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Citation: Huneif, M.A.; Fahad, S.;
Abdulwahab, A.; Alqahtani, S.M.;
Mahnashi, M.H.; Nawaz, A.; Hussain,
F.; Sadiq, A. Antidiabetic,
Antihyperlipidemic, and Antioxidant
Evaluation of Phytosteroids from
Notholirion thomsonianum (Royle)
Stapf. Plants 2023,12, 3591. https://
doi.org/10.3390/plants12203591
Academic Editors: Natália
Cruz-Martins and Christophe Hano
Received: 15 August 2023
Revised: 3 October 2023
Accepted: 7 October 2023
Published: 17 October 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
plants
Article
Antidiabetic, Antihyperlipidemic, and Antioxidant Evaluation
of Phytosteroids from Notholirion thomsonianum (Royle) Stapf
Mohammad A. Huneif 1, Shah Fahad 2, Alqahtani Abdulwahab 1, Seham M. Alqahtani 1,
Mater H. Mahnashi 3, * , Asif Nawaz 4, Fida Hussain 5and Abdul Sadiq 4, *
1Pediatric Department, Medical College, Najran University, Najran 61441, Saudi Arabia;
maalhuneif@nu.edu.sa (M.A.H.); aaalsharih@nu.edu.sa (A.A.); drseham2015@gmail.com (S.M.A.)
2Department of Agronomy, Abdul Wali Khan University Mardan, Mardan 23200, KP, Pakistan;
shahfahad@awkum.edu.pk
3Department of Pharmaceutical Chemistry, College of Pharmacy, Najran University, Najran 61441, Saudi Arabia
4Department of Pharmacy, Faculty of Biological Sciences, University of Malakand,
Chakdara 18000, KP, Pakistan; asifnawaz2446@gmail.com
5Department of Pharmacy, University of Swabi, Swabi 23561, KP, Pakistan; fida2k9@yahoo.com
*Correspondence: matermaha@gmail.com (M.H.M.); sadiquom@yahoo.com (A.S.);
Tel.: +966-508734539 (M.H.M.)
Abstract:
Diabetes mellitus (DM) is a metabolic complication and can pose a serious challenge to
human health. DM is the main cause of many life-threatening diseases. Researchers of natural
products have been continuously engaged in treating vital diseases in an economical and efficient
way. In this research, we extensively used phytosteroids from Notholirion thomsonianum (Royle)
Stapf for the treatment of DM. The structures of phytosteroids
NtSt01
and
NtSt02
were confirmed
with gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR)
analyses. Through
in vitro
studies including
α
-glucosidase,
α
-amylase, and DPPH assays, compound
NtSt01
was found to be comparatively potent. An elevated dose of compound
NtSt01
was also
found to be safe in an experimental study on rats. With a dose of 1.0 mg/kg of NtSt01, the effect on
blood glucose levels in rats was observed to be 519
±
3.98, 413
±
1.87, 325
±
1.62, 219
±
2.87, and
116 ±1.33 mg/dL
on the 1st, 7th, 14th, 21st, and 28th, days, respectively. The
in vivo
results were
compared with those of glibenclamide, which reduced the blood glucose level to 107
±
2.33 mg/dL
on the 28th day. On the 28th day of
NtSt01
administration, the average weights of the rats and vital
organs (liver, kidney, pancreas, and heart) remained healthy, with a slight increase. The biochemical
parameters of the blood, i.e., serum creatinine, blood urea, serum bilirubin, SGPT (or ALT), and
serum alkaline phosphatase, of rats treated with
NtSt01
remained in the normal ranges. Similarly,
the serum cholesterol, triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein
(LDL) levels also remained within the standard ranges. It is obvious from our overall results that
the phytosteroids (specifically
NtSt01
) had an efficient therapeutic effect on the blood glucose level,
protection of vital organs, and blood biochemistry.
Keywords:
Notholirion thomsonianum; diabetes;
in vivo
and
in vitro
studies; phytosteroids; blood
biochemistry
1. Introduction
As a chronic disease, DM is a metabolic disorder in which insulin production by the
pancreas is reduced or the produced insulin may be ineffective, leading to hyperglycemia,
which can cause further damage to other body systems like the circulatory and nervous
systems. Some of the abnormalities that account for DM include defects in the production
of insulin or in its action or secretion and dysfunction in the metabolism of fat, protein, and
carbohydrates [
1
–
3
]. Polyphagia, polyuria, and polydipsia are the some of the symptoms
associated with the state of hyperglycemia [
4
]. There are about 450 million diabetic patients
Plants 2023,12, 3591. https://doi.org/10.3390/plants12203591 https://www.mdpi.com/journal/plants
Plants 2023,12, 3591 2 of 14
around the globe, with this figure expected to reach 690 million by the year 2044 [
5
].
Currently, DM prevalence has been reported as 8.5% of adults globally, and a rapid increase
has been observed in countries with a low or middle income [
6
]. DM is a serious metabolic
disease, and chronic hyperglycemia can cause numerous complications and the dysfunction
of several organs like the kidneys, nerves, eyes, heart, blood vessels, and liver [2,7,8].
There are different types of DM; the common types are type 1 and type 2 DM. Type
1 DM is insulin-dependent and is due to insulin deficiency, along with an impairment
of the
β
cells of the pancreas [
9
], whereas type 2 DM is non-insulin-dependent and is
due to insulin resistance or decreased insulin secretion [
10
]: 95% of diabetic patients have
type 2 DM [
11
]. DM development in people with an impaired tolerance of glucose can be
prevented or managed with the use of antidiabetic drugs or through changing their lifestyle
via exercise, diet control, and/or weight loss [
12
]. The use of antidiabetic drugs can have
number of adverse effects, including hypoglycemia, retention of fluids, osteoporosis, and
heart failure, due to which their use is limited [
13
–
15
]. Hence, the development of new,
effective drugs that have fewer adverse effects is needed to control and manage diabetes. In
the development of antidiabetic drugs that are specific for type 2 DM, several biochemical
approaches can be used. Among the important biochemical pathways, the inhibition of
α
-amylase and
α
-glucosidase is common. Both of these enzymes break down starch and
oligosaccharides into glucose, leading to an increase in the concentration of glucose; hence,
their inhibition is important for decreasing glucose absorption in the intestine [16].
