A novel layered double hydroxide-hesperidin nanoparticles exert antidiabetic, antioxidant and anti-inflammatory effects in rats with diabetes

Abstract

Background
Incidence of diabetes has increased significantly worldwide over recent decades. Our objective was to prepare and characterize a novel nano-carrier of hesperidin to achieve a sustained release of hesperidin and to explore the potency of the novel formula as an antidiabetic agent compared to metformin in type 2 diabetic rats.Methods
Hesperidin was loaded on MgAl-layered double hydroxide (LDH). The formula was characterized using Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), transmission electron microscopy, and dynamic light scattering. The release profile of hesperidin and MgAl-LDH-Hesperidin were studied in vitro. The parameters studied in vivo were blood glucose, glycated hemoglobin (HbA1c), insulin, lipid profile, and liver glycogen levels. We also investigated the levels of interleukin (IL)-17, tumor necrosis factor-Alfa (TNF-α), malondialdehyde (MDA), catalase, and the mRNA expression of peroxisome proliferator-activated receptor-gamma (PPARγ) and nuclear factor erythroid 2-related factor-2 (NrF2).ResultsThere were variations in the XRD patterns and FTIR confirming the physical adsorption of hesperidin on the surface of LDH. The results indicated that the diabetic rats treated with administration of antidiabetic formula, MgAl-LDH-Hesperidin, showed a beneficial effect on the levels of blood glucose, insulin, HbA1c%, and lipid profile, comparing to diabetic control rats. The antidiabetic agent also showed a significant decrease in the levels of TNF-α, IL-17, and MDA, and an increase in the level of catalase. Marked upregulation of the expression levels of mRNA for PPARγ and NrF2 were recorded.Conclusion
The novel nano-hesperidin formula MgAl-LDH-Hesperidin revealed a sustained release of hesperidin and exhibited antidiabetic, antihyperlipidemic, antioxidant, and anti-inflammatory properties, and also is a promising agent for effective delivery of drugs to treat type 2 diabetes.Graphical abstract

Full text

Vol.:(0123456789)
1 3
Molecular Biology Reports
https://doi.org/10.1007/s11033-021-06527-2
ORIGINAL ARTICLE
A novel layered double hydroxide‑hesperidin nanoparticles exert
antidiabetic, antioxidant andanti‑inflammatory effects inrats
withdiabetes
AhmedA.G.El‑Shahawy1· AdelAbdel‑Moneim2 · AbdelazimS.M.Ebeid3· ZienabE.Eldin4· MohamedI.Zanaty3
Received: 16 April 2021 / Accepted: 27 June 2021
© The Author(s), under exclusive licence to Springer Nature B.V. 2021
Abstract
Background Incidence of diabetes has increased significantly worldwide over recent decades. Our objective was to prepare
and characterize a novel nano-carrier of hesperidin to achieve a sustained release of hesperidin and to explore the potency
of the novel formula as an antidiabetic agent compared to metformin in type 2 diabetic rats.
Methods Hesperidin was loaded on MgAl-layered double hydroxide (LDH). The formula was characterized using Fourier
transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), transmission electron microscopy, and dynamic
light scattering. The release profile of hesperidin and MgAl-LDH-Hesperidin were studied invitro. The parameters studied
invivowere blood glucose, glycated hemoglobin (HbA1c), insulin, lipid profile, and liver glycogen levels. We also inves-
tigated the levels of interleukin (IL)-17, tumor necrosis factor-Alfa (TNF-α), malondialdehyde (MDA), catalase, and the
mRNA expression of peroxisome proliferator-activated receptor-gamma (PPARγ) and nuclear factor erythroid 2-related
factor-2 (NrF2).
Results There were variations in the XRD patterns and FTIR confirming the physical adsorption of hesperidin on the surface
of LDH. The results indicated that the diabetic rats treated with administration of antidiabetic formula, MgAl-LDH-Hes-
peridin, showed a beneficial effect on the levels of blood glucose, insulin, HbA1c%, and lipid profile, comparing to diabetic
control rats. The antidiabetic agent also showed a significant decrease in the levels of TNF-α, IL-17, and MDA, and an
increase in the level of catalase. Marked upregulation of the expression levels of mRNA for PPARγ andNrF2 were recorded.
Conclusion The novel nano-hesperidin formula MgAl-LDH-Hesperidin revealed a sustained release of hesperidin and
exhibited antidiabetic, antihyperlipidemic, antioxidant, and anti-inflammatory properties, and also is a promising agent for
effective delivery of drugs to treat type 2 diabetes.
* Adel Abdel-Moneim
adel_men2020@yahoo.com; adel.hassan@science.bsu.edu.eg
1 Materials Science andNanotechnology Department, Faculty
ofPostgraduate Studies forAdvanced Sciences (PSAS),
Beni-Suef University, Beni-Suef62511, Egypt
2 Molecular Physiology Division, Faculty ofScience,
Beni-Suef University, Salah Salem St, Beni-Suef62511,
Egypt
3 Biotechnology andLife Sciences Department, Faculty
ofPostgraduate Studies forAdvanced Sciences (PSAS),
Beni-Suef University, Beni-Suef, Egypt
4 Faculty ofVeterinary Medicine, Beni-Suef University,
Beni-Suef, Egypt

Molecular Biology Reports
1 3
Graphical abstract
Keywords Hesperidin· Layered double hydroxide· Type 2 diabetes· Lipid profile· Oxidative stress
Introduction
Diabetes mellitus is a chronic disease that occurs due to
insufficient insulin production by the pancreas, or inef-
ficient use by the body of the insulin produced [1]. It has
been recorded that 451 million people 18–99years of age
had diabetes in 2017, and nearly 5 million deaths world-
wide have been attributed to diabetic complications [2].
The management of complex illnesses like diabetes still
needs investigation. The axes linking inflammation, insulin
resistance, hyperlipidemia, and oxidative stress also need
more study [3]. Effective management of blood glucose
levels remains the key to controlling type 2 diabetes mel-
litus (T2DM), and strategies to stabilize blood glucose lev-
els play a crucial role in preventing or delaying the onset
and development of diabetic complications. Metformin,
which mitigates insulin resistance, is administered orally,
is the oldest and most commonly prescribed medication
for diabetes today [4]. Metformin, a natural plant product,
is highly pleiotropic making it appropriate for treating
complex metabolic disorders; nevertheless, there are seri-
ous concerns about its potential adverse effects, especially
ketoacidosis [5] and the risk of lactic acidosis [6].
Recently, the development of hypoglycemic agents from
natural products, especially plant material, has received
considerable attention, because plant sources are often low
cost and less toxic [7] than the synthetic counterparts. Fla-
vonoids have emerged as potential treatments for complex
illnesses involving multiple signaling pathways, such as
diabetes [8]. Hesperidin is an abundantly occurring fla-
vanone glycoside in citrus fruits. Hesperidin is also called
a bioflavonoid due to its range of activities [9]. Hesperidin
has pharmacological and biological functions, including
anti-carcinogenic, anti-inflammatory, hypolipidemic, and
antioxidant activities [10]. However, hesperidin has not
been frequently used clinically, because it has a relatively
low solubility in water. Several strategies have been devel-
oped to address this issue, including the development of
effective delivery systems for hesperidin-containing drugs
[11]. Nanosized drug carriers are a promising approach
to overcoming the low oral bioavailability of some sub-
stances. Nanotechnology could be valuable for the delivery
of poorly soluble or short-lived natural products [12]. A
drug delivery system with sustained release technology
could be applied to the treatment of a range of diseases.
Over the last decade, novel nanomaterials have been exam-
ined with respect to their biodegradability, biocompati-
bility, safety, mucoadhesive properties, and specificity of
targeting [13]. Different types of hesperidin-nano-carrier
administration have been used as delivery systems in nano-
medicine. Hesperidin-gold nanoparticles [14] and hesperi-
din-PLGA-Poloxamer [15] have anti-cancer properties.
Green synthesized nanoparticles based on plant gums, in

