pharmaceuticals

Article
LC/MS-MS Analysis of Phenolic Compounds in
Hyoscyamus albus L. Extract: In Vitro Antidiabetic Activity, In
Silico Molecular Docking, and In Vivo Investigation against
STZ-Induced Diabetic Mice
Sabrina Lekmine 1, * , Ouided Benslama 2 , Kenza Kadi 1 , Antonio Ignacio Martín-García 3 ,
Mustafa Abdullah Yilmaz 4 , Salah Akkal 5 , Ali Boumegoura 6 , Abdullah S. Alhomida 7 ,
Mohammad Shamsul Ola 7 and Ahmad Ali 8

1 Biotechnology, Water, Environment and Health Laboratory, Abbes Laghrour University,

Khenchela 40000, Algeria
2 Laboratory of Natural Substances, Biomolecules, and Biotechnological Applications, Department of Natural
and Life Sciences, Larbi Ben M’Hidi University, Oum El Bouaghi 04000, Algeria
3 Estación Experimental del Zaidín (CSIC) Profesor Albareda 1, 18008 Granada, Spain
4 Faculty of Pharmacy, Department of Analytical Chemistry, Dicle University, 21280 Diyarbakir, Türkiye
5 Valorization of Natural Resources, Bioactive Molecules and Biological Analysis Unit, Department of
Chemistry, University of Mentouri Constantine 1, Constantine 25000, Algeria
6 Biotechnology Research Center (C.R.Bt), Ali Mendjeli, Nouvelle Ville, UV 03 BP, Constantine P.O. Box E73, Algeria
7 Department of Biochemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
8 Department of Life Sciences, University of Mumbai, Vidyanagari, Mumbai 400098, India; ahmadali@mu.ac.in
* Correspondence: lekmine.sabrina@univ-khenchela.dz or lekmine.sabrina.40@gmail.com

Citation: Lekmine, S.; Benslama, O.; Abstract: This study aimed to investigate the chemical composition and antidiabetic properties
Kadi, K.; Martín-García, A.I.; Yilmaz, of cultivated Hyoscyamus albus L. The ethanol extract was analyzed using LC-MS/MS, and 18 dis-
M.A.; Akkal, S.; Boumegoura, A.; tinct phenolic compounds were identified. Among these, p-coumaric acid (6656.8 ± 3.4 µg/g),
Alhomida, A.S.; Ola, M.S.; Ali, A. gallic acid (6516 ± 1.7 µg/g), luteolin (6251.9 ± 1.3 µg/g), apigenin (6209.9 ± 1.1 µg/g), and rutin
LC/MS-MS Analysis of Phenolic (5213.9 ± 1.3 µg/g) were identified as the most abundant polyphenolic molecules. In the in vitro
Compounds in Hyoscyamus albus L. antidiabetic experiment, the ability of the plant extract to inhibit α-glucosidase and α-amylase ac-
Extract: In Vitro Antidiabetic Activity,
tivities was examined. The results indicated that the extract from H. albus L. exhibited a higher
In Silico Molecular Docking, and In
inhibitory effect on α-amylase compared to α-glucosidase, with an IC50 of 146.63 ± 1.1 µg/mL
Vivo Investigation against
and 270.43 ± 1.1 µg/mL, respectively. Docking simulations revealed that luteolin, fisetin, and rutin
STZ-Induced Diabetic Mice.
exhibited the most promising inhibitory activity against both enzymes, as indicated by their high
Pharmaceuticals 2023, 16, 1015.
https://doi.org/10.3390/ contrasting inhibition scores. To further investigate the in vivo antidiabetic effects of H. albus L., an
ph16071015 experiment was conducted using STZ-induced diabetic mice. The results demonstrated that the
plant extract effectively reduced the levels of cholesterol and triglycerides. These findings suggest
Academic Editor: Ana Angelica
that H. albus L. may have therapeutic potential for managing hyperlipidemia, a common compli-
Feregrino-Perez
cation associated with diabetes. This highlights its potential as a natural remedy for diabetes and
Received: 22 May 2023 related conditions.
Revised: 9 July 2023
Accepted: 13 July 2023 Keywords: antidiabetic activity; Hyoscyamus albus L.; α-glucosidase; α-amylase; molecular docking
Published: 18 July 2023

1. Introduction
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland. Diabetes mellitus (DM) is a chronic and severe disease resulting from a disturbance
This article is an open access article in glucose metabolism. It has become more prevalent in recent years and poses a serious
distributed under the terms and public health dilemma worldwide [1]. Hyperglycemia, an abnormal postprandial rise in
conditions of the Creative Commons blood sugar levels, is a risk factor for the development of type 2 diabetes [2]. In addition
Attribution (CC BY) license (https:// to macrovascular disorders such as peripheral arterial disease and coronary heart disease,
creativecommons.org/licenses/by/ diabetes can cause other conditions such as peripheral neuropathy, retinopathy, and diabetic
4.0/). kidney disease [3]. The two primary enzymes that catalyze carbohydrate degradation are

Pharmaceuticals 2023, 16, 1015. https://doi.org/10.3390/ph16071015 https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2023, 16, 1015 2 of 17

intestinal alpha-glucosidase and pancreatic alpha-amylase. Prior to being assimilated

into the intestines, starch is digested by α-amylases into oligosaccharides, which are then
hydrolyzed to glucose by intestinal α-glucosidase [4].
One of the treatments used to control type 2 diabetes is to limit glucose absorption
by inhibiting the enzymes that catalyze the hydrolysis of polysaccharides (α-glucosidase
and α-amylase) [5,6]. Acarbose is among the synthetic agents known to inhibit carbohy-
drate digestive enzymes and represents an appropriate therapeutic method for managing
postprandial glycemia [7]. However, despite the FDA’s approval of acarbose and other
glucosidase inhibitors as effective agents for reducing blood glucose levels, these molecules
have been associated with severe adverse effects, including abdominal distension, diarrhea,
hepatotoxicity, and flatulence [7].
In recent years, significant efforts have been made to identify new naturally efficient
α-amylase and α-glucosidase inhibitors to develop new treatments. Medicinal plants are
great resources for inhibitor substances [8]. Therefore, plant-based enzyme inhibitors are
now highly recommended, especially in developed countries that sometimes lack access to
modern therapy and due to concerns about the side effects of synthetic drugs [8].
White henbane (Hyoscyamus albus L.) is a Mediterranean plant member of the Solanaceae
family that possesses an interesting composition containing alkaloids, terpenoids, and phe-
nolic compounds (polyphenols, flavonoids, and tannins) [9]. This plant has been used for
a long time as an analgesic and diuretic and has been traditionally associated with the
treatment of insomnia, asthma, whooping cough, infectious ulcers, and epilepsy [10].
The aim of this study is to identify the phytochemical constituents of an ethanolic
extract of cultivated H. albus using liquid chromatography with tandem mass spectrometry
(LC-MS/MS). Herein, we report the in vitro enzyme inhibition tests and in vivo antidiabetic
activity of the H. albus ethanolic extract, along with docking studies. Furthermore, the
detected phenolic compounds were evaluated as ligands to assess their inhibitory efficacy
against α-amylase and α-glucosidase receptors to understand their biological mechanisms.