For thousands of years, medicinal plants and natural products have been reported
for the treatment of many diseases, including diabetes, especially type 2 DM [
17
–
19
]. The
crude extracts of medicinal plants and their bioactive compounds have been found to be
useful in many pharmacological activities [
20
–
22
]. Approximately 400 plants have been
demonstrated to have antidiabetic activity, but only some of them have been evaluated for
their efficacy [
23
]. A number of natural products of plant origin have been shown to have an
antidiabetic activity. The most important reported phytochemicals include alkaloids, carbo-
hydrates, peptidoglycan, amino acids, glycosides, steroids, glycopeptides, galactomannan
gum, terpenoids, hypoglycans, guanidine, and inorganic ions [
24
]. Different plants and
microorganisms produce
α
-glucosidase and
α
-amylase inhibitors for the regulation of such
enzyme activities [
25
]. Synthetic compounds are also being developed in parallel in order
to create
α
-glucosidase and
α
-amylase inhibitors [
26
,
27
]. Inhibitors of
α
-amylase enzymes
reduce the conversion of starch into glucose usually after eating a meal, which results in a
decrease in the level of glucose in the blood. Therefore,
α
-amylase inhibitors are needed to
control the glucose levels of diabetic patients.
Notholirion thomsonianum is a small bulbous plant of the family Liliaceae. This small
lilium-like medicinal plant has been studied for various pharmacological effects [
28
]. The
plant can be used to improve the digestive system and in the management of microbial
infections [
29
]. Our group has been exploring the medicinal aspects of this species for a
decade. We have previously explored its crude extract for antibacterial, antifungal, and
analgesic effects [
29
,
30
]. In recent years, we explored the potential of this plant for the
management of diabetes mellitus using its various fractions and some bio-guided bioactive
compounds following multitarget
in vitro
and in silico approaches [
31
]. Based on our
previous experience with this plant, its hydroalcoholic extract contains phytosteroids,
which have been identified. This current study is extensive compared with our previously
published work. In this study, we extensively used these phytosteroids for investigating
in vitro
and
in vivo
antidiabetic targets. Furthermore, the beneficial effects of the identified
phytosteroids were also explored on the vital organs of the body like the liver, kidney,
pancreas, and heart and on blood biochemistry.
2. Results
2.1. Phytochemistry
In this research, we initially purified and identified two different phytosteroids (
NtSt01
and
NtSt02
, as shown in Figure 1). The isolated amount of
NtSt01
was 830 mg as a white
Plants 2023,12, 3591 3 of 14
powder, while 375 mg of
NtSt02
was isolated as a yellowish-brown solid. The structures
of these two isolated compounds were initially confirmed with GCMS analysis. The re-
tention time of compound
NtSt01
was 56.964 min, with a base peak value of 55.1 (Table
S1 Supporting Information). The fragmentation pattern of compound
NtSt01
is shown
in the Figure S1 of the Supporting Information. The spectrum and its fragmentation pat-
tern were compared with the library spectrum and the difference spectrum, as shown in
Figures S2 and S3 of the Supporting Information, respectively. The chemical name of the
identified compound
NtSt01
is 3-
β
-Acetoxystigmasta-4,6,22-triene with an IUPAC name
of (E)-17-(5-ethyl-6-methylhept-3-en-2-yl)-10,13-dimethyl-2,3,8,9,10,11,12,13,14,15,16,17-
dodecahydro-1H-cyclopenta[a]phenanthren-3-yl acetate. Similarly, from the same GCMS
analysis, the compound
NtSt02
was observed at a retention time of 57.812 min, with a major
peak at m/z135 (Table S2 of Supporting Information). The fragmentation pattern of the com-
pound
NtSt02
is shown in Figure S4 of the Supporting Information. This spectrum and its
fragmentation pattern were compared with the library spectrum and difference spectrum,
as shown in Figures S5 and S6 of the Supporting Information, respectively. The chemi-
cal name of the identified compound
NtSt02
is 4,6-cholestadien-3beta-ol, benzoate, and
the IUPAC name is 10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,8,9,10,11,12,13,14,15,16,17-
dodecahydro-1H-cyclopenta[a]phenanthren-3-yl benzoate.
Plants 2023, 12, x FOR PEER REVIEW 3 of 15
2. Results
2.1. Phytochemistry
In this research, we initially puried and identied two dierent phytosteroids
(NtSt01 and NtSt02, as shown in Figure 1). The isolated amount of NtSt01 was 830 mg as
a white powder, while 375 mg of NtSt02 was isolated as a yellowish-brown solid. The
structures of these two isolated compounds were initially conrmed with GCMS analysis.
The retention time of compound NtSt01 was 56.964 min, with a base peak value of 55.1
(Table S1 Supporting Information). The fragmentation paern of compound NtSt01 is
shown in the Figure S1 of the Supporting Information. The spectrum and its fragmenta-
tion paern were compared with the library spectrum and the dierence spectrum, as
shown in Figures S2 and S3 of the Supporting Information, respectively. The chemical
name of the identied compound NtSt01 is 3-β-Acetoxystigmasta-4,6,22-triene with an
IUPAC name of (E)-17-(5-ethyl-6-methylhept-3-en-2-yl)-10,13-dimethyl-
2,3,8,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl acetate.
Similarly, from the same GCMS analysis, the compound NtSt02 was observed at a reten-
tion time of 57.812 min, with a major peak at m/z 135 (Table S2 of Supporting Information).
The fragmentation paern of the compound NtSt02 is shown in Figure S4 of the Support-
ing Information. This spectrum and its fragmentation paern were compared with the
library spectrum and dierence spectrum, as shown in Figures S5 and S6 of the Support-
ing Information, respectively. The chemical name of the identied compound NtSt02 is
4,6-cholestadien-3beta-ol, benzoate, and the IUPAC name is 10,13-dimethyl-17-(6-
methylheptan-2-yl)-2,3,8,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phe-
nanthren-3-yl benzoate.
Figure 1. Chemical structures of the identied phytosteroids in Notholirion thomsonianum.