Molecular Biology Reports
1 3
conjunction with hesperidin can be used as novel antimi-
crobial agents [16], and zinc oxide-hesperidin nanoparti-
clesprotect against oxidative damage in the rat liver [17].
Layered double hydroxides (LDHs) may intercalate bio-
active molecules via anion exchange, making them potential
targeted carriers for anti-inflammatory agents and antibiot-
ics, [18, 19]. The lamellar architecture of these molecules
produces a high surface area to volume ratio, allowing
therapeutic agents to be intercalated and released only at
the target site, thereby reducing their side effects. Valuable
characteristics, such as ease of preparation, low cost, high
biocompatibility, and low cytotoxicity make these nanoma-
terials promising for biomedical application [20]. Several
types of organic and nonorganic anions may be inserted into
the hydroxide layer area by ion-exchange and co-precipita-
tion [21]. Hence, the LDH structure has garnered attention
as a drug carrier and a platform for the controlled delivery
of hesperidin. However, this is a relatively new technology,
and new insights are still required in this field. There have
been few investigations addressing the beneficial effects of
hesperidin-nano carrier delivery on different metabolic dis-
eases, especially diabetes. In this study, we introduced a new
LDH-Hesperidin nanoparticle formula to achieve a sustain
release of hesperidin. Besides, this study was conducted to
evaluate the efficacy of hesperidin and a novel MgAl-LDH-
Hesperidin as an antidiabetic, antihyperlipidemic, and anti-
inflammatory agent against streptozotocin-induced type 2
diabetes mellitus in albino rats.
Materials andmethods
Materials
Hesperidin and streptozotocin (STZ) were purchased from
Sigma chemicals Co., St. Louis, MO, and stored at 4°C.
Aluminum nitrate [Al (NO3)3.9H2O], magnesium nitrate
[Mg(NO3)2.6H2O], Hydrochloric acid (HCl), and sodium
hydroxide (NaOH) were purchased from El Gomhouria
Company for Trading Chemicals and Medical Appliances
(Cairo, Egypt). All other chemicals were of analytical grade
and were purchased from a standard commercial supplier.
Preparation ofMgAl‑ LDH andMgAl‑
LDH‑Hesperidin
Mg (NO3)2.6H2O (0.045 mol) and Al (NO3)3.9H2O
(0.015mol) at an Mg: Al molar ratio of 3:1 was dissolved
in 100ml of distilled water [22]. 320ml NaOH solution
(0.15M) had added dropwise until completely precipitated
at pH 8.5. The precipitate was stirred for 20h at 65°C,
filtered, washed several times with distilled water, and
dried at 40°C. MgAl-NO3 Layered double hydroxides
(LDH)-hesperidin was processed by repeating the same
methods and adding 0.03mol of hesperidin to the medium
before precipitation, so the molar ratio was 2:3:1 hesperidin:
Mg (NO3)2: Al (NO3)3. The MgAl-NO3 LDH-Hesperidin
precipitate suspension was stirred at 60°C for 20h, filtered,
washed, and dried at 40°C.
Characterization
X‑ray diffraction
The method was used to characterize the materials’ crystal-
linity, using Cu K α (Ţ = 1,54Å) radiation. The device uses
a 30mA current, a functioning voltage of 40kV (power
1200W), and a scanning speed of 2/min (step size = 0.050
and step time = 1.5s) in the 10–70 scanning range (2ϴ
scale). The crystalline phases have been characterized by
the corresponding International Center for Diffraction Data
(ICDD).
Fourier Transform Infrared Spectroscopy
The functional groups were determined using FTIR. The
FTIR spectra were obtained using a Bruker (Vertex 70 FTIR-
FT Raman) spectrometer. The absorption spectral of MgAl-
LDH, hesperidin, and MgAl-NO3 LDH-Hesperidin ranged
from 4500 to 500 cm−1. In the scans, the resolution was
1 cm−1, and the spectra of three scans were averaged.
Morphology
The surface morphology and particle size of the prepared
MgAl-LDH nanoparticles and MgAl-LDH-Hesperidin were
investigated using a high-resolution transmission electron
microscope (HR-TEM) (JEM 1400, Japan) operated at
300kV. A field emission scanning electron microscope
(FESEM; using Philips-XL30 device, The Netherlands)
equipped with energy dispersive X-ray microanalysis hard-
ware was also used. The microscopy samples were prepared
by the dispersal of the samples in deionized water, which
was diluted 1:5 (v/v) at room temperature.
Zeta Sizer andZeta potential measurements
The Zeta potential, hydrodynamic size, and colloidal stabil-
ity of the dispersed MgAl-LDH nanoparticles and MgAl-
LDH-Hesperidin particles were measured using photon
correlation spectroscopy or dynamic light scattering (DLS)
using a ZS90 Zetasizer instrument (Malvern, UK). The
change in the Zeta potential of MgAl- LDH nanoparticles
and MgAl-LDH-Hesperidin was measured in water disper-
sant with a refractive index of 1.330.

Molecular Biology Reports
1 3
UV–Vis spectra
The loading efficiency of the MgAl-LDH nanoparticles
was carried out using a UV–Vis spectrometer (CARY100,
Germany). The hesperidin loading and release percentage
were calculated at the absorption peak of hesperidin, λ
max = 285nm.
Entrapment efficiency
As in previous work [23], we determined the hesperidin
entrapment efficiency of MgAl-LDH nanoparticles. The for-
mulation was centrifuged at 14,000rpm at 4°C for 45min, to
separate the supernatant that contained free hesperidin. The
concentration of non-bound hesperidin was measured using
aUV–VISspectrophotometer at λ max = 285nm. All meas-
urements were executed in triplicate (n = 3). The hesperidin
entrapment efficiency (EE %) was calculated as follows:
Hesperidin invitro release
The release of hesperidin from MgAl-LDH-Hesperidin was
assayed according to dialysis bag method [24]. Five mil-
liliters of MgAl-LDH-Hesperidin nanoparticle suspension,
equivalent to 20mg of hesperidin, was placed in a dialysis
bag with a cellophane membrane, and a molecular weight
cutoff of 10,000–12,000Da. The dialysis bag was tied and
placed into 200ml of phosphate buffer (pH 6.8). The dialysis
was performed in a shaking incubator maintained at 37°C at
150rpm. Regarding the solubility, as we know that hesperi-
dinis sparinglysolublein aqueousbuffers, and to achieve a
maximum solubility in the aqueous buffer, the PBS release
media was mixed with a solution of DMSO [25], taking into
account that hesperidinhas asolubilityof approximately
0.5mg/ml in a 1:5solutionof DMSO: PBS(pH 7.2). At
different time intervals, samples were withdrawn and the
solution replenished with fresh buffer. The concentration
of hesperidin was measured using a spectrophotometer at λ
max = 285nm. The free hesperidin released under the same
conditions was assessed as a control. All measurements were
performed in triplicate.
Regarding the mechanism and kinetics of drug release,
the results of the invitro release study were fitted with vari-
ous kinetic equations, like zero order, first order, Higuchi’s
model, Hixson’s model, and Kor’s model, with calculations
to get the Correlation coefficient (R2) value for each one
(TableS1).
EE
%=
Total amount of Hesperidin −free amount of Hesperidin
Total amount of Hesperidin
×
100
In vitro cytotoxicity
The cytotoxicity of MgAl-LDH-Hesperidin toward WI38
normal lung cells was measured using MTT assays, invitro,
according to Mardani etal. [26]. The cells were cultured for
24h in a 96 96-well plate and then starved for six hours. The
cells were then treated with nanoparticles at concentrations of
0–100μM. MTT was added to each well after 48h of incuba-
tion and incubated for another four hours at a final concentra-
tion of 0.5mg/mL. A stop solution of 150 μL of dimethyl
sulfoxide was added to each well. A micro-platform reader
(Bio-Rad Type 680, Hercules, CA, USA) at 570nm was used
to measure the absorption, and the percent viability was cal-
culated. The percent of viable cells was calculated as follows:
The OD refers to the optical density.
In vivo study
Animals andinduction ofdiabetes
White male albino rats, aged four weeks and weighing
200–300g, were purchased from the Institute of Animal
Health, Beni-Suef. The rats were housed in special trans-
parent cages at a controlled temperature of 37°C, with a
12:12h light: dark cycle over a two-week adaptation period.
Type 2 diabetes mellitus was experimentally induced as fol-
lows: rats were fasted for 16h and then injected intraperi-
toneally with a single dose of nicotinamide (110mg/kg b.
w dissolved in 0.9% normal saline). After 15min, all rats
were injected intraperitoneally again with a dose of STZ
(65mg/kg body weight freshly dissolved in 5mmol/l citrate
buffer pH 4.5) [27]. One week after STZ injection, glucose
levels were monitored in all rats [28]. In this experiment,
rats fasted overnight for 12h were administered a dose of
glucose (3g/kg body weight.) by gastric intubation. After
two hours, blood samples were taken from a lateral tail vein
into a sodium fluoride tube, centrifuged, and the plasma
glucose level was then estimated. Rats with plasma glu-
cose ≥ 200mg/dL were used in the experiment and the others
were excluded. Experimental animals were signed and man-
aged according to the Institutional Animal Care Committee
(IACUC), Beni-Suef University, Egypt, (BSU/2018/11/17),
in compliance with the guidelines of the National Institute of
Health (NIH) for the use of laboratory animals.
Experimental design
Sixty diabetic rats were divided into six groups, (n = 10
in each group) and treated as follows: The first group was
normal, the second group comprised diabetic rats treated
Cell viability %=(OD of treated cells∕OD of untreated cells)×100