2. Results
2.1. Total Phenolic, Flavonoid, and Tannin Contents
Table 1 presents the quantities of total phenolic compounds (TPC), flavonoid com-
pounds (TFC), and tannin compounds (TTC) in the ethanolic extracts of cultivated H. albus.
The TPC was determined using the gallic acid standard graph with the equation
y = 0.0127x − 0.0106 (R2 = 0.9988). Similarly, the TFC was calculated using the quercetin
calibration curve with the equation y = 0.0334x − 0.1031 (R2 = 0.9382). The TTC content
was determined using the catechin calibration curve with the equation y = 0.133x − 0.0183
(R2 = 0.9663).

Table 1. Contents of the ethanolic extract of H. albus in terms of total phenols, flavonoids, and tannins.

Sample TPC (µgEAG/mg E) TFC (µg EQ/mgE) TTC (µg ECT/mgE)

H. albus ethanolic extract 245.20 ± 0.53 a 120.55 ± 0.56 a 60 ± 0.42 b
Significant differences between the samples are denoted by letters (a, b) (p < 0.05), indicating statistically significant
variations among the groups.

2.2. Screening and Measurement of Phenolic Compounds

Figure 1 illustrates the base peak chromatogram (BPC) of the ethanolic extract of
cultivated H. albus, while Table 2 provides the quantitative results. A total of 18 phenolic
compounds with diverse chemical structures were identified. There were significant differ-
ences in the concentrations of these chemicals. The most abundant phenolic compounds
in the cultivated H. albus extract, as determined by LC-MS/MS, were p-coumaric acid
(6656.8 ± 3.4 µg/g), gallic acid (6516 ± 1.7 µg/g), luteolin (6251.9 ± 1.3 µg/g), apigenin
(6209.9 ± 1.1 µg/g), and rutin (5213.9 ± 1.3 µg/g). Additionally, the extract exhibited
relatively high levels of hyperoside (2123 ± 1.2 µg/g), naringenin (923 ± 2.1 µg/g), hes-
 (124.26 ± 2.15 µg/g), vanillin (76.7 ± 1.2 µg/g), and salicylic acid (43.71 ± 3.3 µg/g) and
negligible amounts of tr-caffeic acid (12.71 ± 1.37 µg/g). Regarding non-phenolic sub-
stances, the extract exhibited a high concentration of quinic acid (114.7 ± 4.3 µg/g) and a
lower amount of malic acid (3.6 ± 2.3 µg/g). Additionally, tannic acid (970 ± 1.6) and cou-
Pharmaceuticals 2023, 16, 1015 marin (0.8 ± 2.3), which are two subclasses of phenolic compounds, were present 3 of 17in the
extract (Figure 1 and Table 2). The base peak chromatogram (BPC) of the LC-MS/MS chro-
matograms of a 250 ppb standard mix is presented in Figure S1 (Supplementary Materi-
als). The analytical
peretin (883 ± 1.7 parameters of standard
µg/g), quercetin compounds
(293 ± 6.2 µg/g), andare presented(212
kaempferol in Table
± 2.1 S1 (Supple-
µg/g)
mentary Materials).
as flavonoids.

Figure 1. 1.
Figure LC-MS/MS
LC-MS/MSchromatogram of cultivated
chromatogram of cultivatedH.H.albus
albus ethanolic
ethanolic extract.
extract.

Table 2. Amounts (µg/g dry plant) of selected phytochemicals in cultivated H. albus ethanolic extract.

Compound Number RT [M-H]− MS2 (Collision Energy) Cultivated H. albus (µg dry/g Extract)
1 Quinic acid 3.32 191.0 85 (22), 93 (22) 114.7 ± 4.3
2 Malic acid 3.54 133.1 115 (14), 71 (17) 3.6 ± 2.3
3 tr-Aconiticacid 4.13 172.9 85 (12), 129 (9) N.D.
4 Gallic acid 4.29 169.1 125 (14), 79 (25) 6516 ± 1.7
5 Chlorogenic acid 5.43 353.0 191 (17) N.D.
6 Protocatechuicacid 5.63 153.0 109 (16), 108 (26) N.D.
7 Tannic acid 6.46 183.0 124 (22), 78 (34) 970.0 ±1.6
8 tr-Caffeic acid 7.37 179.0 135 (15), 134 (24), 89 (31) 12.71 ± 1.3
9 Vanillin 8.77 151.1 136 (17), 92 (21) 76.7 ± 1.2
10 p-Coumaric acid 9.53 163.0 119 (15), 93 (31) 6656.8 ± 3.4
11 Rosmarinic acid 9.57 358.9 161 (17), 133 (42) N.D.
12 Rutin 10.16 608.1 300 (37), 271 (51), 301 (38) 5213.9 ± 1.3
13 Hesperidin 9.69 611.1 303, 465 N.D.
14 Hyperoside 10.53 459.1 300, 301 2123.0 ± 1.2
15 4-OHBenzoicacid 11.72 137.0 93, 65 N.D.
16 Salicylicacid 11.72 137.0 93, 65, 75 43.71 ± 3.3
17 Myricetin 11.94 317.0 179, 151, 137 N.D.
18 Fisetin 12.61 285.0 135, 121 124.26 ± 2.1
19 Coumarin 12.52 147.0 103, 91, 77 0.8 ± 2.3
20 Quercetin 14.48 300.9 179, 151, 121 293.0 ± 6.2
21 Naringenin 14.66 271.0 151, 119, 107 923.0 ± 2.1
22 Hesperetin 15.29 301.0 164, 136, 108 883.0 ± 1.7
Pharmaceuticals 2023, 16, 1015 4 of 17

Table 2. Cont.

Compound Number RT [M-H]− MS2 (Collision Energy) Cultivated H. albus (µg dry/g Extract)
23 Luteolin 15.43 285.0 175, 151, 133 6251.9 ± 1.3
24 Kaempferol 15.43 285.0 217, 133, 151 212.0 ± 2.1
25 Apigenin 16.31 269.0 151, 117 6209.9 ± 1.1
N.D.: Compound not detected above the LOD.

Furthermore, the cultivated H. albus extract contained moderate amounts of fisetin

(124.26 ± 2.15 µg/g), vanillin (76.7 ± 1.2 µg/g), and salicylic acid (43.71 ± 3.3 µg/g)
and negligible amounts of tr-caffeic acid (12.71 ± 1.37 µg/g). Regarding non-phenolic
substances, the extract exhibited a high concentration of quinic acid (114.7 ± 4.3 µg/g) and
a lower amount of malic acid (3.6 ± 2.3 µg/g). Additionally, tannic acid (970 ± 1.6) and
coumarin (0.8 ± 2.3), which are two subclasses of phenolic compounds, were present in
the extract (Figure 1 and Table 2). The base peak chromatogram (BPC) of the LC-MS/MS
chromatograms of a 250 ppb standard mix is presented in Figure S1 (Supplementary
Materials). The analytical parameters of standard compounds are presented in Table S1
(Supplementary Materials).

