for toxic metal cleanup
using Suaeda fruticosa. J. Hazard Mater. 186, 443–450.
Bennani-Kabchi, N., Kehel, L., Fdhil, H., Marquie, G., 1999. Effect of Suaeda fruticosa
aqueous extract in the hypercholesterolaemic and insulin-resistant sand rat.
Therapie 54, 725–730.
Bhandurge, P., Rajarajeshwari, N., Alagawadi, K., Agrawal, S., 2010. Antidiabetic and
hyperlipaemic effects of Citrus Maxima Linn fruits on alloxan-induced diabetic rats.
Int. J. Drug Dev. Res. 2, 273–278.
Crozier, A., Clifford, M.N., Ashihara, H., 2008. Plant Secondary Metabolites: Occurrence,
Structure and Role in the Human Diet. John Wiley & Sons.
Duh, P.-D., 1998. Antioxidant activity of burdock (Arctium lappa Linne): its scavenging
effect on free-radical and active oxygen. J. Am. Oil Chem. Soc. 75, 455–461.
García-Ayllón, M.-S., Small, D.H., Avila, J., Sáez-Valero, J., 2011. Revisiting the role of
acetylcholinesterase in Alzheimer’s disease: cross-talk with P-tau and β-amyloid.
Front. Mol. Neurosci. 4.
Goodall, D.W., Perry, R.A., Howes, K.M.W., 2009. Arid Land Ecosystems: Volume 2,
Structure, Functioning and Management. Cambridge University Press.
Grata, E., Guillarme, D., Glauser, G., Boccard, J., Carrupt, P.-A., Veuthey, J.-L., Rudaz, S.,
Wolfender, J.-L., 2009. Metabolite profiling of plant extracts by ultra-high-pressure
liquid chromatography at elevated temperature coupled to time-of-flight mass
spectrometry. J. Chromatogr. A 1216, 5660–5668.
Grochowski, D.M., Uysal, S., Aktumsek, A., Granica, S., Zengin, G., Ceylan, R.,
Locatelli, M., Tomczyk, M., 2017. In vitro enzyme inhibitory properties, antioxidant
activities, and phytochemical profile of Potentilla thuringiaca. Phytochemistry Letters
20, 365–372.
Halliwell, B., 1990. How to characterize a biological antioxidant. Free Radic. Res.
Commun. 9, 1–32.
Halliwell, B., 1995. Antioxidant characterization: methodology and mechanism.
Biochem. Pharmacol. 49, 1341–1348.
Hameed, A., Hussain, T., Gulzar, S., Aziz, I., Gul, B., Khan, M.A., 2012. Salt tolerance of a
cash crop halophyte Suaeda fruticosa: biochemical responses to salt and exogenous
chemical treatments. Acta Physiol. Plant. 34, 2331–2340.
Irwin, J.J., Sterling, T., Mysinger, M.M., Bolstad, E.S., Coleman, R.G., 2012. ZINC: a free
tool to discover chemistry for biology. J. Chem. Inf. Model. 52, 1757–1768.
Jalas, J., Suominen, J., 1988. Atlas Florae Europaeae: Volume 3: Distribution of Vascular
Plants in Europe. Cambridge University Press.
Food and Chemical Toxicology 154 (2021) 112348
Karker, M., Falleh, H., Msaada, K., Smaoui, A., Abdelly, C., Legault, J., Ksouri, R., 2016.
Antioxidant, anti-inflammatory and anticancer activities of the medicinal halophyte
Reaumuria vermiculata. EXCLI journal 15, 297.
Karlsson, D., 2013. Biomolecular Screening for Inhibitors of Butyrylcholinesterase:
Identification and Characterization Using in Vitro and in Silico Tools.
Ksouri, R., Falleh, H., Megdiche, W., Trabelsi, N., Mhamdi, B., Chaieb, K., Bakrouf, A.,
Magné, C., Abdelly, C., 2009. Antioxidant and antimicrobial activities of the edible
medicinal halophyte Tamarix gallica L. and related polyphenolic constituents. Food
Chem. Toxicol. 47, 2083–2091.