Molecular Biology Reports
1 3
with saline, the third group included diabetic rats treated
with MgAl-LDH, the fourth group contained diabetic rats
treated with hesperidin, the fifth group comprised diabetic
rats treated with MgAl-LDH-Hesperidin, and the sixth
group included diabetic rats treated with metformin. The
dose of each compound was 50mg/kg body weight, in the
form of an aqueous suspension [28], and was administrated
orally for 30 successive days. The weights of all rats were
recorded at the beginning and end of the study period. The
doses were changed each week according to the variations
in body weight.
Blood sample collection andbiochemical assay
andinvivo parameters
Blood samples were collected from all rats following over-
night fasting. The blood samples were obtained from the
retro-orbital venous plexus using a capillary pipette method.
The blood was collected into three tubes; the first tube con-
tained EDTA, the second tube contained sodium fluoride,
and the third tube was plain. The levels of plasma glucose,
serum cholesterol, high-density lipoprotein (HDL), and tri-
glycerides were estimated using kits obtained from Spinre-
act (Barcelona, Spain). Low-density-lipoprotein (LDL) and
very-low-density-lipoprotein (vLDL) were calculated using
Friendewald’s equation [29]. Free fatty acid was measured
using kits from Abcam (Cambridge, UK). Serum insulin
levels were determined using enzyme-linked immunosorbent
assay (ELISA) kits manufactured by Linco Research (Saint
Charles, MO), and glycosylated hemoglobin (HbA1c) per-
centage was estimated using kits from Helena Laboratories
(Beaumont, TX), according to the manufacturer’s protocol.
The liver glycogen was assessed using a method published
by Shokri-Afra etal. [30].
The homeostatic model assessment of insulin resist-
ance (HOMA-IR) and homeostasis model assessment
of β-cell function (HOMA β), were calculated accord-
ing to the following equations:[31]
The current study investigated catalase (CAT), malondial-
dehyde (MDA), tumor necrosis factor-alpha (TNF α), and
interleukin (IL)-17 in liver clearhomogenate samplesusing
ELISA kits purchased from MyBioSource (San Diego, CA),
according to the manufacturer’s instructions.
HOMA-IR =fasting insulin(U/L)×fasting glucose(mg/dL)∕405
(1)
HOMA
𝛽
=fasting insulin (U∕L)x 360]∕fasting glucose (mg∕dL)−63
Detection ofPPAR‑γ andNrF2 gene expression
byreal‑time PCR
Total RNA was extracted from frozen-liver samples using
TRIzol Reagent (MBI Fermentas, Germany) according to
the manufacturer’s instructions. cDNA synthesis was per-
formed using High-Capacity cDNA Reverse Transcription
Kits, (Invitrogen, Carlsbad, CA) according to the manu-
facturer’s instructions. Real-time PCR was conducted on a
20 μL system comprising 10 μL of 1 × SsoFast EvaGreen
Supermix (Bio-Rad, Hercules, CA), 2 μL of cDNA, 6 μL
of RNase/DNase-free water and 500nM of the primer pair
sequences: PPAR-g; F: 5′-AAG CCA TCT TCA CGA TGC
TG-3′, R: 5′-TCA GAG GTC CCT GAA CAG TG-3′; NRF2.
F: 5′-CAT TGA GGT GTA TTT CAC GG-3′, R: 5′-GGC AAG
TGG CCA TT G TG T TC-3′. B-actin, F: 5′-T G T TTG AGA CCT
TCA ACA CC-3′ R: 5′-CGC TCA TTG CCG ATA GTG AT-3′.
The conditions of the thermal cycler were as follows: 30s at
95°C, then 40 cycles of 5s at 95°C and 10s at 60°C. For
each reaction, a 65–95°C ramp was performed, with a melt-
ing curve study. With each process, the threshold duration at
which the fluorescent signal exceeded an arbitrarily defined
threshold close to the middle of the log-linear amplification
step was estimated, and the relative amount of mRNA was
calculated. The amplification data were analyzed following
the 2-∆∆Ct method of Livak and Schmittgen [32] using the
manufacturer’s software, and the values were normalized
to β-actin.
Histopathological studies
Histopathological microscopic examination was performed.
The rats were euthanized with a ketamine-xylazine mix at a
ratio of 1: 1 and a dosage of 0.1ml/100g at the end of the
study. The tissue of the pancreas was excised and preserved
in 10% formalin for 72h at room temperature. Sections of
4–5μm in thickness were dyed with Hematoxylin and Eosin
for examination [33].
Statistical analysis
Statistical analysis was conducted using the Statistical Pack-
age for Social Sciences (IBM SPSS for WINDOWS7, version
22, SPSS Inc., Chicago). Data were analyzed using one-way
analysis of variance (ANOVA). The results were expressed
as mean ± standard error (SE) and values of P < 0.05 were
considered to indicate significance. The correlations among
the parameters were determined, and the Pearson method
was used to calculate the degree of dependence between vari-
ables, to produce a simple linear correlation analysis.

Molecular Biology Reports
1 3
Results anddiscussion
Qualitative analysis ofXRD
Figure1(a) shows the XRD of MgAl-LDH, hesperidin, and
MgAl-LDH-Hesperidin. The XRD spectrum of the MgAl-
LDH nanoparticles was well-matched with the ICDD card
No 00–054-1030, reflecting a pure phase with a rhombo-
hedral crystal lattice structure and a space group R-3m.
The lattice parameters were (a) 3.0300, (b) 3.0380 and (c)
22.0060Å, (α) 90°, (β) 90°, and (γ) 120°. The figure showed
three strong diffraction peaks at 2θ° [11.86°, 23.35°, 35.09°]
with corresponding diffraction planes (003), (006), (012).
The high diffraction signal intensity reflected the high crys-
tallinity of the synthesized MgAl-LDH nanoparticles [34].
The basal diffraction peaks of the plane (003) and (006)
are distinct peaks produced by the MgAl-LDH nanoparti-
cles. The highest basal diffraction peak (003) reflected the
interlayer anion of the synthesized MgAl-LDH nanoparticles
[35]. The increased width of the diffracted peaks reflected
the decrease of the crystallite size. The crystallite size of
the pure MgAl-LDH, calculated according to Eq.1, was ≈
74.54nm.
The same figure shows the crystal form of a neat hes-
peridin drug. The obtained XRD spectrum of hesperidin
agreed with the findings of Sansoneet al. [36]. The spec-
trum was well-matched with the ICDD reference code No.
00–0,050,287. The calculated crystallite size of the pure
drug was ≈ 40.01543nm. The figure covers an abundance
of diffracted peaks reflecting numerous structural plans,
and plentiful electron densities of its chemical formula
C12H34O16. The high signal intensity and narrow width of
the diffracted peaks denoted a good crystallinity state.
The MgAl-LDH-Hesperidin spectrum included the dif-
fraction peaks of hesperidin and MgAl-LDH, with a tenuous
peak shifting to the right. Shifting of the diffracted peak
position occurs as a result of factors such as substitution
doping, temperature, and stress [37]. The shifting here was
due to differences in the angle of interaction as a result of
the alteration of the arrangement of the hesperidin structure
planes during the loading process. The observed change
in the peak position of the diffracted peak 003 was due to
changes in the interatomic distance of the MgAl-LDH [38].
The plane arrangement and the differences in interatomic
distance were caused by the intercalation of the hesperidin
between MgAl-LDH layers, or the adsorption of hesperidin
on the surface of the Mgal-No3 LDH. The basal spacing
of the 003 plane decreased from 7.45400Å in the case of
MgAl-LDH to 4.50047Å in MgAl-LDH-Hesperidin. Hence,
the observed similarity of the diffracted peaks between hes-
peridin and LDH-hesperidin reflected the chemical structure
integrity of the hesperidin after loading.
The lowering of the signal intensity of the MgAl-LDH-
Hesperidin diffracted peaks compared to neat hesperidin sig-
nified a decrease in the crystallinity. The coincided diffracted
peak (006) in the MgAl-LDH, hesperidin, and MgAl-LDH-
Hesperidin spectra indicated a good combination between
MgAl-LDH and hesperidin. There were some apparently
weak diffracted peaks (arrowhead); the weakness of the peak
in the XRD pattern indicates either destructive interference
among the diffracted X-ray photons, or that the electron
density distribution that interacts with the X-ray photons is
low. The spectrum of MgAl-LDH-Hesperidin revealed the
integrity structure of the hesperidin and MgAl-LDH after
loading, and also showed slight changes in the shape, the
position of the signals, and the relative integrated inten-
sity of the diffracted peaks in the given range of 2θ angle.
These alterations implied hesperidin loading on MgAl-LDH
(Fig.1a).
Fig. 1 (a) Exhibits the XRD of MgAl-LDH, hesperidin, and MgAl-
LDH-Hesperidin, (b) The vibration modes of functional groups