2.3. In Vitro Test Methods for Inhibiting Enzymes

Table 3 shows that the plant extract exhibited a higher inhibitory effect on α-amylase
compared to α-glucosidase. Notably, the observed inhibitory potency of the plant extract
on both enzymes is relatively similar to that of the standard molecule acarbose. Acarbose
displayed IC50 values of 146.63 ± 1.1 µg/mL for α-amylase and 270.43 ± 1.1 µg/mL
for α-glucosidase.

Table 3. In vitro antidiabetic assay of H. albus extracts.

IC50 of Enzyme Inhibitory Assay (µg/mL)

Sample
Alpha-Amylase Alpha-Glucosidase
Cultivated H. albus 120.5 ± 1.3 243.2 ± 1.3
Acarbose 146.63 ± 1.1 270.43 ± 1.1
The values are calculated by the mean and standard deviation of five independent experiments.

2.4. Molecular Docking Studies

Docking simulations were conducted on the 18 phenolic compounds of H. albus to
evaluate their affinity and binding mode with two target enzymes, alpha-amylase and alpha-
glucosidase. To ensure the accuracy of the docking procedure within the target enzymes,
the docking of the co-crystallized inhibitor, acarbose, had beenpreviously established.
Acarbose exhibited spatial conformations closely aligned with their corresponding co-
crystallized structures, with root-mean-square deviation (RMSD) values below 1, validating
the accuracy of the docking configuration.
The results of the docking simulations revealed that the top three phenolic compounds
demonstrated favorable binding within the catalytic pocket of both tested enzymes. The
theoretical binding modes of these compounds are depicted in Figures 2 and 3. These
specific molecules were selected based on their notable differential inhibition scores against
the two enzymes, as outlined in Tables 4 and 5.
 sponding co-crystallized structures, with root-mean-square deviation (RMSD) values be-
low 1, validating the accuracy of the docking configuration.
The results of the docking simulations revealed that the top three phenolic com-
pounds demonstrated favorable binding within the catalytic pocket of both tested en-
zymes. The theoretical binding modes of these compounds are depicted in Figures 2 and
Pharmaceuticals 2023, 16, 1015 3. These specific molecules were selected based on their notable differential inhibition 5 of 17
scores against the two enzymes, as outlined in Tables 4 and 5.

Figure
Figure 2. 2. Two-dimensionalpredicted
Two-dimensional predicted binding
bindingmode
modeforforacarbose
acarboseand thethe
and best toptop
best phenolic
phenolicligands
ligands of
of H. albus with α-glucosidase (5NN8). In the center, a 3D view of the binding gorge with an ex-
H. albus with α-glucosidase (5NN8). In the center, a 3D view of the binding gorge with an expanded
panded picture at the active site showing acarbose, fisetin, luteolin, and rutin as cyan,
Pharmaceuticals 2023, 16, x FOR PEER REVIEW
yellow, or-
6 of 18
picture
ange,at
andthegreen
active siterespectively,
sticks, showing acarbose,
bound tofisetin, luteolin,
the surface and rutin as cyan, yellow,
of α-glucosidase. orange, and
green sticks, respectively, bound to the surface of α-glucosidase.

Figure 3. Two-dimensional predicted binding mode for acarbose and the five best top polyphenolic
Figure 3. Two-dimensional predicted binding mode for acarbose and the five best top polyphenolic
ligands of H. albus with α-amylase (2QV4). In the center, a 3D view of the binding gorge with an
expanded
ligands picture
of H. albus at theα-amylase
with active site shows acarbose,
(2QV4). fisetin,
In the rutin, aand
center, 3Dquercetin
view ofasthe
cyan, bleu, ma-gorge with an
binding
genta,
expanded and yellow
picture sticks,
at the respectively,
active site showsbound to the surface
acarbose, of α-amylase.
fisetin, rutin, and quercetin as cyan, bleu, magenta,
and yellow
Table 4.sticks, respectively,
The topthree results forbound
dockingto
H.the
albussurface
phenolicof α-amylase.
ligands with α-glucosidase.

Binding Force Contacts with

Hydrophobic Van der Waals Electrostatic
(Energy) Hydrogen
Interactions Interactions Interactions
(kcal/mol) (Interactions)
Asp404, His674, Trp618, Ala284, Leu405, Leu650,
Trp376, Trp481,
Acarbose −8.6 Asp616, Arg600, Ile441, Trp516, Asp518, Arg672, -
Phe649
Met519, Asp282 Trp613, Gly615, Phe525
Pharmaceuticals 2023, 16, 1015 6 of 17

Table 4. The topthree results for docking H. albus phenolic ligands with α-glucosidase.

Binding Force Contacts with

Hydrophobic Van der Waals Electrostatic
(Energy) Hydrogen
Interactions Interactions Interactions
(kcal/mol) (Interactions)
Asp404, His674, Trp618, Ala284, Leu405, Leu650,
Trp376, Trp481,
Acarbose −8.6 Asp616, Arg600, Ile441, Trp516, Asp518, Arg672, -
Phe649
Met519, Asp282 Trp613, Gly615, Phe525
Asp282, Arg600, Phe525, Phe649, Ser523, Met519, Trp376, Ile441,
Luteolin −8.2 Asp616
His674 Trp481 Asp404, Trp516, Trp613, Asp518
Ile441, Trp516, Arg672, Trp613,
Asp616, Met519,
Fisetin −8.2 His674, Asp404 Trp481, Phe649 Trp376, Leu650, Phe525, Ser523,
Asp518
Asp282, Arg600
Asn524, Ala555, Arg281, Leu283,
Ala284, Phe525, Met519, Trp481,
Rutin −8.0 Asp282, Ser523 Leu678, Leu650 -
Trp618, Arg600, Asp616, trp376,
Phe649, Ser676

Table 5. The topthree results for the docking of H. albus phenolic ligands with α-amylase.