2.2. Alpha Glucosidase Inhibition
The in vitro α-glucosidase inhibitory results of both phytosteroids of N. thomsonia-
num (NtSt01 and NtSt02) are shown in Table 1. The percent inhibitions were recorded on
all concentrations in triplicate. NtSt01 was three times more potent than NtSt02, as ob-
served from their respective IC50 values. NtSt01 exhibited inhibitions of 85.00 ± 1.52, 81.52
± 1.85, 77.63 ± 1.56, 68.78 ± 1.02, and 61.22 ± 0.85% at experimental concentrations of 500,
250, 125, 62.50, and 31.25 μg/mL, respectively. The IC50 values of NtSt01 and NtSt02 were
7.34 and 22.87 μg/mL, respectively, in comparison to the standard drug, acarbose, with an
IC50 value of 2.14 μg/mL.
Table 1. Alpha-glucosidase inhibitions of the phytosteroids.
Comp/Standard
Conc (μg/mL)
Percent Inhibition
(Mean ± SEM)
IC50 (μg/mL)
NtSt01
500
250
85.00 ± 1.52 ***
81.52 ± 1.85 ***
7.34
Figure 1. Chemical structures of the identified phytosteroids in Notholirion thomsonianum.
2.2. Alpha Glucosidase Inhibition
The
in vitro α
-glucosidase inhibitory results of both phytosteroids of N. thomsonianum
(
NtSt01
and
NtSt02
) are shown in Table 1. The percent inhibitions were recorded on all
concentrations in triplicate.
NtSt01
was three times more potent than
NtSt02
, as observed
from their respective IC
50
values.
NtSt01
exhibited inhibitions of 85.00
±
1.52, 81.52
±
1.85,
77.63
±
1.56, 68.78
±
1.02, and 61.22
±
0.85% at experimental concentrations of 500, 250,
125, 62.50, and 31.25
µ
g/mL, respectively. The IC
50
values of
NtSt01
and
NtSt02
were 7.34
and 22.87
µ
g/mL, respectively, in comparison to the standard drug, acarbose, with an IC
50
value of 2.14 µg/mL.
2.3. Alpha Amylase Inhibition
The
in vitro α
-amylase activities of
NtSt01
and
NtSt02
were also analyzed in compari-
son to the standard acarbose, as shown in Table 2. Likewise,
NtSt01
was found with very
practical result which was eleven folds more potent than
NtSt02
. The IC
50
value of
NtSt01
and
NtSt02
were 4.17 and 46.73
µ
g/mL respectively in comparison to the standard drug
acarbose with the IC
50
value of 1.96
µ
g/mL. The
NtSt01
demonstrated percent inhibitions
of 80.03
±
2.11, 75.52
±
0.96, 71.63
±
0.92, 67.63
±
2.51 and 62.35
±
1.78% at experimental
concentrations of 500, 250, 125, 62.50 and 31.25 µg/mL respectively.
Plants 2023,12, 3591 4 of 14
Table 1. Alpha-glucosidase inhibitions of the phytosteroids.
Comp/Standard Conc (µg/mL) Percent Inhibition
(Mean ±SEM) IC50 (µg/mL)
NtSt01
500
250
125
62.50
31.25
85.00 ±1.52 ***
81.52 ±1.85 ***
77.63 ±1.56 ***
68.78 ±1.02 ***
61.22 ±0.85 ***
7.34
NtSt02
500
250
125
62.50
31.25
77.56 ±3.22 ***
70.63 ±2.45 ***
64.52 ±3.15 ***
59.98 ±1.88 ***
52.63 ±1.52 ***
22.87
Standard Drug
500
250
125
62.50
31.25
92.65 ±0.55
89.53 ±1.45
83.89 ±2.65
78.63 ±1.98
70.52 ±2.63
2.14
All the values are expressed as mean
±
SEM compared with the standard. Two-way ANOVA followed by
Dunnett’s test was applied. ***, significantly different (p< 0.001) compared with standard drug.
Table 2. Alpha amylase inhibitions of the phytosteroids.
Comp/Standard Conc (µg/mL) Percent Inhibition
(Mean ±SEM) IC50 (µg/mL)
NtSt01
500
250
125
62.50
31.25
80.03 ±2.11 **
75.52 ±0.96 **
71.63 ±0.92 **
67.63 ±2.51 **
62.35 ±1.78 **
4.17
NtSt02
500
250
125
62.50
31.25
74.99 ±1.53 ***
64.32 ±1.85 ***
60.04 ±0.86 ***
53.10 ±2.05 ***
46.84 ±0.67 ***
46.73
Standard Drug
500
250
125
62.50
31.25
91.01 ±1.36
87.79 ±1.27
82.33 ±1.00
75.63 ±0.86
71.07 ±1.82
1.96
All the values are expressed as mean
±
SEM compared with the standard. Two-way ANOVA followed by
Dunnett’s test was applied. ** = p< 0.01, *** = p< 0.001 compared with standard drug.
2.4. Antioxidant Assay
Antioxidant activity is a very important supplementary activity in antidiabetic studies.
Antioxidants combat excessive free radicals within the body. This concept applies to the
reduction of free radicals in the pancreas, which partially protects the pancreas from damage
and helps with diabetic control. Keeping this concept in mind, we also determined the
antioxidant activity of the phytosteroids. Though both of our phytosteroids demonstrated
low antioxidant activity profiles, they were both notable as supplementary targets. The
observed IC
50
values for
NtSt01
and
NtSt02
were 142.76 and 223.43
µ
g/mL, respectively,
as shown in Table 3.
Plants 2023,12, 3591 5 of 14
Table 3. Antioxidant potential of the phytosteroids.
Comp/Standard Conc (µg/mL) Percent Inhibition
(Mean ±SEM) IC50 (µg/mL)
NtSt01
500
250
125
62.50
31.25
61.05 ±2.05 ***
55.79 ±0.92 ***
47.08 ±2.41 ***
42.53 ±0.69 ***
37.25 ±2.66 ***
142.76
NtSt02
500
250
125
62.50
31.25
59.12 ±1.96 ***
52.01 ±1.07 ***
42.99 ±2.70 ***
33.08 ±1.17 ***
28.52 ±2.63 ***
223.43
Standard Drug
500
250
125
62.50
31.25
91.52 ±2.52
85.04 ±0.63
81.54 ±1.28
77.87 ±1.49
75.37 ±1.69
0.74
All the values are expressed as mean
±
SEM compared with the standard. Two-way ANOVA followed by
Dunnett’s test was applied. *** = p< 0.001 compared with the standard drug.