Molecular Biology Reports
1 3
FTIR spectra analysis
The prepared pearly white LDH is composed of hydrous
aluminum, magnesium hydroxide, and nitrates, and has the
formula Mg6 Al2 (OH) 16(NO3)0.4H2O. The FTIR spec-
trum shown in Fig.1(b) revealed the vibration modes of the
functional groups. Weak broadband OH of the interlayered
water molecules was present at 3250 cm−1, and the weak
band observed at 1636 cm−1 reflected the bending vibration
of interlayered H-OH present in the hydrotalcite structure
[39]. Two types of nitrate anion may present in the LDH
structure: free or non-hydrogen bonded nitrate, and nitrate
hydrogen bonded to the interlayer water and the ‘brucite-
like’ hydroxyl surface. Moving to the left, the strong band at
1378 cm−1 reflected the anti-symmetric stretchingv 3vibra-
tion modes and supported the presence of interlayered nitrate
anions [40]. The band at 618cm −1 represented the stretching
vibration of M–O (M metal representing Mg and Al and O
oxygen) and the bending vibration of the M- hydroxyl group
(OH) hydrotalcite layer [41]. The hesperidin (C28H34O15) IR
spectrum was the same as the standard in the literature, and
showed a strong band of OH at 3335 cm−1; CH (aliphatic) at
3077 cm−1; CH (aromatic) at 2922 cm−1; C = C (aromatic) at
1601, 1514, 1465 and 1364 cm−1; a strong absorption band
at 1644 cm−1 corresponding to its carbonyl stretching vibra-
tion of C = O at 1636 cm−1; and C-O at 1280 and 1195 cm−1
[15]. The FTIR spectrum of MgAl-LDH-Hesperidin did not
display any new chemical bond formation or any differences
in the peak patterns, confirming the absence of an evident
chemical interaction between the MgAl-LDH and hesperi-
din. The spectrum presented a slight shift to the left in the
position of the transmitted peaks, reinforcing the suggestion
of the physical adsorption of hesperidin on the surface of
MgAl-LDH, either by an electrostatic interaction or by the
formation of weak hydrogen bonds.
Electron microscopy study
In the current study, we used high-resolution transmission
electron microscopy (HRTEM) to investigate the morphol-
ogy of the materials. Figure2 shows the HRTEM images
of MgAl-LDH, and MgAl-LDH-Hesperidin. Figure2(a)
shows the rhombohedral shape of the prepared MgAl-LDH,
an observation which is in agreement with the XRD results.
The particle size ranged from 330 to 380nm. Figure2(b)
shows agglomerated stacked layers of MgAl-LDH loaded
Fig. 2 (a) Shows a rhombo-
hedra shape of the prepared
MgAl-LDH, (b) Agglomerated
stacked layers of Mg–Al-LDH
with loaded hesperidin, (c)
Presented a FESEM image of
MgAl-LDH-Hesperidin

Molecular Biology Reports
1 3
with hesperidin, which reflected the dark signal intensity
in the image. Figure2(c) shows a FESEM image of MgAl-
LDH-Hesperidin, revealing accumulated layers in the form
of lamellar or sheet structures (red circles), and a pore distri-
bution (blue circles) with various volumes and sizes.
Zetasizer andZeta potential measurements
Dynamic light scattering (DLS) is an extremely useful
approach to characterization. This approach supports the
analysis of the properties of particles in solution, under
physiologically relevant conditions [42]. In DLS, the fluc-
tuations in the intensity of light scattered by a colloidal dis-
persion are observed over time, and the analysis of the self-
correlated data yields information about the hydrodynamic
radius of the sample, which indicates the way in which the
particles behave in a fluid. The fluctuations in scattering
intensity initially originate from the Brownian motion of
the particles [43]. In the current research, Fig.3 (a) shows
the size distribution by intensity. The intensity distribution
size and the cumulant fit size were similar. The Z-average,
cumulants mean, or mean hydrodynamic diameter size dis-
tribution of MgAl-LDH-Hesperidin nanoparticles deter-
mined by DLS was 398.5nm, which was slightly bigger
than that determined by HRTEM. This discrepancy arises
because a number distribution from electron microscopy
will be much smaller than an intensity distribution from
DLS. The particle size determined by DLS represents its
hydrodynamic diameter, whereas that obtained by HRTEM
is the real diameter. The PDI value of 0.5 estimated a small
Fig. 3 (a) Shows the size
distribution report by intensity,
(b) Displayed the measured zeta
potential