Contacts with
Binding Force Van der Waals
Hydrogen Hydrophobic Interactions
(Energy) (kcal/mol) Interactions
(Interactions)
Gly164, Thr163, Ala106,
Asn105, Trp59, Val107, His305, Trp58, Ala198, Asp197,
Acarbose −9.2 His101,Gln63, Arg195, - Lys200, His201, Ile235, Tyr151,
Glu233, Asp300, Leu162, Leu165, Ile51, Gly104
Tyr62, His299
Trp58, His299, Arg195, Glu233,
Asp197, Asp300,
Fisetin −9.3 Tyr62, Trp59 Ala198, Leu162,
Gln63, His305
His101, Leu165
Leu162, Leu165, Ala198,
Quercetin −9.2 Gln63, Asp197, Asp300 Tyr62, Trp59 His101, Arg195, Glu233, Trp58,
His299, His305
Leu165, Trp59, Trp58, Ala198,
Tyr151, His201, Ile235,
Rutin −9.1 Thr163 Tyr62, His101, Asp197, Arg195,
Lys200, Leu162
Asp300, Glu233

2.5. Drug Similarity and the ADMET Profile

In terms of the pharmacokinetic and ADMET (absorption, distribution, metabolism,
excretion, and toxicity) profile, an ideal drug candidate should comply with Lipinski’s Rule
of Five, which is a set of criteria for optimal oral administration, and it should also be free
of potential toxicity [11]. Therefore, conducting pharmacokinetic and ADMET profiling of
the identified promising hit molecules is crucial in the drug development process to assess
their bioavailability and potential adverse effects. The pharmacokinetic and drug-likeness
characteristics of the top active ligands from H. albus are summarized in Tables S2 and S3
(Supplementary Materials).

2.6. In Vivo Antidiabetic

2.6.1. Acute Oral Toxicity
Table 6 presents the results of biochemical markers, including ASAT (aspartate amino-
transferase), ALAT (alanine aminotransferase), creatinine, and urea. Elevated levels of
aminotransferase activity (ALAT and ASAT) are typically indicative of hepatotoxicity [12].
However, upon comparing the treated mice with the control group, no significant impact on
Pharmaceuticals 2023, 16, 1015 7 of 17

the biochemical parameters was observed, even at the highest dosage of 2000 mg/kg body
weight. These results suggest that the H. albus extract does not cause any hepatotoxicity. On
the other hand, an increase in creatinine and urea concentrations is commonly associated
with renal dysfunction [13].

Table 6. Blood examination of mice, 14 days following treatment with H. albus extracts.

Groups Parameters for the Renal Function

ASAT ALAT Urea (g/L) Creatinine (mg/L)
Group 1 (1000 mg/kg) 156 ± 2.2 47.2 ± 3.5 0.34 ± 1.3 <0.37
Group 2(1500 mg/kg) 159.3 ± 1.2 49.6 ± 3.6 0.43 ± 1.3 <0.37
Group 3 (2000 mg/kg) 162.5 ± 1.4 48.3 ± 2.5 0.42 ± 1.5 <0.37
Control 157.2 ± 5.2 50.0 ± 2.3 0.59 ± 1.4 <0.37
ASAT:Aspartate-Amino-Transferase; ALAT: Alanine-Amino-Transferase.

The LD50 (median lethal dose) for the extract was determined to be above 2000 mg/kg,
indicating a relatively safe profile. Additionally, administering various concentrations of
the extract did not result in any notable changes in renal parameters and liver enzymes,
suggesting that the phenolic compounds identified by LC-MS/MS are non-toxic and do
not have adverse effects on internal organs at the concentrations present in the extract.
Moving on to the hypoglycemic activity, an oral glucose tolerance test (OGTT) was
conducted in healthy mice, and the results are presented in Table 7. The hypoglycemic
activity of the H. albus extract was evaluated through an oral glucose tolerance test (OGTT)
conducted in healthy mice. The results, presented in Table 7, indicate a significant reduction
in blood glucose levels in all experimental groups compared to the control group. The
effect of the reference drug, glibenclamide, was found to be significantly greater than that
of the H. albus extract. However, it is worth noting that the administration of 20 mg/kg of
the H. albus extract led to a noticeable decrease in blood sugar levels in mice, as evident
from the data in Table 7.

Table 7. Effect of H. albus extract on the oral glucose tolerance test (OGTT) in good-health mice.

Amount of Blood Glucose (g/L)

Groups 0 min 30 min 1h 1 h and 30 min 2h
H. albus 10 mg/kg 0.93 ± 0.7 2.4 ± 1.3 2.1 ± 1.7 1.9 ± 2.0 1.8 ± 1.3
H. albus 20 mg/kg 0.93 ± 1.1 2.01 ± 1.8 1.5 ± 2.2 0.9 ± 1.2 0.8 ± 0.6
Glibenclamide20 mg/kg 0.95 ± 0.8 0.62 ± 0.5 0.56 ± 0.2 0.47 ± 0.5 0.41 ± 0.3
Control 0.9 ± 1.2 2.3 ± 1.5 2.01 ± 2.1 1.82 ± 2.1 1.17 ± 1.4

2.6.2. Antidiabetic Activity in Streptozotocin-Induced Hyperglycemia Model

As shown in Table 8, H. albus extract significantly reduced blood glucose concentrations
in the streptozotocin (STZ)-induced mice model compared to controls and glibenclamide.

Table 8. Effect of oral administration of H. albus extract on hyperglycemia in STZ-diabetic mice.

Amount of Blood Glucose (g/L), Body Weight (g), and Lipid Profile
Groups 0 Day 5 Day 10 Day 15 Day 20 Day
BGL BW BGL BW BGL BW BGL BW BGL BW TC HDL TG
H. albus 10 mg/kg 3.97 26.3 2.61 25.2 2.52 25.6 2.32 25.3 2.11 25.3 0.85 ± 1.2 0.45 ± 1.1 0.46 ± 1.3
H. albus 20 mg/kg 2.86 27.4 1.59 27.6 1.65 26.5 1.24 27.9 1.32 26.6 0.91 ± 2.2 0.51 ± 2.1 0.41 ± 2.3
Glibenclamide
3.61 27.5 3.93 26.3 3.43 26.1 2.91 25.9 2.82 25.3 / / /
20 mg/kg
Untreated
3.51 28.2 4.53 24.3 4.31 22.1 4.62 22.6 4.35 22.3 1.83 ± 1.6 0.21 ± 1.4 0.89 ± 1.1
diabetic mice
Normal 1.08 26.6 1.02 26.5 0.98 26.1 1.12 26.8 1.10 27.4 0.95 ± 1.3 0.51 ± 2.2 0.34 ± 1.6
BGL: Blood glucose level; BW: Body weight; HDL: High-density lipoprotein; TC: Total cholesterol; TG: Triglycerides.
Pharmaceuticals 2023, 16, 1015 8 of 17

After 20 days of treatment, mice administered the H. albus plant extract at both tested
doses (10 mg/kg and 20 mg/kg) exhibited nearly identical blood glucose concentrations
compared to non-diabetic mice. In contrast, the glibenclamide group (20 mg/kg) did
not significantly lower blood sugar levels compared to the control group. The most
significant reduction in blood glucose levels was observed at the concentration of 20 mg/kg.
Therefore, after 20 days of treatment, the cultivated H. albus extract effectively reduced
hyperglycemia in both STZ-diabetic and healthy mice, leading to the achievement of normal
blood sugar levels.
In addition to hyperglycemia, diabetes is characterized by polydipsia (increased drink-
ing), polyuria (excessive urine production), and weight loss with polyphagia (increased
appetite). Table 8 displays the variations in body weight of diabetic mice from the beginning
of the experiment to the final day. Compared to the normal control group, the injection of
streptozotocin caused a significant decrease in body weight in all diabetic mice. However,
in contrast to the normal model group, treatment with H. albus extracts did not significantly
increase the body weight of treated mice. On the contrary, the glibenclamide-treated group
experienced significant weight loss after 20 days at a concentration of 20 mg/kg.
Following streptozotocin injection, diabetic control mice exhibited a substantial in-
crease in total serum cholesterol and triglyceride levels, along with a decrease in the
high-density lipoprotein (HDL) ratio compared to normal mice (Table 8). In contrast, ad-
ministration of the studied extracts at doses of 10 mg/kg and 20 mg/kg to diabetic mice
resulted in a significant reduction in cholesterol and triglyceride levels, accompanied by
a remarkable increase in HDL levels. These findings indicate that the extracts effectively
regulated the lipid profiles, restoring them to levels comparable to those observed in normal
control animals.