2.5. In Vivo Results
Based on the enzymatic assays, we observed that
NtSt01
is a potential inhibitor of
α
-glucosidase and
α
-amylase. With these results, we further analyzed the compound
NtSt01
in
in vivo
studies using experimental rats following ethical guidelines. The dose
of
NtSt01
was started at 200 and was increased up to 2000 mg/kg body weight. The rats
remained healthy; no morbidity, mortality or irritation was observed for the tested doses
during the acute toxicity studies.
Phytosteroid
NtSt01
(1.0 mg/kg) was administered to alloxan-induced fasting diabetic
rats, and the blood glucose levels were observed until 28 days, as shown in Table 4.
The blood glucose level in the normal control group remained within the normal range
during the observational time. In contrast, the blood glucose level of the diabetic control
group remained elevated throughout the experiment. At 1.0 mg/kg of
NtSt01
, the effect
on the blood glucose levels in rats were 519
±
3.98, 413
±
1.87, 325
±
1.62, 219
±
2.87
and
116 ±1.33 mg/dL
on days 1, 7, 14, 21, and 28, respectively. In comparison, in the
glibenclamide control group (0.5 mg/kg), the blood glucose level dropped from 517
±
0.77
(day 1) to 103 ±1.70 mg/dL (day 28).
Table 4.
Observational changes in blood glucose level in alloxan-induced fasting diabetic rats in
mg/dL.
Groups Day 1 Day 7 Day 14 Day 21 Day 28
Normal control 102 ±2.36 106 ±0.98 105 ±1.77 110 ±0.91 109 ±1.07
Diabetic control 521 ±1.28 517 ±2.06 526 ±1.00 524 ±2.66 529 ±2.80
Glibenclamide (0.5 mg/kg) 517 ±0.77 398 ±2.08 302 ±1.38 207 ±1.91 103 ±1.70
NtSt01 (1.0 mg/kg) 519 ±3.98 413 ±1.87 325 ±1.62 219 ±2.87 116 ±1.33
During the observational time period, the effect on the body weight (in grams) of
the rats was also closely observed (as shown in Table 5). In the normal control group, the
body weight of the rats remained the same throughout the four weeks. In the diabetic
control group, we observed a body weight loss of 18 g of rats at four weeks. With the
administration of phytosteroid
NtSt01
at a concentration of 1.0 mg/kg, the rats became
healthy, and there was a gain in body weight. The body weight changed from 238
±
0.33 g
Plants 2023,12, 3591 6 of 14
(day 1) to 246
±
0.11 g (day 28) in our sample. In comparison, the weight of the rats in the
glibenclamide control group also increased slightly.
Table 5. The effect of NtSt01 on body weight in grams of fasting rats.
Groups Day 1 Day 7 Day 14 Day 21 Day 28
Normal control 226 ±0.98 227 ±1.27 228 ±0.63 229 ±1.67 233 ±0.48
Diabetic control 232 ±2.69 229 ±1.98 225 ±1.39 217 ±2.69 214 ±3.09
Glibenclamide (0.5 mg/kg) 235 ±1.36 236 ±1.37 239 ±0.67 244 ±2.38 249 ±3.18
NtSt01 (1.0 mg/kg) 238 ±0.33 240 ±0.49 242 ±1.04 245 ±1.11 246 ±0.11
Diabetes mellitus is a perilous disease, and it affect all the vital organs of the body.
After the
in vivo
experiments, the rats from the different groups were euthanized following
the ethical procedure. The vital organs were isolated, and the weights were recorded, as
shown in Table 6. The weights of the liver, kidney, pancreas, and heart were
9.83 ±0.92
,
1.01
±
0.15, 0.92
±
0.12, and 1.04
±
0.13 g, respectively, in the
NtSt01
-administered group.
In diabetic control group, we observed a drastic effect on the average weights of the
vital organs.
Table 6. The effect of NtSt01 on vital organ weights in grams of fasting rats.
Groups Liver Kidney Pancreas Heart
Normal control 9.16 ±0.45 0.97 ±0.13 0.88 ±0.02 1.02 ±0.11
Diabetic control 6.38 ±0.10 1.26 ±0.37 0.71 ±0.13 0.83 ±0.08
Glibenclamide (0.5 mg/kg) 9.21 ±0.66 0.99 ±0.16 0.84 ±0.21 0.90 ±0.02
NtSt01 (1.0 mg/kg) 9.83 ±0.92 1.01 ±0.15 0.92 ±0.12 1.04 ±0.13
The results of the renal function and liver function tests are summarized in Table 7.
The serum creatinine, blood urea, serum bilirubin, ALT, and serum alkaline phosphatase of
the normal, diabetic, and standard (glibenclamide) groups were compared with the results
of phytosteroid
NtSt01
. In the normal group, all the parameters were within the normal
range. In the diabetic group, elevated blood urea, ALT, and serum alkaline phosphatase
levels were observed. In a pattern like that of the standard group, the phytosteroid
NtSt01
group exhibited normal serum creatinine, blood urea, and serum bilirubin values. However,
the ALT and serum alkaline phosphatase values were near the upper normal limit. These
overall results showed that compound
NtSt01
was effective in protecting the vital organs
from damage over the four weeks of the experiment.
Table 7. Biochemical profile of blood in alloxan-induced diabetic rats.