Molecular Biology Reports
1 3
width, and reflected the homogeneous size distribution that
was confirmed by HRTEM images. The intercept value of
0.979 indicated a high signal-to-noise ratio. The measured
Zeta potential (Fig.3b), was − 35.5 mv, indicating moderate
stability, and explaining the aggregation state that appeared
in the SEM images.
Entrapment efficiency, release profile
andcytotoxicity assays
According to the optimized conditions of the preparation, as
previously described, the average entrapment efficiency of
hesperidin was 89.1%. Figure4 showed the release profile
of hesperidin from MgAl-LDH at a constant temperature of
37 ± 0.5°C and PH of 6.8 for 12h. Overall, the hesperidin
cumulative release percent increased over time. However,
the figure indicated a variation in the release rate between
free hesperidin and MgAl-LDH-Hesperidin. Also, the figure
exhibited a difference in the initial releasing rate between
free hesperidin and MgAl-LDH-Hesperidin within the first
60min. The release of free hesperidin was fast and increased
consistently over time, and the cumulative release was about
98.8% 12h after the start. While the curve of MgAl-LDH-
Hesperidin showed a sustained release pattern of 66.4%.
Drug release is the drug solutes’ migration from the ini-
tial position in their containing material system to the release
medium. This simple process is affected by multiple com-
plex factors such as the physicochemical properties of the
drug solutes (solubility, stability, charges), the characteris-
tics of the material system (composition, structure, swelling,
degradation), release environment (pH, temperature, ionic
strength and enzymes), and the interactions between these
factors. Diffusion, swelling, and degradation of the material
system are the key driving forces for drug solute transport
[44].
The invitro release data applied to various kinetics mod-
els to predict the drug release mechanism and kinetics, as
recorded in TableS1. The values of R2 revealed that the
best fitting mechanism was the first order, with an R2 value
(0.9921). As mentioned above, we enforced the physical
adsorption of hesperidin on the surface of MgAl-LDH,
either by an electrostatic interaction or by weak hydrogen
bonds formation. Our assumption is that the transport mech-
anism of hesperidin from LDH was Fickian diffusion, fol-
lowing Fick’s second law of diffusion.
About the toxicity, our data suggested that hesperidin or
MgAl-LDH-Hesperidin at low and high doses did not pro-
duce any cytotoxicity in WI38 normal lung cells. The liver
and kidney consistently exhibited a normal pattern of func-
tion, an observation which indicates an absence of cytotoxic
effects of the antidiabetic agents in diabetic rats (TableS2).
No abnormal signs or adverse behavioral reactions were
observed in the treated rats compared with the untreated rats.
In vivo study
Hypoglycemic andhypolipidemic study
Figure5 shows that the diabetic group had a significant
increase in plasma glucose level compared to the normal
rat group. Oral administration of metformin, hesperidin, or
MgAl-LDH-Hesperidin produced a significant (P < 0.001)
amelioration of plasma glucose, insulin, and HbA1c% lev-
els compared with the diabetic group. MgAl-LDH acts as a
host of the drug with no significant denaturation of the drug
molecules [45]. Our finding was in keeping with the results
obtained by Abdel-Moneim etal. [46] who reported that
hesperidin alleviated the decreased levels of serum insulin
in high-fat-fed/STZ-induced diabetic rats. The current study
found that pancreatic sections of diabetic control rats showed
atrophied Islets of Langerhans, with associated degeneration
and necrosis of β-cells (Fig. S1-B), compared with normal
pancreatic sections (Fig. S1-A). The diabetic group treated
with hesperidin (Fig. S1-D) or MgAl-LDH-Hesperidin (Fig.
S1-E) had pancreases with intact histological structure, and
restoration of the pancreatic Islets of Langerhans, while the
diabetic group treated with metformin showed moderate
shrinkage of the Islets of Langerhans, with degeneration
and necrosis of some cells, and congestion of blood vessels
(Fig. S1-F). Consistently, partial destruction of β-cells can
be explained by the significant increase in blood glucose
levels and decrease in serum insulin levels in the diabetic
group compared with the normal group. In contrast, oral
administration of hesperidin and its nano-formula induced
a significant amelioration in damage to the structure of β
Fig. 4 Displays the invitro release profile of hesperidin from MgAl-
LDH at a constant temperature

Molecular Biology Reports
1 3
cells and the level of production of insulin. The liver gly-
cogen content was markedly depleted in the diabetic group
compared to the normal group (Table1). Our data are in
agreement with the findings of Dhananjayan etal. [47], who
reported that diabetic rats showed a depletion in glycogen
content which may be due to glycogenolysis as a result of
a marked decrease in insulin levels. This study found that
treatment with hesperidin and its nano-formula produced
a marked amelioration in the liver glycogen content when
compared to the diabetic group. LDH may increase the anti-
diabetic efficiency of hesperidin by increasing its concentra-
tion in the target tissue, acting as an antioxidant agent, and
protecting pancreatic β-cells from the cytotoxicity of STZ,
thereby increasing the production of insulin, increasing liver
glycogen content, and finally, improving glycemic status.
With respect to lipid profile, the data in Fig.6 show a sig-
nificant (P < 0.001) elevation in serum cholesterol, triglyc-
erides, and LDL-cholesterol in the diabetic group compared
Fig. 5 Effect of free hesperidin, metformin, and MgAl-LDH-Hes-
peridin nanoparticles on serum glucose, insulin, HbA1c %, and
HOMA-β. Values significantly different compared to the control
group: P < 0.05. Results are mean ± SE (n = 10). Values that share the
same superscript symbol are not significantly different. HbA1c Gly-
cosylated hemoglobin, HOMA-IR Homeostatic Model Assessment of
Insulin Resistance

Molecular Biology Reports
1 3
with the control group, while HDL-C showed different
behavior. These data were in agreement with the results of
Ahmed etal. [28], who reported that hesperidin produced
a noticeable decrease in the serum levels of cholesterol, tri-
glycerides, and LDL, and a marked increase in the serum
level of HDL. The hesperidin-nano-formula induced a sig-
nificant amelioration in serum cholesterol level when com-
pared with the free hesperidin. As shown in Table1, the free
fatty acid (FFA) concentration was significantly increased
in the diabetic group. Previous studies have reported that
FFAs were elevated in diabetic rats [15, 28]. FFAs play a
crucial role in the expression of several genes involved in
the metabolism of carbohydrates and lipids. Oral adminis-
tration of hesperidin and MgAl-LDH-Hesperidin induced
a marked decrease in the level of FFAs compared to the
diabetic group. Our finding was in agreement with previous
reports which found that hesperidin ameliorates the high
level of lipids and FFAs in type 2 diabetic rats [46]. LDH
may increase the anti-hyperlipidemic efficiency of hesperi-
din by increasing the production of insulin, which has lipo-
genesis and hypolipidemic effects [48, 49].
Figure7 C shows a significant downregulation of PPARγ
mRNA expression levels in the diabetic control group com-
pared to the normal control group; however, the treated
groups showed a significant upregulation in expression
compared to the diabetic group. The rats treated with MgAl-
LDH-Hesperidin showed a significant (P < 0.001) increase
in the levels of PPARγ mRNA. Our data also indicated a
significant correlation between blood glucose level and
PPARγ expression (Fig. S2). In agreement with our results,
Mahmoud etal. [50] reported that hesperidin can ameliorate
dyslipidemia and hyperglycemia in diabetic rats by activat-
ing PPARγ mRNA expression
PPARγ activation can modulate several genes in adipo-
cytes, causing a noticeable increase in adipogenesis and the
uptake of lipids, and regulating glucose metabolism [51].
Hence, PPARγ improves insulin sensitivity by increasing the
storage of fatty acids in fat cells, thus lowering lipotoxicity,
and by increasing the production of adiponectin from fat
cells [53]. PPARγ is considered to be an important anti-
oxidative and anti-inflammatory pharmacotherapy target in
several diseases and is commonly used as an antidiabetic
target. The upregulation of PPARγ induces activation of
anti-inflammatory and antioxidant biomarkers by nuclear
factor-κB suppression, lowering the production of ROS,
and upregulating the expression of antioxidant enzymes.
Hesperidin can induce anti-inflammatory activity via the
upregulation of PPARγ, which increases insulin sensitivity,
glucose uptake from the liver, and alleviates hyperglycemia
[50]. Activation of PPARγ can enhance insulin sensitivity
through the modulation of pro-inflammatory cytokines and
adipocyte hormones [53]. MgAl-LDH-Hesperidin can exert
antidiabetic and hypolipidemic effects by enhancing the pro-
duction of insulin from β-cells, increasing insulin sensitiv-
ity, activating PPARγ mRNA expression, and elevating liver
glycogen content and lipogenesis.
Oxidative stress study
The current study revealed an elevation in lipid peroxida-
tion (MDA), together with antioxidant activity (CAT), as
shown in Fig.7 and Table1. As expected, there was a sig-
nificant (P < 0.001) increase in MDA levels in the diabetic
control group compared to the normal control group. Oral
treatment with free hesperidin or MgAl-LDH-Hesperidin
produced a noticeable enhancement in CAT activity with
the reduction of the level of MDA. Fig. S2 shows a signifi-
cant correlation between blood glucose level with each of
MDA, CAT, and NrF2 levels. Our results were in agree-
ment with those of Ebaid etal. [54] who reported that
MDA levels increased significantly in STZ-induced type
2 diabetes, with a decrease in antioxidant enzyme levels.
The decrease in CAT activity in our study may be attribut-
able to an increase in ROS and lipid peroxidation levels.
The author also reported that in hyperglycemia, the anti-
oxidant enzyme CAT had significantly reduced activity,
Table 1 Effect of antidiabetic agents on MDA, CAT, vLDL, FFA, and Liver glycogen in normal, diabetic, and diabetic treated groups
Data are expressed as mean ± SEM (n = 10). Values significantly different compared to the control group: P < 0.05. Values that share the same
superscript symbol are not significantly different. LDH Layered double hydroxides, MDA Malondialdehyde, CAT catalase, vLDL very-density
lipoprotein, FFA Free fatty acids
Parameter group MDA (nmol/g. tissue) CAT (U/g. tissue) vLDL (mg/dl) FFA (mmol/L) Liver glycogen
(mg/g. tissue)
Normal 2.25 ± 0.15d115.60 ± 0.60a10.88 ± 0.31d0.57 ± 0.03d26.88 ± 0.52a
Diabetic Control 4.55 ± 0.10a78.86 ± 0.59d37.30 ± 0.37a1.66 ± 0.08a11.49 ± 0.0.46e
Diabetic rats + MgAl-LDH 4.09 ± 0.07b81.01 ± 0.77c36.40 ± 0.35a1.57 ± 0.07a12.38 ± 0.32e
Diabetic rats + hesperidin 2.86 ± 0.10c113.16 ± 0.55b13.65 ± 0.30c1.02 ± 0.07b18.50 ± 0.73c
Diabetic rats + MgAl-LDH-Hesperidin 2.32 ± 0.12d115.05 ± 0.43a13.28 ± 0.30c0.79 ± 0.04c21.75 ± 0.49b
Diabetic rats + Metformin 3.01 ± 0.06c112.53 ± 0.67b23.28 ± 0.30b1.55 ± 0.09a14.13 ± 0.55d
F-Prob P < 0.001 P < 0.001 P < 0.001 P < 0.001 P < 0.001