3. Discussion
The presence of phenolic compounds in medicinal plants has sparked significant
interest due to their potential antioxidant effects in various diseases, including cancer,
heart disorders, stroke, and inflammation [14]. LC-MS/MS has been widely utilized in
quantitative applications, offering high selectivity and sensitivity compared to other LC
methods [15]. In this study, LC-MS/MS analysis of cultivated H. albus ethanol extracts was
performed, leading to the identification of 18 phenolic chemicals (Table 1).
The analysis revealed a rich profile of flavonoids in the H. albus ethanolic extract,
including apigenin and luteolin, which have demonstrated various therapeutic proper-
ties [16]. Additionally, rutin, kaempferol, and quercetin, known for their anti-inflammatory
properties, were found in relatively high concentrations in H. albus, suggesting its potential
for mitigating and treating chronic human conditions [17]. Our findings showed a higher
phenolic content compared to a previous study by Tlili [18], who employed LC-ESI-MS
and identified only 11 phenolic compounds.
Our research represents a novel contribution to the field, building upon previous
studies on the therapeutic effects of H. albus. In particular, a study by Bourebaba et al. [19]
investigated the antidiabetic effects of calystegines extracted from H. albus seeds, high-
lighting their potential as antihyperglycemic and hypolipidemic agents. In our study,
the H. albus extract demonstrated strong inhibition against α-amylase compared to α-
glucosidase, which is likely attributed to the presence of phenolic compounds synthesized
by the plant. Phenols have been shown to reduce blood glucose levels through various
mechanisms, including the downregulation of carbohydrate digestion and intestinal glu-
cose uptake, activation of pancreatic insulin production, stimulation of hepatic glucose
release, and facilitation of glucose assimilation in insulin-sensitive tissues [20].
Regarding the α-glucosidase enzyme, the docking analysis demonstrated that the top
three phenols interacted with key residues in the enzyme’s active site, namely Asp616,
Asp518, Asp404, His674, and Arg600. In α-glucosidase, the basic residues His674 and
Arg600 and the acidic residues Asp616, Asp518, Asp404, and Glu521 are shown to have
a critical catalytic role [21]. The lead-selected compounds bind at the entrance zone of
Pharmaceuticals 2023, 16, 1015 9 of 17

α-glucosidase’s active site (Figure 2), either acting as a competitive inhibitor by preventing
the substrate from entering the active site or as an uncompetitive inhibitor by blocking the
exit of the product from the enzyme [22].
With the lowest binding energy value (−8.6 kcal/mol), acarbose interacts with the
α-glucosidase catalytic site by establishing 10 hydrogen bonds with the amino acids Asp404,
His674, Asp616, Arg600, Met519, and Asp282. The analysis of interactions with 5NN8
predicted that the top five phenols occupy the same location as acarbose, as depicted in the
expanded view of the active site in Figure 2. Their binding affinities are as follows: luteolin
(−8.2 kcal/mol), fisetin (−8.2 kcal/mol), and rutin (−8.0 kcal/mol).
Based on the binding scores, the two flavonoids, luteolin and fisetin, exhibit the
strongest binding to α-glucosidase. The docked view shows them deeply embedded within
the binding cavity of α-glucosidase (Figure 2). According to Vaya et al. [23], the presence
of hydroxyl radicals on the flavonoid B ring likely facilitates the dispersion of electronic
states, enabling easier hydrogen donation and formation of hydrogen bonds with the
α-glucosidase catalytic site residues.
The docking study conducted by Lim et al. [24] on selected compounds, including
fisetin and other flavonoids, with α-glucosidase provides valuable insights into the binding
interactions of these compounds, complementing our findings on the phenolic compounds
from H. albus. Luteolin forms three hydrogen bonds with the residues Asp282, Arg600, and
His674. Regarding fisetin, His674 and Asp404 provide hydrogen bonding interactions with
this molecule (Table 3 and Figure 2).
In the case of α-amylase, substrate hydrolysis involves the participation of key residues
Asp197 and Glu233, which contain carboxylic acids and form a covalent glycosyl-enzyme
intermediate. Asp197 acts as the catalytic nucleophile, targeting the aglycon portion of the
substrate during the formation of the covalent intermediate. Glu233, on the other hand, acts
as an acid/base catalyst, facilitating the protonation of the leaving group and deprotonation
of water, which breaks the covalent bond between the substrate and Asp197 and attaches
an OH group to it [25]. Another important residue involved in the substrate hydrolysis
mechanism is Asp300, which anchors the conjugation of bound substrates [26].
Table 5 presents the binding affinity represented by the docking score and the catalytic
residue involved in hydrogen bonding for the top three phenols with α-amylase (2QV4).
The co-crystallized ligand acarbose exhibited a binding energy of −9.2 kcal/mol. A detailed
examination of its interaction with the amylase revealed 13 hydrogen interactions with
Gly164, Thr163, Ala106, Asn105, Trp59, Val107, His101, Gln63, Arg195, Glu233, Asp300,
Tyr62, and His299, which is consistent with the active residues reported earlier [27]. The top
three compounds selected based on their binding score are fisetin (−9.3 kcal/mol), quercetin
(−9.3 kcal/mol), and rutin (−9.1 kcal/mol). They were found to exhibit interesting modes
of interaction, interfering with the three key amino acids of the enzyme’s active site through
various hydrogen, hydrophobic, and van der Waals interactions, as depicted in Figure 3.
Based on the docking score and conformation, fisetin appears to have the highest
affinity toward the active site of α-amylase. This ligand forms four hydrogen bonds,
two of which are with the key amino acids Asp197 and Asp300. Indeed, hydrogen bond
interactions with receptors are crucial as they provide the required organization for distinct
folding and selectivity, facilitating intermolecular interactions within the protein–ligand
complex [28]. Except for rutin, the other compounds form at least one hydrogen bond with
one of the key amino acids of the catalytic triad (Asp197, Glu233, and Asp300).
The modification of α-amylase activity has significant implications for carbohydrate
utilization as an energy source. Natural α-amylase inhibitors have been found in various
medicinal herbs and have been explored for their potential in treating diabetes [29,30]. In
previous studies, several plant compounds, including quercetin [31], luteolin [32], and
fisetin [33], were identified as inhibitors of α-amylase, consistent with our findings.
For a bioactive molecule to be effectively absorbed through biological membranes, it
must satisfy Lipinski’s Rule of Five criteria. These criteria include having a maximum of
5 hydrogen bond donors, 10 hydrogen bond acceptors, a molecular weight below 500 g/mol,
Pharmaceuticals 2023, 16, 1015 10 of 17