Test N.Control D.Control Standard NtSt01
(1.0 mg/kg) Unit Reference
Range
S.Creatinine 0.42 ±0.02 0.96 ±0.10 0.43 ±0.01 0.6 ±0.02 mg/dL 0.4–0.8
Blood Urea 19.1 ±0.17 187 ±3.65 20.2 ±0.33 19.1 ±2.36 mg/dL 15–22
S.bilurubin 0.71 ±0.24 0.96 ±0.01 0.81 ±0.03 0.72 ±0.77 mg/dL Up to 1.0
SGPT(ALT) 29.4 ±1.07 268 ±1.37 37 ±1.11 32 ±0.25 U/L 17–30
S.ALK.Phosphatase 114.7 ±0.39 159 ±1.07 125 ±1.22 134 ±2.33 U/L 30–130
The lipid profile is a major concern in the evaluation of serum triglycerides, total
cholesterol, high-density cholesterol (HDL), and low-density cholesterol (LDL). The results
Plants 2023,12, 3591 7 of 14
of lipid profile of all the experimental groups are summarized in Table 8. Except for the
serum cholesterol, all other values were within the normal ranges.
Table 8. Antilipidemic effects of NtSt01 on alloxan-induced diabetic rats.
Groups S.Cholesterol S.Triglycerides HDL LDL
Normal control 52.06 ±1.04 93.54 ±1.37 43.50 ±2.35 23.88 ±0.82
Diabetic control 280.63 ±2.33 456 ±3.21 40.76 ±1.49 165.5 ±3.65
Glibenclamide (0.5 mg/kg) 59.98 ±0.99 112 ±0.99 32.55 ±2.35 34.16 ±2.66
NtSt01 (1.0 mg/kg) 77.45 ±1.72 125 ±3.65 38.10 ±1.64 48.85 ±1.08
References Range 10–54 26–145 Up to 50 10–54
2.6. Molecular Docking Studies
For the determination of the pharmacological parameters of the ligand with targeted
protein moieties, using the fit model theory with ligand–enzyme interactions, docking
studies were performed. These docking results were examined to understand the different
interaction parameters.
NtSt01
was docked with the targeted macromolecules to analyze
the binding affinity. It was found to have excellent binding affinity with an energy of
−
8.84 Kcal/mol against
α
-glucosidase 7K9Q and -8.9 Kcal/mol against
α
-amylase 4W93.
The results of the interaction with
α
-glucosidase are depicted in Figure 2. The prominent
interactions are Arg 500 and Asn 551, respectively.
Plants2023,12,xFORPEERREVIEW8of16
Tab l e8.AntilipidemiceffectsofNtSt01onalloxan-induceddiabeticrats.
GroupsS.CholesterolS.TriglyceridesHDLLDL
Normalcontrol52.06±1.0493.54±1.3743.50±2.3523.88±0.82
Diabeticcontrol280.63±2.33456±3.2140.76±1.49165.5±3.65
Glibenclamide(0.5mg/kg)59.98±0.99112±0.9932.55±2.3534.16±2.66
NtSt01(1.0mg/kg)77.45±1.72125±3.6538.10±1.6448.85±1.08
ReferencesRange10–5426–145Upto5010–54
2.6.MolecularDockingStudies
Forthedeterminationofthepharmacologicalparametersoftheligandwithtargeted
proteinmoieties,usingthefitmodeltheorywithligand–enzymeinteractions,docking
studieswereperformed.Thesedockingresultswereexaminedtounderstandthedifferent
interactionparameters.NtSt01wasdockedwiththetargetedmacromoleculestoanalyze
thebindingaffinity.Itwasfoundtohaveexcellentbindingaffinitywithanenergyof−8.84
Kcal/molagainstα-glucosidase7K9Qand-8.9Kcal/molagainstα-amylase4W93.There-
sultsoftheinteractionwithα-glucosidasearedepictedinFigure2.Theprominentinter-
actionsareArg500andAsn551,respectively.
Figure2. The(a)3Dand(b)2DvisualizationsofNtSt01withα-glucosidase.
NtSt01alsodisplayedexcellentresults,withgreatbindingaffinitiesagainstα-amyl-
ase.TheresultsaredepictedinFigure3.Theprominentinteractionswerefoundtobewith
Trp59,His101,Leu162,His201,Ala198,andIle235,respectively.
Figure 2. The (a) 3D and (b) 2D visualizations of NtSt01 with α-glucosidase.
NtSt01
also displayed excellent results, with great binding affinities against
α
-amylase.
The results are depicted in Figure 3. The prominent interactions were found to be with Trp
59, His 101, Leu 162, His 201, Ala 198, and Ile 235, respectively.
Plants 2023,12, 3591 8 of 14
Plants2023,12,xFORPEERREVIEW9of16
Figure3. The(a)3Dand(b)2DvisualizationofNtSt01withα-amylase.
3.MaterialsandMethods
3.1.Phytochemistry
WecollectedrhizomesofNotholirionthomsonianum,whichwerealsousedinourpre-
viousresearch[29–31].Inthisstudy,wesubjectedthehydroalcoholicextractofN.thom‐
sonianumtoisolation.Initially,weusedalarge-diametergravitycolumntopartiallypurify
thesample.Thesolventsystemusedinthischromatographyincludedn-hexaneandethyl
acetate.Initially,thecolumnwasstartedwithpuren-hexanefora100mLelution.Then,
thepolarityofsolventwasgraduallyincreasedbyadding5%ethylacetateeachtime,and
thefractionswerecollected.Thefractionswerepreliminarycheckedviathin-layerchro-
matography(TLC)analysis.Oneofthefractionscontainingtwomajorandafewminor
spotswasconcentratedandsubjectedtoasmallcolumnforfurtherpurification.Theelu-
tionfractionsweremonitoredcloselyandcollectedinseparatedvials.Thevialswithma-
jorcoelutedspotswerecombinedanddriedviaarotaryevaporator.Thetwomajorspots
weresubjectedtoGCMSanalysisfortheidentificationofthecomponentsasperthepre-
viouslydescribedprocedure[32].
3.2.Alpha‐GlucosidaseInhibition
Thealpha-glucosidaseinhibitoryeffectsofourcompoundsweredeterminedviaa
reportedprotocol[31].Sampledilutions(50µL)and100µLofα-glucosidasesolution(0.5
U/mL)weremixed.Then,600µLofphosphatebuffer(0.1M,pH6.9)wasadded,andthe
mixturewasincubatedat37°Cfor15min.Afterthis,5mMsubstrate(p-nitrophenylα-D-
glucopyranoside)solution(20µL)in0.1Mphosphatebuffer(pH6.9)wasaddedandagain
incubatedunderthesameconditions.Sodiumcarbonatewasaddedtostopthereaction;
at405nm,theabsorbancewasnotedusingaspectrophotometer.Amixturewithoutα-
glucosidaseservedastheblank,andamixturewithoutthetestcompoundservedasthe
control.Thepercentenzymeinhibitionwascalculatedas:
%Alphaglucosidaseinhibition . .