Molecular Biology Reports
1 3
to attenuate the harmful effects of reactive oxygen spe-
cies. Our finding was in agreement with the results of Liu
etal. [55], who reported that hesperidin can regulate the
concentration of glucose and improve oxidative stress by
decreasing MDA content and increasing both superoxide
dismutase and CAT activities in retinal ganglion cells.
The current study revealed that hesperidin was physically
adsorbed on the surface of MgAl-LDH and MgAl-LDH
exhibited sustained release of hesperidin for 12h, which
reinforced the efficiency of hesperidin as an antioxidant
agent. Hu and Liu [56] reported that nano-fabricated ther-
apeutic agents can improve redox homeostasis through
several mechanistic pathways, such as the introduction of
reactive oxygen species-generating agents and/or inhibi-
tion of innate anti-oxidation systems.
Figure7 shows a significant (P < 0.001) downregulation
of NrF2 mRNA expression levels in the diabetic control
group compared to the normal control group, However, the
groups treated with the agents tested had significant upreg-
ulation of expression compared to the diabetic group. In
Fig. 6 Effect of free hesperidin, metformin, and MgAl-LDH-Hes-
peridin nanoparticles on serum cholesterol, Triglycerides, LDL, and
HDL. Values significantly different compared to the control group:
P < 0.05. Results are mean ± SE (n = 10). Values that share the same
superscript symbol are not significantly different. HDL High-density-
lipoprotein, LDL Low-density-lipoprotein, LDL

Molecular Biology Reports
1 3
particular, the rats treated with MgAl-LDH-Hesperidin had
a highly significant increase. The transcription factor Nrf2
is considered to be the main regulator of many antioxidant
genes. Nrf2 has a high electrophile-sensing property, and
reactive oxygen species can bind with the cysteine residues
of Kelch-like ECH-associated protein1 (KEAP1), resulting
in the release of Nrf2, which is subsequently transported
inside the nucleus. Subsequently, Nrf2 binds to antioxi-
dant response elements (AREs) to induce the upregulation
of many antioxidant and cytoprotective genes, including
superoxide dismutase, CAT, glutathione peroxidase-1, and
NAD(P)H quinone oxidoreductase-1. Nrf2/ARE/antioxi-
dants are considered to be involved in pathways contributing
to the control of inflammation [57]. MgAl-LDH-Hesperidin
upregulation induced by MgAl-LDH-Hesperidin was associ-
ated with decreased oxidative stress and enhanced antioxi-
dant defenses. These findings were strongly supported by
previous studies in which stimulation of Nrf2 increased the
Fig. 7 Effect of free hesperidin, metformin, and MgAl-LDH-Hesperi-
din nanoparticles on TNF-a and IL-17 levels and PPARγ and NrF2
expression. Values significantly different compared to the control
group: P < 0.05. Results are mean ± SE (n = 10). Values that share the
same superscript symbol are not significantly different. TNF α Tumor
necrosis factor-alpha, IL-17 Interleukin (IL), PPAR-γ Peroxisome
proliferator-activated receptor gamma, NRF2 Nuclear factor erythroid
2-related factor 2

Molecular Biology Reports
1 3
production and activity of the antioxidant enzymes superox-
ide dismutase and CAT, metabolized toxic oxidative stress
intermediates, and protected cells such as β-cells against
stress-induced damage [58]. Therefore, the antioxidant activ-
ity of Nrf2 has a crucial role in preventing tissue injury.
We hypothesized that the MgAl-LDH-Hesperidin has the
potential to activate antioxidant enzyme productionto modu-
late oxidative stress, thereby ameliorating several metabolic
disorders.
Inflammatory cytokine study
The present study showed that both the pro-inflammatory
cytokines TNF-α and IL-17 were increased significantly
(P < 0.001) in the diabetic group, as shown in Fig.7. A
significant (P < 0.001) positive correlation was observed
between blood glucose levels and levels of TNF-α and
IL-17 (Fig.7). Moreover, Fig. S2 shows a significant posi-
tive correlation between blood glucose level with IL-17
and TNF-α. Tilg and Moschen [59] reported a positive
correlation between T2DM and the increase in inflamma-
tory cytokines such as TNF-α, IL-6, and CRP. IL-17 gene
expression in patients with T2DM was shown to be associ-
ated with TNF-α gene expression [60]. IL-17 promotes the
NF-κB pathway,which up-regulates the pro-inflammatory
cytokinesIL-1β, IL-6, and TNF-α, therebyinducing insu-
lin resistance, and finally leading to the establishment of
T2DM [61]. Thus, treatment agents that target IL-17 can
control diabetes by mitigating the inflammation which leads
to insulin resistance and T2DM. The group treated with our
novel form of hesperidin showed greater anti-inflammatory
properties compared with other treated groups. Treatment
with anti-IL-17 neutralizing antibodies elevated serum adi-
ponectin concentration, reduced serum levels of TNF-α, and
enhanced adipocyte differentiation markers [62]. It has been
suggested that therapeutic strategies that regulate inflam-
mation and reduce inflammatory markers are a promising
tool, because of the association between insulin resistance
and inflammation [63]. Both hesperidin and MgAl-LDH-
Hesperidin treatments considerably lowered the TNF-α and
IL-17 levels that represent the anti-inflammatory activity
of these agentsand contribute to the sensitizing effects of
insulin, subsequently leading to modulation of the glycemic
and lipidemic status.
Although, the current study succeeded to reveal the
potency of MgAl-LDH-Hesperidin as an antidiabetic;
nevertheless, the study had some limitations. The study
did not subject to the invivo pharmacokinetic to the for-
mula to investigate its bioavailability and the solubility.
Besides, different cellular molecular investigations should
be considered.
Conclusion
In summary, a novel MgAl-LDH-Hesperidin formula
was an effective nano-carrier for the sustained delivery
of hesperidin. MgAl-LDH nano-carrier is of consider-
able interest, particularly for drugs with low solubility
and permeability, such as hesperidin. We found that the
novel MgAl-LDH-Hesperidin formula ameliorates hyper-
glycemia and hyperlipidemia in treated diabetic rats by
enhancing the production of insulin from β-cells, elevat-
ing liver glycogen content, and increasing lipogenesis.
MgAl-LDH-Hesperidin can also increase insulin sensitiv-
ity and glucose uptake by alleviating the oxidative stress,
by increasing the expression of PPARγ mRNA, and the
activation of the Nrf2/ARE/antioxidant pathways, as well
as controlling inflammation by decreasing the levels of
the pro-inflammatory cytokines TNF-α, and IL-17 (Fig.
S3). The enhanced biological activity of LDH-Hesperidin
formula may be due to entrapment efficiency of LDH, hes-
peridin nanostructure, and achieving the sustained release.
The study suggests that the MgAl-LDH-Hesperidin for-
mula could be considered as a promising approach to the
management of diabetes.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s11033- 021- 06527-2.
Acknowledgements The authors are thankful to all members at Materi-
als Science and Nanotechnology Determent, and Biotechnology and
life sciences Department, Faculty of Postgraduate Studies for Advanced
Sciences (PSAS), Beni-Suef University Egypt for supporting the prepa-
ration of the nanomaterials.
Authors’ contributions All authors contributed to the study’s concep-
tion and design. Material preparation, data collection and analysis
were performed by Ahmed El-Shahawy, Mohamed Zanaty, Abdelazim
Ebeid, and Zienab Eldine. The first draft of the manuscript was written
by Adel Abdel Moneim, Ahmed El-Shahawy, and Mohamed Zanaty
and all authors commented on previous versions of the manuscript. All
authors read and approved the final manuscript.
Funding No funding was received for conducting this study.
Availability of data and materials All data generated or analysed during
this study are included in this published article.
Declarations
Conflict of interest The authors declare that they have no competing
interests.
Ethical approval The Institutional Animal Care Committee of Beni-
Suef University, Egypt, approved the entire conducted procedures
(BSU/2018/11/17).