and a LogP value (partition coefficient) not exceeding 5 [34]. Among the compounds tested,
acarbose and rutin were the only ones that violated Lipinski’s rule, indicating potential
limitations in their absorption and bioavailability. The other compounds in our study
met Lipinski’s criteria, suggesting their favorable characteristics for biological membrane
permeability and potential as bioactive molecules (Table S2).
Indeed, aqueous solubility and log S are crucial factors that impact the oral absorption
and bioavailability of a compound. These properties determine the substance’s ability
to dissolve in water and interact with enzymes and transporters in the intestinal wall,
which ultimately affect its absorption [35,36]. In this study, all the bioactive compounds
demonstrated good solubility, falling within the appropriate range.
Caco-2 cell permeability is another important criterion used to assess the permeability
of compounds across the gut–blood barrier [37]. The results indicated that the three
flavonoids, luteolin, and fisetin exhibited excellent permeability, suggesting their potential
for effective absorption.
Molecular flexibility, as determined by the number of rotatable bonds (nRB), is another
chemical characteristic that influences the bioavailability of a molecule [37]. The selected
bioactive molecules in this study had nRB values ranging from 1 to 7, which aligns with
the reported range for the majority of drug molecules (1–10) [38]. Importantly, none of
the promising molecules have the ability to cross the blood–brain barrier (BBB). This is
a desirable characteristic for antidiabetic compounds, as it eliminates the possibility of
adverse effects on the central nervous system.
Regarding their interactions with enzymes, the phenols luteolin, fisetin, and quercetin
are predicted to potentially inhibit CYP1A2, CYP2C9, and CYP3A4 enzymes. Cytochrome
P450 (CYP) enzymes play a vital role in metabolizing various substances in the body,
including drugs and xenobiotics [39]. Inhibition of these enzymes can affect drug clearance
and increase the risk of toxicity [40]. Additionally, the evaluation of toxic behavior suggests
that none of the promising compounds are hERG blockers, which eliminates the risk of
cardiac toxicity. However, rutin may pose a potential risk of immunotoxicity, while only
quercetin, fisetin, and luteolin have the potential to behave as estrogen receptor disruptors.
It is important to note that these predictions and evaluations are based on computational
analysis and should be further validated through in vitro and in vivo studies for a more
comprehensive understanding of the compounds’ safety and pharmacological profiles.
Before conducting pharmacological research and developing phytopharmaceutical
products, it is important to determine the acute toxicity of any medicinal herb [41]. The liver
and kidney are vital organs involved in metabolism and waste elimination, respectively.
Biochemical assessments of these organs can provide insights into the toxic effects of
substances without sacrificing animals [42]. In this study, the renal parameters and liver
enzymes showed no signs of alteration in the functioning of these organs after administering
varying amounts of H. albus extracts. These findings suggest that the plant may be a
relatively safe candidate for drug development, but further toxicological research on
different models and time scales is necessary to confirm its safety profile.
In terms of the oral glucose tolerance test, the results indicate that the anti-enzymatic
activities of H. albus extracts may prevent a rise in postprandial blood glucose levels. Ad-
ditionally, H. albus extracts are rich in phenolic compounds known for their antidiabetic
effects [43]. Several phenols, such as caffeic acid, quercetin, tannic acid, and naringenin,
have been shown to affect glucose uptake by inhibiting glucose carriers (sodium-glucose
transporter-1) and SGLT2 (sodium-glucose transporter-2) in enterocytes [44]. These mecha-
nisms contribute to the regulation of blood glucose levels.
The results obtained from the oral glucose tolerance test (OGTT) indicate a significant
reduction in blood glucose levels in all experimental groups compared to the control group,
suggesting the potential hypoglycemic activity of the H. albus extract. Although the effect
of the reference drug, glibenclamide, was observed to be significantly greater than that
of the H. albus extract, it is important to consider the context of this comparison. Gliben-
clamide is a well-established antidiabetic drug known for its potent hypoglycemic effects.
Pharmaceuticals 2023, 16, 1015 11 of 17

Therefore, its superior performance in reducing blood glucose levels is expected. However,
the observed decrease in blood sugar levels following the administration of 20 mg/kg of
the H. albus extract is noteworthy. This suggests that the extract has the potential to exert a
hypoglycemic effect, albeit to a lesser extent than the reference drug.The presence of bioac-
tive phenolic compounds identified through LC-MS/MS analysis supports the hypothesis
that these compounds may contribute to the observed hypoglycemic activity. However, it is
important to acknowledge that the exact mechanisms underlying the hypoglycemic effect
of H. albus extract require further investigation. Future studies should focus on elucidating
the specific phenolic compounds responsible for the hypoglycemicactivity and exploring
their mechanisms of action. Additionally, investigating the dose–response relationship and
conducting long-term studies could provide valuable insights into the optimal dosage and
duration of treatment.Furthermore, it is worth considering the potential synergistic effects
of the different components present in the H. albus extract. The combination of multiple
bioactive compounds might contribute to the overall hypoglycemic activity observed.
The stability of weight observed in mice administered with H. albus extract suggests
that the synthesis of phenolic compounds by the plant may impact neuro-endocrine regu-
lation, which is directly influenced by hyperglycemia. This implies that H. albus has the
potential to improve weight reduction in diabetic mice by regulating blood glucose levels.
Hyperlipidemia, characterized by elevated levels of LDL, cholesterol, and triglycerides
(TRG) and reduced levels of HDL, is commonly associated with diabetes. Insulin deficiency
in diabetes can inhibit enzymes involved in fatty acid transformation, leading to increased
lipid levels [45,46]. Insulin treatment is known to reduce plasma triglycerides and stimulate
lipoprotein lipase, thereby decreasing lipid levels in diabetic individuals [47]. The tested
H. albus extracts significantly reduced lipid levels in diabetic mice, suggesting a possible
partial recovery of insulin production. Further research, including immunocytochemical
staining for β-cells and insulin level testing, should be conducted to optimize and confirm
these findings.

4. Materials and Methods

4.1. Instruments and Chemicals
The chemical composition of H. albus was investigated by LC–MS/MS technique
(Shimadzu, Kyoto, Japan). All standards were obtained from Aldrich (Sigma-Aldrich,
Darmstadt, Germany). The α-amylase of microbial origin was extracted from the fungi
Aspergillus oryzae.