. 100(1)
3.3.Alpha‐AmylaseInhibition
Figure 3. The (a) 3D and (b) 2D visualization of NtSt01 with α-amylase.
3. Materials and Methods
3.1. Phytochemistry
We collected rhizomes of Notholirion thomsonianum, which were also used in our
previous research [
29
–
31
]. In this study, we subjected the hydroalcoholic extract of N.
thomsonianum to isolation. Initially, we used a large-diameter gravity column to partially
purify the sample. The solvent system used in this chromatography included n-hexane and
ethyl acetate. Initially, the column was started with pure n-hexane for a 100 mL elution.
Then, the polarity of solvent was gradually increased by adding 5% ethyl acetate each time,
and the fractions were collected. The fractions were preliminary checked via thin-layer
chromatography (TLC) analysis. One of the fractions containing two major and a few
minor spots was concentrated and subjected to a small column for further purification. The
elution fractions were monitored closely and collected in separated vials. The vials with
major coeluted spots were combined and dried via a rotary evaporator. The two major
spots were subjected to GCMS analysis for the identification of the components as per the
previously described procedure [32].
3.2. Alpha-Glucosidase Inhibition
The alpha-glucosidase inhibitory effects of our compounds were determined via a
reported protocol [
31
]. Sample dilutions (50
µ
L) and 100
µ
L of
α
-glucosidase solution
(
0.5 U/mL
) were mixed. Then, 600
µ
L of phosphate buffer (0.1 M, pH 6.9) was added, and
the mixture was incubated at 37
◦
C for 15 min. After this, 5 mM substrate (p-nitrophenyl
α
-D-glucopyranoside) solution (20
µ
L) in 0.1 M phosphate buffer (pH 6.9) was added
and again incubated under the same conditions. Sodium carbonate was added to stop
the reaction; at 405 nm, the absorbance was noted using a spectrophotometer. A mixture
without
α
-glucosidase served as the blank, and a mixture without the test compound
served as the control. The percent enzyme inhibition was calculated as:
% Alpha glucosidase inhibition =
Control abs.−Sample abs.
Control abs.×100 (1)
3.3. Alpha-Amylase Inhibition
The inhibitory activity of our compounds against alpha-amylase enzymes was deter-
mined as per the previously reported protocols [
31
]. Test samples of 100
µ
L were mixed
with an enzyme solution (200
µ
L) and 100
µ
L of phosphate buffer (pH 6.9, 2 mM). The
mixture was then incubated at 25
◦
C for 20 min, followed by the addition of 100
µ
L of
Plants 2023,12, 3591 9 of 14
starch solution (1%). The same procedure was followed for positive controls, where phos-
phate buffer was added instead of enzyme solution (200
µ
L). After 5 min of incubation,
3,5-dinitrosalicylic acid reagent (500
µ
L) was added to the test samples and control group.
The mixtures were incubated again for 10 min; at 580 nm, the absorbance was recorded
using a spectrophotometer. The alpha-amylase percent inhibition was calculated as:
% Amylase inhibition =1−Test sample abs.
Control abs.×100 (2)
3.4. Antioxidant Assay
To evaluate the antioxidant potential of our compounds, the DPPH free radical scav-
enging assay developed by Brand William was used [
33
]. The DPPH solution was made
by dissolving 20 mg of DPPH in 100 mL of methanol, and its absorbance at 517 nm was
adjusted to 0.75. Then, 2 mL of DPPH solution was added to 2 mL of sample dilutions
ranging from 31.25 to 500 µg/mL, and the mixture was incubated at room temperature in
the dark for 15 min. The absorbance was noted at 517 nm, and the following formula was
used to determine the % inhibition of DPPH free radicals.
% Inhibition =
Control abs.−Sample abs.
Control abs.×100 (3)
3.5. Molecular Docking Studies
The docking studies were performed using Auto Dock Vina 1.2.2. PyRx. The three-
dimensional structure of ligand
NtSt01
was drawn in ChemDraw 20.0 software and saved
as a Mol.file. The structure was modified by adding polar hydrogen using Discovery
Studio Visualizer and saved in PDB format. The three-dimensional structures of both
targeted proteins,
α
-glucosidase and
α
-amylase, were acquired from the RCSB protein data
bank (http://www.rcsb.org accessed on 22 September 2023) as PDB id 4W93 and 7K9Q,
respectively, and saved in PDB format. Before starting the computational studies on the
ligand, the docking process protocols were validated through a redocking process. These
ligands and targeted protein structures were permitted for energy minimization through
the Charm force field factor, which detached the unwanted crystallographic observations.
The ligand and targeted protein structures were opened in Autodock Vina and converted
to ligands and macromolecules as Pdbqt molecules. The grid box was adjusted as center X:
10.112, Y: 68.356, and Z: 32.853, with dimensions (Angstrom) X: 75.2435, Y: 105.3254, and Z:
103.4758. The results were visualized through Discovery Studio Visualizer software 2017
R2 [22].
3.6. In Vivo Experiments
3.6.1. Experimental Animals and Ethical Approval
The experimental rats were purchased from the Breeding House of National Institute
Health, Islamabad, Pakistan. The average weights of these rats ranged from 220 to 250 g.
The animals were handled as per the guidelines of the University of Malakand animals
By-Laws 2008 (Scientific Procedure Issue I) under the approval of the ethical committee via
letter No. DREC-140/B. The food, water, and light/dark cycles provided to animals were
observed by the ethical committee [
34
]. The animals were euthanized as per the AVMA
guidelines version 2020. The animals were subjected to slow exposure of halothane vapors
to induce anesthesia. There was a gradual increase in the dosage of halothane vapors,
which eventually euthanized the animals [35].
3.6.2. Acute Toxicity
In experimental animals, acute toxicity studies were performed to determine the safe
dose of our test compounds for
in vivo
studies. Test compounds were administered at
increasing doses of up to 2000 mg/kg, and rats were observed for any aberrant behavior or
lethality [34].