Molecular Biology Reports
1 3
References
1. Sarwar N, Gao P, Seshasai SR etal (2010) Emerging Risk Factors
Collaboration Diabetes mellitus, fasting blood glucose concentra-
tion, and risk of vascular disease: a collaborative meta-analysis of
102 prospective studies. Lancet 375:2215–2222
2. Cho N, Shaw JE, Karuranga S etal (2018) IDF Diabetes Atlas:
Global estimates of diabetes prevalence for 2017 and projections
for 2045. Diabetes Res Clin Pract 138:271–281
3. Chatterjee C, Sparks DL (2011) Hepatic lipase, high density lipo-
proteins, and hypertriglyceridemia. Am J Pathol 178:1429–1433.
https:// doi. org/ 10. 1016/j. ajpath. 2010. 12. 050
4. Bailey CJ (2015) The current drug treatment landscape for
diabetes and perspectives for the future. Clin Pharmacol Ther
98:170–184
5. Schwetz V, Eisner F, Schilcher G etal (2017) Combined met-
formin-associated lactic acidosis and euglycemic ketoacidosis.
Wien Klin Wochenschr 129:646–649. https:// doi. org/ 10. 1007/
s00508- 017- 1251-6
6. Reeker W, Schneider G, Felgenhauer N etal (2000) Metformin-
induced lactacidosis Dtsch Medizinische Wochenschrift 125:249–
251. https:// doi. org/ 10. 1055/s- 2007- 10240 85
7. Patel DK, Prasad SK, Kumar R, Hemalatha S (2012) An overview
on antidiabetic medicinal plants having insulin mimetic property.
Asian Pac J Trop Biomed 2:320–330. https:// doi. org/ 10. 1016/
S2221- 1691(12) 60032-X
8. Al-Ishaq RK, Abotaleb M, Kubatka P etal (2019) Flavonoids
and their anti-diabetic effects: Cellular mechanisms and effects
to improve blood sugar levels. Biomolecules. https:// doi. org/ 10.
3390/ biom9 090430
9. Horáková L (2011) Flavonoids in prevention of diseases with
respect to modulation of Ca-pump function. Interdiscip Toxicol
4:114–124. https:// doi. org/ 10. 2478/ v10102- 011- 0019-5
10. Li C, Schluesener H (2017) Health-promoting effects of the citrus
flavanone hesperidin. Crit Rev Food Sci Nutr 57:613–631
11. Ficarra R, Tommasini S, Raneri D et al (2002) Study of
flavonoids/β-cyclodextrins inclusion complexes by NMR, FT-IR,
DSC, X-ray investigation. J Pharm Biomed Anal 29:1005–1014
12. Bilia AR, Isacchi B, Righeschi C etal (2014) Flavonoids loaded
in nanocarriers: an opportunity to increase oral bioavailability and
bioefficacy. Food Sci Nutr 5(13):1212–1227
13. Duttagupta DS, Jadhav M, V, J Kadam V, (2015) Chitosan: a pro-
pitious biopolymer for drug delivery. Curr Drug Deliv 12:369–381
14. Sulaiman GM, Waheeb HM, Jabir MS, Khazaal SH, Dewir YH,
Naidoo Y (2020) Hesperidin loaded on gold nanoparticles as a
drug delivery system for a successful biocompatible, anti-cancer,
anti-inflammatory and phagocytosis inducer model. Sci Rep
10(1):9362
15. Ali SH, Sulaiman GM, Al-Halbosiy MMF etal (2019) Fabrication
of hesperidin nanoparticles loaded by poly lactic co-Glycolic acid
for improved therapeutic efficiency and cytotoxicity. Artif Cells,
Nanomedicine Biotechnol 47:378–394. https:// doi. org/ 10. 1080/
21691 401. 2018. 15591 75
16. Anwar A, Masri A, Rao K etal (2019) Antimicrobial activities
of green synthesized gums-stabilized nanoparticles loaded with
flavonoids. Sci Rep 9(1):3122
17. Ansar S, Abudawood M, Alaraj ASA, Hamed SS (2018) Hesperi-
din alleviates zinc oxide nanoparticle induced hepatotoxicity and
oxidative stress. BMC Pharmacol Toxicol 19(1):65
18. Rojas R, Palena MC, Jimenez-Kairuz AF etal (2012) Modeling
drug release from a layered double hydroxide–ibuprofen com-
plex. Appl Clay Sci 62:15–20
19. El-Shahawy A, Abdel-Moneim A, Eldin Z etal (2020) Char-
acterization and efficacy of a novel formula of insulin chitosan
LDH-nanohybrid for oral administration. Nano Biomed Eng
12:297–305
20. Ameena Shirin VK, Sankar R, Johnson AP et al (2021)
Advanced drug delivery applications of layered double hydrox-
ide. J Control Release 330:398–426
21. Conterosito E, Gianotti V, Palin L etal (2018) Facile prepara-
tion methods of hydrotalcite layered materials and their struc-
tural characterization by combined techniques. Inorganica Chim
Acta 470:36–50
22. El-Shahawy AAG, El-Ela FIA, Mohamed NA etal (2018) Syn-
thesis and evaluation of layered double hydroxide/doxycycline
and cobalt ferrite/chitosan nanohybrid efficacy on gram positive
and gram negative bacteria. Mater Sci Eng C 91:361–371
23. Abdel-Moneim A, El-Shahawy A, Yousef AI etal (2020)
Novel polydatin-loaded chitosan nanoparticles for safe and
efficient type 2 diabetes therapy: In silico, invitro and invivo
approaches. Int J Biol Macromol 154:1496–1504. https:// doi.
org/ 10. 1016/j. ijbio mac. 2019. 11. 031
24. Kuo YC, Chung JF (2011) Physicochemical properties of nevi-
rapine-loaded solid lipid nanoparticles and nanostructured lipid
carriers. Colloids Surf B Biointerfaces 83:299–306
25. Padilla de la Rosa JD, Ruiz-Palomino P, Arriola-Guevara E
etal (2018) A green process for the extraction and purifica-
tion of hesperidin from mexican lime peel (Citrus aurantifo-
lia Swingle) that is extendible to the citrus genus. Processes
6(12):266. https:// doi. org/ 10. 3390/ pr612 0266
26. Mardani R, Hamblin MR, Taghizadeh M etal (2020) Nanomi-
cellar-curcumin exerts its therapeutic effects via affecting angio-
genesis, apoptosis, and T cells in a mouse model of melanoma
lung metastasis. Pathol Pract 216:153082
27. Ghasemi A, Khalifi S, Jedi S (2014) Streptozotocin-nicotina-
mide-induced rat model of type 2 diabetes. Acta Physiol Hung
101:408–420
28. Ahmed OM, Mahmoud AM, Abdel-Moneim A, Ashour MB
(2012) Antidiabetic effects of hesperidin and naringin in type 2
diabetic rats. Diabetol Croat 41:53–67
29. Friedewald WT, Levy RI, Fredrickson DS (1972) Estimation
of the concentration of low-density lipoprotein cholesterol in
plasma, without use of the preparative ultracentrifuge. Clin
Chem 18:499–502
30. Shokri-Afra H, Ostovar-Ravari A, Rasouli M (2016) Improve-
ment of the classical assay method for liver glycogen fractions:
ASG is the main and metabolic active fraction. Eur Rev Med
Pharmacol Sci 20:4328–4336
31. Onishi Y, Hayashi T, Sato KK etal (2010) Fasting tests of
insulin secretion and sensitivity predict future prediabetes in
Japanese with normal glucose tolerance. J Diabetes Inves-
tig 1(5):191–195. https:// doi. org/ 10. 1111/j. 2040- 1124. 2010.
00041.x
32. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expres-
sion data using real-time quantitative PCR and the 2− ΔΔCT
method. Methods 25:402–408
33. Drury R (1983) Theory and practice of histological techniques. J
Clin Pathol 36(5):609
34. Nejati K, Mokhtari A, Khodam F, Rezvani Z (2015) Syntheses of
Mg-Al-NO3 layered double hydroxides with high crystallinity in
the presence of amines. Can J Chem 94:66–71. https:// doi. org/ 10.
1139/ cjc- 2015- 0265
35. Shafiei SS, Solati-Hashjin M, Rahim-Zadeh H, Samadikuchak-
saraei A (2013) Synthesis and characterisation of nanocrystalline
Ca-Al layered double hydroxide {[Ca2Al(OH)6]NO3·nH 2O}:
Invitro study. Adv Appl Ceram 112:59–65. https:// doi. org/ 10.
1179/ 17436 76112Y. 00000 00045
36. Sansone F, Rossi A, Gaudio P etal (2009) Hesperidin gastroresist-
ant microparticles by spray-drying: Preparation, characterization,