4.2. Plant Material

Seeds of H. albus were sourced from the Wilaya of Mila and cultivated in 3 kg pots.
After one month of germination, the seedlings were transplanted individually into separate
pots in a controlled greenhouse environment with irrigation maintained at half of the field
capacity. Upon reaching the final growth stage in June, the plants were harvested, dried,
ground into a powder, and stored for future use.

4.3. Total Bioactive Compound Determination (TPC, TFC, and TTC)

The total amount of phenolic compounds (TPC) was determined using the modified
Folin–Ciocalteu method [48,49]. The results were expressed as micrograms of gallic acid
equivalents per milligram of extract (µg GAE/mg). Gallic acid equivalents were calculated
based on a standard curve generated using gallic acid as a reference compound [48]. The
total flavonoid content (TFC) was determined following the method described by Topcu
et al. [50]. The results were reported as micrograms of quercetin equivalents per milligram
of extract (µg QE/mg). Quercetin equivalents were calculated using a standard curve
constructed with quercetin as a reference compound [50]. The total tannin content (TTC) of
the various extracts was calculated according to the procedure by Oueslati et al. [51]. The
results were expressed in terms of milligrams of tannic acid equivalent per gram of dry
Pharmaceuticals 2023, 16, 1015 12 of 17

plant material (mg CAE/g). Tannic acid equivalents were determined using a standard
curve generated with tannic acid as a reference compound [51].

4.4. Plant Extract Preparation for LC-MS/MS and an Enzyme Inhibitory Test
The hydro-alcoholic extracts (70% ethanol) were prepared using 10 g of powdered
plant material for each treatment. The plant material was combined with ethanol and
allowed to dissolve for 24 h at room temperature. The mixture was then filtered, and the
filtrate was concentrated using a rotary evaporator (Hahnvapor) at 40 ◦ C. The resulting
residue of the hydroalcoholic extract was collected and stored at 4 ◦ C for further use.

4.5. Polyphenolic Detection and Quantitation

The LC–MS/MS technique with a tandem MS system in conjunction with the UHPLC
(Nexera, Shimadzu Corporation, Kyoto, Japan) was used for the analysis of our samples
as described by Lekmine et al. [52]. The LC–MS analysis was performed using a set of
specific instruments, including LC-30AD binary pumps, a CTO-10ASvp column oven, a
DGU-20A3R degasser, and a SIL-30AC autosampler. For the separation of compounds,
we employed a reversed-phase C18 Inertsil ODS-4 analytical column with dimensions
of 150 mm × 4.6 mm × 3 µm, operating at a temperature of 40 ◦ C. The elution gradient
involved the use of two mobile phases: mobile phase A composed of H2 O, ammonium
formate (5 mM), and formic acid (0.1%) and mobile phase B consisting of methanol, ammo-
nium formate (5 mM), and formic acid (0.1%). The elution of compounds was achieved
using a gradient of these mobile phases.
To maintain the flow rate, the solvent flow was set at 0.5 mL/min, and a fixed injection
volume of 4 µL was used for sample introduction. The electrospray ionization (ESI) source
was employed for air pressure ionization. To optimize the ESI conditions, we determined
the following parameters: a desolvation line (DL) temperature of 250 ◦ C, an interface
temperature of 350 ◦ C, a heat block temperature of 400 ◦ C, a drying gas flow rate of
15 L/min, and a nebulizing gas flow rate of 3 L/min.
For quantification, a multiple-reaction monitoring (MRM) strategy was adopted, and
three transitions per compound were used. The first transition served for quantification,
while the subsequent transitions were utilized for confirmation purposes.
To ensure the reliability of the LC–MS/MS method, comprehensive method validation
parameters were determined. The analytical characteristics of reference compounds, includ-
ing linearity ranges and rectilinear regression estimates, were previously documented in
the literature [53]. All calibration curves for the studied compounds exhibited linearity and
reproducibility, with correlation coefficients exceeding 0.991. Additionally, the limit of de-
tection (LOD) and limit of quantitation (LOQ) for the analyzed compounds were reported
by Ertas and Yener [53]. LOD values ranged from 0.05 to 25.8 g/L, while LOQ values
ranged from 0.17 to 85.9 g/L. Moreover, the recovery percentages of phenolic compounds
ranged from 96.9% to 106.2%.
In this study, the identification of the analyzed molecules was carried out through
a rigorous approach combining multiple techniques. Initially, library MS/MS matching
was performed by comparing the acquired spectra with a comprehensive database of
known phytochemicals. Additionally, relevant literature references were consulted to cross-
reference retention times, mass spectra, and fragmentation patterns. Furthermore, accurate
mass measurement, isotopic pattern analysis, and, whenever possible, comparison with
authentic standards were employed for further confirmation. This integrated approach
ensured the reliable identification of the targeted phytochemicals.

4.6. Enzymatic Inhibitory Assay

The enzymatic inhibitory assays were conducted in 96-well microplates, and the
absorbance readings were taken using a Multimode Plate Reader. Acarbose, a well-known
inhibitory molecule, was used as the standard for comparison in this study. All experiments
were performed in triplicate to ensure accuracy and reproducibility of the results. For the
Pharmaceuticals 2023, 16, 1015 13 of 17

evaluation of anti-alpha-amylase activity, the iodine/potassium iodide method [54] was

employed. The absorbance was measured at 630 nm using a spectrophotometer. The
inhibitory activity of the test samples was compared to that of acarbose, and an IC50 value
was determined as a measure of the potency of the samples in inhibiting alpha-amylase.
The alpha-glucosidase inhibitory test was carried out using the Nampoothiri technique [55].
Again, acarbose was used as a positive control in this assay. The absorbance readings
were recorded, and the inhibitory activity of the test samples against alpha-glucosidase
was evaluated.

4.7. Computational Docking Methodology

The 2D structures of 18 phenolic compounds from H. albus were retrieved in SMILES
strings from the PubChem database. The Build Structure module of UCSF Chimera 1.15
was used to generate 3D structures of the compounds. To prepare the ligands for docking,
hydrogen atoms were added, and atom valences were adjusted. The 3D structures of α-
glucosidase (PDB ID: 5NN8) and α-amylase (PDB ID: 2QV4) complexed with the inhibitor
acarbose were obtained from the Protein Data Bank (PDB) with resolutions of 2.45 Å and
1.97 Å, respectively. The protein structures were prepared using the Dock Prep wizard
of UCSF Chimera software. This involved removing water molecules, other heteroatoms,
and co-crystallized ligands. Charges were corrected using the AMBER ff14SB force field,
and polar hydrogen atoms were added.To determine the binding sites for docking, the co-
crystallized inhibitor acarbose was used as a reference. The grid generation was established
based on the localization of acarbose within the active sites of both enzymes. The grid
parameters, including size and spacing, were set to ensure comprehensive sampling of the
binding site. Molecular docking simulations were performed using the Autodock Vina
program. The scoring function in Autodock Vina estimates ligand binding affinities based
on empirical potentials. The search algorithm systematically explores the conformational
space of the ligands within the defined binding sites to identify favorable binding modes.