Plants 2023,12, 3591 10 of 14
3.6.3. Induction of Diabetes
Alloxan monohydrate (Sigma Aldrich; Steinhein, Switzerland) was used to induce
diabetes at a dose of 160 mg/kg in the experimental animals [
36
]. The experimental animals
were kept in fasting mode for 8–12 h but allowed water only before being subjected to the
bioassay. The level of blood glucose was checked after 48 h of administration of alloxan
using a glucometer, and only the animals that were diabetic were considered for the study,
having a blood glucose level of more than 200 mg/dL [34].
3.6.4. Experimental Design
The antidiabetic activity of our test compounds was studied in alloxan-induced dia-
betic animals. All the experimental animals were placed into four different groups, with
6 animals in each group.
Group 1:
Only normal saline i.p was given to this nondiabetic group throughout the
experimental period.
Group 2:
Alloxan was administered i.p to this group for diabetes induction and rats
were under observation without any treatment throughout the experiment.
Group 3:
This group was the alloxan-induced diabetic group, which was treated with
0.5 mg/kg of the standard drug, glibenclamide.
Group 4:
This group was the alloxan-induced diabetic group, which was treated with
1.0 mg/kg of NtSt01 intraperitoneally.
3.6.5. Lipid Profile
Standard methods were used for the analysis of total cholesterol, serum triglycerides,
low-density lipoprotein (LDL), and high-density lipoprotein (HDL). Briefly, 10 mL of serum
sample was added to 1000 mL solution of triglyceride. The mixture was incubated for
10 min
at 37
◦
C and the absorbance was recorded at 546 nm. To monitor blood cholesterol,
diagnostic kits were utilized following the specifications of the manufacturer. A total
of 10
µ
L of serum sample was added to 1000
µ
L of cholesterol solution, followed by
incubation for 10 min at 37
◦
C; the absorbance against blank was noted at 546 nm. For
HDL concentration determination, 200
µ
L of serum sample and 500
µ
L of HDL solution
were combined, and the mixture was allowed to stand at room temperature for 5 min.
Again, the mixture was mixed and subjected to further centrifugation for 5 min, followed
by supernatant collection. Then, 50
µ
L of supernatant of HDL was added to 500
µ
L of
solution of cholesterol; the mixture was incubated at 37
◦
C for 5 min. Then, at 546 nm, the
sample absorbance was measured. Similarly, LDL was determined in all groups following
the manufacturer’s specifications [37].
LDL = Total cholesterol + HDL −Triglycerides/5 (4)
3.6.6. Renal Functions Tests
Renal functions tests were performed to determine blood urea and serum creati-
nine [
38
]. To determine blood urea, enzyme reagent 1 (1000
µ
L) and 10
µ
L of serum were
mixed together and incubated for 5 min at 25
◦
C, followed by the addition of 1000
µ
L of
reagent 2 to the mixture. After 5 min, the absorbance was recorded at 578 nm. The serum
creatinine was measured very carefully, as it is highly sensitive reaction to temperature.
Then, 500
µ
L of reagent and 50
µ
L of serum were mixed, followed by incubation for 1 min
at 37 ◦C, and the absorbance was noted at 500 nm.
3.6.7. Liver Functions Tests
Serum glutamate pyruvate transaminase (SGPT/ALT), alkaline phosphatase (ALP),
and bilirubin tests were performed following standard protocols using micro lab 300 and
tecno plus biochemistry analyzers [
39
]. To perform the SGPT/ALT test, 50
µ
L of serum
sample was added to 500 µL of reagent (400 µL of reagent 1 and 100 µL of reagent 2). The
mixture was incubated at 37
◦
C for 30 s, and the absorbance was noted at 340 nm. To
Plants 2023,12, 3591 11 of 14
determine ALP, a kit was used following the manufacturer’s specifications: 10
µ
L of serum
sample was added to 500 µL of reagent (400 µL of reagent 1 and 100 µL of reagent 2). The
mixture was then incubated at 37
◦
C for 30 s, and the absorbance was noted at 405 nm.
Similarly, the concentration of bilirubin was evaluated using standard protocols. Four
types of reagents, R1, R2, R3, and R4, were used in this test. R2 (25
µ
L) was added to R1
(
100 µL
). After this,
100 µL
of serum sample and 500
µ
L of R3 were added and allowed to
stand at
25 ◦C
for 5 min. Then, 500
µ
L of R4 was added; at 25
◦
C for 5 min, the mixture
was incubated. The absorbance of the sample was recorded at 546 nm.
3.6.8. Statistical Analysis
Two-way ANOVA followed by Dunnett’s post-test were applied for the comparison of
the positive control with the test groups using GraphPad prism 8.0.1 software. pvalues less
than or equal to 0.05 were considered statistically significant. The findings of the statistical
analysis are shown as mean ±SEM.
4. Discussion
For thousands of years, number of medicinal plants and natural products have been re-
ported for the treatment of many diseases including diabetes [
40
–
42
]. Metformin, obtained
from Galega officinalis, has been the first-line drug used for 60 years for the treatment of type
2 diabetes [
43
]. Plant-based drugs have minimal or no side effects compared with synthetic
drugs; therefore, researchers have mainly focused on natural products for developing new
drugs. In DM, the main treatment strategy involves controlling high glucose levels in the
blood. Apart from this,
α
-amylase and
α
-glucosidase enzymes inhibitions are important
strategies for controlling hyperglycemia, as these enzymes convert starch into glucose,
resulting in increased levels of blood glucose [
44
]. For Notholirion thomsonianum, the
in vitro
antidiabetic properties of the
in vitro
targets of
α
-amylase,
α
-glucosidase, and tyrosine
phosphatase 1B, and the antioxidant potential have been studied using free radical assays
of ABTS, DPPH, and H
2
O
2
[
1
]. In this study, we determined the antidiabetic potential of
Notholirion thomsonianum through
in vitro
(
α
-amylase and
α
-glucosidase inhibition) and
in vivo studies.
The
in vitro
antidiabetic properties of Notholirion thomsonianum were determined
against
α
-amylase and
α
-glucosidase.