Mollica, A., Zengin, G., Durdagi, S., Ekhteiari Salmas, R., Macedonio, G., Stefanucci, A.,
Dimmito, M.P., Novellino, E., 2019. Combinatorial peptide library screening for
discovery of diverse α-glucosidase inhibitors using molecular dynamics simulations
and binary QSAR models. J. Biomol. Struct. Dyn. 37, 726–740.
Mollica, A., Zengin, G., Locatelli, M., Stefanucci, A., Mocan, A., Macedonio, G.,
Carradori, S., Onaolapo, O., Onaolapo, A., Adegoke, J., 2017. Anti-diabetic and anti-
hyperlipidemic properties of Capparis spinosa L.: in vivo and in vitro evaluation of its
nutraceutical potential. Journal of Functional Foods 35, 32–42.
Naija, D.S., Bouzidi, A., Boussaada, O., Helal, A.N., Mahjoub, M.A., Echafai, N.,
Mighri, Z., 2014. The antioxidant and free-radical scavenging activities of Tamarix
boveana and Suaeda fruticosa fractions and related active compound. European
Scientific Journal, ESJ 10.
Nemudzivhadi, V., Masoko, P., 2014. In vitro assessment of cytotoxicity, antioxidant, and
anti-inflammatory activities of Ricinus communis (Euphorbiaceae) leaf extracts. Evid.
base Compl. Alternative Med. 2014.
Parihar, M., Hemnani, T., 2004. Alzheimer’s disease pathogenesis and therapeutic
interventions. J. Clin. Neurosci. 11, 456–467.
Parvez, S., Kang, M., Chung, H.S., Cho, C., Hong, M.C., Shin, M.K., Bae, H., 2006. Survey
and mechanism of skin depigmenting and lightning agents. Phytother Res.: An
International Journal Devoted to Pharmacological and Toxicological Evaluation of
Natural Product Derivatives 20, 921–934.
Qasim, M., Gulzar, S., Khan, M.A., 2011. Halophytes as Medicinal Plants. NAM Meeting
in Denizli, Turkey.
Querfurth, H.W., LaFerla, F.M., 2010. Mechanisms of disease. N. Engl. J. Med. 362,
329–344.
Ritter, J.M., 2012. Drugs for Alzheimer’s disease. Br. J. Clin. Pharmacol. 73, 501–503.
Saleem, H., Zengin, G., Locatelli, M., Mollica, A., Ahmad, I., Mahomoodally, F.M.,
Abidin, S.A.Z., Ahemad, N., 2019. In vitro biological propensities and chemical
profiling of Euphorbia milii Des Moul (Euphorbiaceae): a novel source for bioactive
agents. Ind. Crop. Prod. 130, 9–15.
van der Watt, E., Pretorius, J.C., 2001. Purification and identification of active
antibacterial components in Carpobrotus edulis L. J. Ethnopharmacol. 76, 87–91.
Weber, D., Ansari, R., Gul, B., Khan, M.A., 2007. Potential of halophytes as source of
edible oil. J. Arid Environ. 68, 315–321.
Wickens, G.E., Field, D.V., Goodin, J.R., 2012. Plants for Arid Lands: Proceedings of the
Kew International Conference on Economic Plants for Arid Lands Held in the Jodrell
Laboratory, Royal Botanic Gardens, Kew, England, 23–27 July 1984. Springer
Science & Business Media.
Yadav, R., Agarwala, M., 2011. Phytochemical analysis of some medicinal plants.
J. Phytol.
Zengin, G., Atasagun, B., Aumeeruddy, M.Z., Saleem, H., Mollica, A., Bahadori, M.B.,
Mahomoodally, M.F., 2019. Phenolic profiling and in vitro biological properties of
two Lamiaceae species (Salvia modesta and Thymus argaeus): a comprehensive
evaluation. Ind. Crop. Prod. 128, 308–314.
Zengin, G., Nithiyanantham, S., Locatelli, M., Ceylan, R., Uysal, S., Aktumsek, A.,
Selvi, P.K., Maskovic, P., 2016. Screening of in vitro antioxidant and enzyme
inhibitory activities of different extracts from two uninvestigated wild plants:
Centranthus longiflorus subsp. longiflorus and Cerinthe minor subsp. auriculata.
European Journal of Integrative Medicine 8, 286–292.