4.8. Pharmacokinetic and ADMET Profile

Pharmacokinetic profiling, comprising drug-likeness, was performed using the web-
based application Swiss-ADME server (https://www.swissadme.ch (accessed on 5 April
2023)). The web-based applications ADMETlab 2.0 (https://admetmesh.scbdd.com/ (ac-
cessed on 5 April 2023)) and ProTox-II (https://tox-new.charite.de (accessed on 5 April
2023)) were also used to perform the toxicity evaluation. For each active compound, several
parameters were calculated, including human intestinal absorption, bioavailability, brain
penetration, carcinogenicity, mutagenicity, and immunotoxicity. The SMILES of the ligands
were uploaded to the used computational tools, and their results were computed.

4.9. In Vivo Antidiabetic Activity

4.9.1. Animals
Male albino mice weighing approximately 20 g and 28 g were obtained from the
Paster Institute in Algeria. The mice were housed in standard climate-controlled cages
with a humidity level of 50% and a temperature maintained at 25 ◦ C. They were provided
with a standard diet for a period of 10 days and then subjected to a 12h fasting period
before screening.

4.9.2. Acute Toxicity

The male albino mice were divided into four groups, each consisting of four mice.
They were administered a single oral dose of 1000 mg/kg, 1500 mg/kg, and 2000 mg/kg of
H. albus extract, respectively. The control group received a saline solution. Detailed obser-
vations of the mice were conducted during the first 4 h after treatment and at least twice
daily for the subsequent 14 days. At the end of the study, the animals were anesthetized
and sacrificed, and blood samples were collected in heparinized tubes for renal and hepatic
screening tests.
Pharmaceuticals 2023, 16, 1015 14 of 17

4.9.3. Hypoglycemic Activity

The oral glucose tolerance test was conducted on four groups of healthy mice (n = 4).
Prior to the experiment, all animals underwent a 12h fasting period. H. albus extract
was administered at doses of 10 mg/kg and 20 mg/kg, respectively, 30 min before oral
glucose administration at a dose of 2 g/kg. In the control group, glibenclamide at a dose of
20 mg/kg was used as the reference. Blood glucose levels were measured at 0, 30, 60, 90,
and 120 min following glucose administration.

4.9.4. Antihyperglycemic Activity in Streptozotocin-Induced Hyperglycemia Model

Male albino mice were fasted overnight prior to the experiment and divided into
five groups, each consisting of four mice. Experimental diabetes was induced by a sin-
gle intraperitoneal injection of streptozotocin solution at a concentration of 130 mg/kg
(dissolved in 0.1 M citrate buffer, pH 4.5). Mice that exhibited diabetes symptoms after
72 h, with blood glucose levels equal to or greater than 250 mg/dL, were selected for
further examination. For a duration of 20 days, the experimental diabetic mice received the
following oral treatments:
• Group 1: normal control mice;
• Group 2: diabetic control mice;
• Group 3: diabetic mice treated with H. albus at a concentration of 10 mg;
• Group 4: diabetic mice treated with H. albus at a concentration of 20 mg;
• Group 5: diabetic mice administered glibenclamide at a dose of 20 mg/kg.
Blood glucose levels and body weight changes were measured every five days. At the
end of the experiment, all animals were anesthetized and sacrificed, and blood samples
were collected to determine the levels of total cholesterol, HDL, and triglycerides.

5. Conclusions
This research investigates the antidiabetic activity of Hyoscyamus albus, a medicinal
herb of pharmaceutical involvement. To our knowledge, this is the first research that
reported the efficacy of H. albus ethanol extract as an inhibitor of the two regulatory
enzymes α-amylase and α-glucosidase, which are implicated in diabetes. The combination
of the in vitro and in vivo tests confirms the great potential of H. albus for inhibiting these
key enzymes and demonstrates its antihyperglycemic action. The insilico results agreed
with the in vitro and in vivo findings and produced clear evidence of the strong molecular
interactions between the phytochemicals and α-amylase and α-glucosidase. Despite this, a
possibility of a synergistic inhibitory effect between the different compounds should not be
neglected because this can have a more significant inhibition impact with a different mode
of interaction.
In the present study, flavonoids, particularly fisetin and rutin, exhibited significant
binding affinity with both enzymes. The insights derived from this research have the
potential to contribute to the development of new functional foods rich in flavonoids for the
treatment of diabetes. These functional foods could target the inhibition of both α-amylase
and α-glucosidase, aiming to address metabolic dysregulation associated with diabetes.
Overall, our study focused on investigating the phenolic compounds present in H. albus
using a negative-mode LC-MS/MS approach. While this analysis provided valuable
insights into the phenolic profile, we acknowledge the limitations of our approach in
not exploring the alkaloid composition. Further investigations employing alternative
techniques are necessary to comprehensively elucidate the complete phytochemical profile
of H. albus, including its alkaloid constituents. Future studies addressing these aspects
will contribute to a more comprehensive understanding of the phytochemical composition
and its potential therapeutic applications. Furthermore, our study made a significant
contribution to the understanding of H. albus and its potential hypoglycemic activity.
However, it is important to recognize the limitations associated with the small sample
size employed in our study. The sample size of n =4 in each group may have limited the
statistical power and generalizability of our findings. To address this limitation, future
Pharmaceuticals 2023, 16, 1015 15 of 17

research endeavors should aim to conduct studies with larger sample sizes, enabling a
more robust statistical analysis and enhancing the reliability and applicability of the results.

Supplementary Materials: The following supporting information can be downloaded at https://www.

mdpi.com/article/10.3390/ph16071015/s1, Figure S1: LC MS/MS chromatograms of 250 ppb standard
mix; Table S1: Analytical parameters of LC MS/MS method; Table S2: Drug-likeness characteristics of
the acarbose and the top active ligands of H. albus; Table S3: ADMET properties of the acarbose and
the top active ligands of H. albus.
Author Contributions: Methodology and writing, S.L., O.B. and A.I.M.-G.; visualization, K.K., S.A.,
M.A.Y., A.A., A.S.A., M.S.O. and A.B. All authors have read and agreed to the published version of
the manuscript.
Funding: Researchers Supporting Project Number (RSPD2023R710), King Saud University, Riyadh,
Saudi Arabia.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is available within article and Supplementary Materials.
Acknowledgments: The authors wish to express their thanks to the Algerian Ministry of Higher
Education and Scientific Research (MESRS, DGRSDT) and the National Centre for Biotechnology
Research (C.R.B.T) for their respective financial and material support and Experimental del Zaidín
(CSIC) Professor Albareda for their scientific contributions. The authors would like to thank the
funding of the Researchers Supporting Project Number (RSPD2023R710), King Saud University,
Riyadh, Saudi Arabia.
Conflicts of Interest: The authors declare no conflict of interest.

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