α
-Amylase converts starch into disaccharides and
oligosaccharides, whereas
α
-glucosidase hydrolyzes disaccharides into glucose [
45
].
α
-
Amylase and
α
-glucosidase enzymes, if inhibited, decrease the glucose level via the break-
down of starch in GIT, which is retarded via the inhibition of these enzymes, which
ameliorates hyperglycemia in diabetic patients. In the
α
-glucosidase inhibition assay, the
compounds
NtSt01
and
NtSt02
exhibited inhibitions of 85.00
±
1.52 and 77.56
±
3.22%
at the highest concentration (500
µ
g/mL), with IC
50
values of 7.34 and 22.87
µ
g/mL,
respectively. Compounds
NtSt01
and
NtSt02
exhibited 80.03
±
2.11 and 74.99
±
1.53%
inhibition at 500
µ
g/mL against
α
-amylase with IC
50
values of 4.17 and 46.73
µ
g/mL,
respectively. However, at the same tested concentration (500
µ
g/mL), the standard drug
acarbose demonstrated 92.65
±
0.55% inhibition against
α
-glucosidase and 91.01
±
1.36%
inhibition against α-amylase with IC50 values of 2.14 and 1.96 µg/mL, respectively.
In the blood glucose test, diabetes induction with alloxan was confirmed, and the blood
glucose levels in the diabetic group were observed as 521
±
1.28, 517
±
2.06,
526 ±1.00
,
524
±
2.66, and 529
±
2.80 mg/dL on days 1, 7, 14, 21 and 28, respectively. Treatment of
diabetic animals with
NtSt01
at a dose of 1.0 mg/kg produced a decline in blood glucose
levels, i.e., 519
±
3.98, 413
±
1.87, 325
±
1.62, 219
±
2.87, and 116
±
1.33 mg/dL on the 1st,
7th, 21st, and 28th days of the treatment, respectively.
No statistical differences in the weights of the liver, kidney, pancreas, and heart
were observed among all groups after completion of the experiments. The kidney func-
tion profile, lipid profile, and biochemical profile of the test animals in all groups were
also determined. In the biochemical tests, blood urea, serum bilirubin, serum creatinine,
SGPT(ALT), and serum alkaline phosphatase levels were high in the diabetic disease group
Plants 2023,12, 3591 12 of 14
and decreased after treatment with 1.0 mg/kg
NtSt01
, as shown in Table 7. In the lipid tests,
considering serum cholesterol, serum triglycerides, LDL, and HDL, a significant decline
was demonstrated in our sample (
NtSt01
1.0 mg/kg) in comparison with the diabetic
animals, as shown in Table 8. Both the
in vitro
and
in vivo
antidiabetic results for
NtSt01
revealed the potential of this compound for the management of DM and as a multitarget
antidiabetic agent.
Molecular docking is an important approach to determine the binding energies and
interactions of a drug molecule for its target. We docked our isolated compounds against
α
-
glucosidase and
α
-amylase enzymes. The molecular docking studies revealed encouraging
binding interactions with the target proteins for our compounds.
Though the whole of our work is based on the development of an antidiabetic drug
from medicinal plant source, significant efforts have also been made by synthetic organic
chemists for the discovery of new antidiabetic agents [
46
]. Among the synthetic approaches,
the modification of immunosugar has been identified to develop potential inhibitors of
alpha-glucosidase [47–49].
5. Conclusions
Herein, we explored the antidiabetic potential of phytosteroids from Notholirion thom-
sonianum. In an attempt to combat diabetes and its consequences, weave used phytosteroid
NtSt01
as a natural drug. In the
in vitro α
-glucosidase,
α
-amylase, and DPPH assays,
compound
NtSt01
was found to be potent enough to be tested in experimental animals.
The compound was initially found safe in experimental rats and was then found effective in
combating induced diabetes over the four weeks of the experiment. After the experiment,
we also observed that the weights of the liver, kidney, pancreases, and heart and their
functions were within the allowed limits. Our overall results show that phytosteroids
(specifically
NtSt01
) have an efficient therapeutic effect on the blood glucose level and
other protective effects on organs like the liver, kidney, pancreases, and heart, so may serve
as a multitarget antidiabetic drug.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/plants12203591/s1, Table S1: GCMS results of compound
NtSt01
.
Figure S1: MS spectrum and fragmentation pattern of
NtSt01
. Figure S2: Library spectrum and
fragmentation pattern of
NtSt01
. Figure S3: Difference spectrum of
NtSt01
. Figure S4:
1
H NMR
spectrum of
NtSt01
. Table S2: GCMS results of compound
NtSt02
. Figure S5: MS spectrum and
fragmentation pattern of
NtSt02
. Figure S6: Library spectrum and fragmentation pattern of
NtSt02
.
Figure S7: Difference spectrum of NtSt02. Figure S8: 1H NMR spectrum of NtSt02.
Author Contributions:
M.A.H., S.F., A.A., S.M.A., A.N. and F.H.: conceptualization, methodology,
software, validation, formal analysis, investigation, resources, data curation, writing—original draft
preparation, and writing—review and editing; M.H.M. and A.S.: Conceptualization, methodology,
validation, investigation, resources, writing—original draft preparation, supervision, writing—review
and editing, visualization, and funding acquisition. All authors have read and agreed to the published
version of the manuscript.
Funding:
This research obtained funding under the National Research Priorities and Najran Area
Research funding program grant code number NU/NRP/MRC/12/40.
Institutional Review Board Statement:
The animals were handled as per the guidelines of the
University of Malakand animals By-Laws 2008 (Scientific Procedure Issue I) under the approval of
the ethical committee via letter No. DREC-140/B.
Data Availability Statement:
The data are presented in the manuscript and Supporting Information.
Acknowledgments:
The authors acknowledge support from the Deanship of Scientific Research, Najran
University, Kingdom of Saudi Arabia, for funding this work under the National Research Priorities and
Najran Area Research funding program grant code number NU/NRP/MRC/12/4). We are also grateful
to the Department of Pharmacy, University of Malakand, Pakistan, for the laboratory facilities.
Conflicts of Interest: The authors declare no conflict of interest.
Plants 2023,12, 3591 13 of 14
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