Molecular Biology Reports
1 3
and dissolution profiles. AAPS PharmSciTech 10:391–401.
https:// doi. org/ 10. 1208/ s12249- 009- 9219-0
37. Ahad A, Taher MA, Das MK etal (2019) Effect of Y substitution
on magnetic and transport properties of Ba 0. 95 La 0. 05 Ti 1–x
Y x O 3 ceramics. Results Phys 12:1925–1932. https:// doi. org/ 10.
1016/j. rinp. 2019. 01. 072
38. Modrogan C, Căprărescu S, Dăncilă AM etal (2020) Mixed oxide
layered double hydroxide materials: Synthesis, characterization
and efficient application for Mn2+ removal from synthetic waste-
water. Materials (Basel). https:// doi. org/ 10. 3390/ ma131 84089
39. Conroy M, Soltis JA, Wittman RS etal (2017) Importance of
interlayer H bonding structure to the stability of layered minerals.
Sci Rep 7:1–10. https:// doi. org/ 10. 1038/ s41598- 017- 13452-7
40. Tao Q, Yuan J, Frost RL etal (2009) Effect of surfactant concen-
tration on the stacking modes of organo-silylated layered double
hydroxides. Appl Clay Sci 45:262–269. https:// doi. or g/ 10. 1016/j.
clay. 2009. 06. 007
41. Madej D, Tyrała K (2020) Insitu spinel formation in a smart nano-
structured matrix for no-cement refractory castables. Materials
(Basel). https:// doi. org/ 10. 3390/ ma130 61403
42. Ye Z, Jiang X, Wang Z (2012) Measurements of particle size
distribution based on Mie scattering theory and Markov chain
inversion algorithm. J Softw 7:2309–2316. https:// doi. org/ 10.
4304/ jsw.7. 10. 2309- 2316
43. Malm AV, Corbett JCW (2019) Improved Dynamic Light Scatter-
ing using an adaptive and statistically driven time resolved treat-
ment of correlation data. Sci Rep 9:1–11. https:// doi. org/ 10. 1038/
s41598- 019- 50077-4
44. Fu Y, Kao WJ (2010) Drug release kinetics and transport mecha-
nisms of non-degradable and degradable polymeric delivery sys-
tems. Expert Opin Drug Deliv 4:429–444. https:// doi. org/ 10. 1517/
17425 24100 36022 59
45. Khan AI, Ragavan A, Fong B etal (2009) Recent developments
in the use of layered double hydroxides as host materials for the
storage and triggered release of functional anions. Ind Eng Chem
Res 48:10196–10205. https:// doi. org/ 10. 1021/ ie901 2612
46. Abdel-Moneim A, Ashour MB, Mahmoud AM, Ahmed OM
(2011) Insulin sensitizing effects of hesperidin and naringin in
experimental model of induced type 2 diabetes in rats: focus on
tumor necrosis factor-alpha and resistin. Nat Sci 7:134–141
47. Dhananjayan I, Kathiroli S, Subramani S, Veerasamy V (2017)
Ameliorating effect of betanin, a natural chromoalkaloid by modu-
lating hepatic carbohydrate metabolic enzyme activities and gly-
cogen content in streptozotocin–nicotinamide induced experimen-
tal rats. Biomed Pharmacother 88:1069–1079
48. Boden G, Shulman GI (2002) Free fatty acids in obesity and type
2 diabetes: defining their role in the development of insulin resist-
ance and β-cell dysfunction. Eur J Clin Invest 32:14–23
49. Abdin AA, Baalash AA, Hamooda HE (2010) Effects of rosiglita-
zone and aspirin on experimental model of induced type 2 diabe-
tes in rats: focus on insulin resistance and inflammatory markers.
J Diabetes Complications 24:168–178
50. Mahmoud AM, Ahmed OM, Abdel-Moneim A, Ashour MB
(2013) Upregulation of PPARγ mediates the antidiabetic effects of
citrus flavonoids in type 2 diabetic rats. Int J Bioassays 2:756–761
51. Ahmadian M, Suh JM, Hah N, Liddle C, Atkins AR, Downes M,
Evans RM (2013) PPARγ signaling and metabolism: the good,
the bad and the future. Nat Med 19(5):557–566
52. Jones JR, Barrick C, Kim K-A etal (2005) Deletion of PPARγ
in adipose tissues of mice protects against high fat diet-induced
obesity and insulin resistance. Proc Natl Acad Sci 102:6207–6212
53. Hammarstedt A, Andersson CX, Sopasakis VR, Smith U (2005)
The effect of PPARγ ligands on the adipose tissue in insulin resist-
ance. Prostaglandins, Leukot Essent Fat acids 73:65–75
54. Ebaid H, Bashandy SAE, Alhazza IM etal (2019) Efficacy of a
methanolic extract of adansonia digitata leaf in alleviating hyper-
glycemia, hyperlipidemia, and oxidative stress of diabetic rats.
Biomed Res Int. https:// doi. org/ 10. 1155/ 2019/ 28351 52
55. Liu WY, Liou S-S, Hong T-Y, Liu I-M (2017) Protective effects
of hesperidin (citrus flavonone) on high glucose induced oxidative
stress and apoptosis in a cellular model for diabetic retinopathy.
Nutrients 9:1312. https:// doi. org/ 10. 3390/ nu912 1312
56. Hu J, Liu S (2020) Modulating intracellular oxidative stress via
engineered nanotherapeutics. J Control Release 319:333–343
57. Lee C (2017) Collaborative power of Nrf2 and PPARγ activators
against metabolic and drug-induced oxidative injury. Oxid Med
Cell Longev. https:// doi. org/ 10. 1155/ 2017/ 13781 75
58. Jung KA, Kwak MK (2010) The Nrf2 system as a potential
target for the development of indirect antioxidants. Molecules
15(10):7266–7291. https:// doi. org/ 10. 3390/ molec ules1 51072 66
59. Tilg H, Moschen AR (2008) Inflammatory mechanisms in the
regulation of insulin resistance. Mol Med 14:222–231
60. Chen C, Shao Y, Wu X, Huang C, Lu W (2016) Elevated Inter-
leukin-17 levels in patients with newly diagnosed type 2 diabetes
mellitus. Biochem Physiol 5:206
61. Abdel-Moneim A, Bakery HH, Allam G (2018) The potential
pathogenic role of IL-17/Th17 cells in both type 1 and type 2
diabetes mellitus. Biomed Pharmacother 101:287–292
62. Ohshima K, Mogi M, Jing F etal (2012) Roles of interleukin
17 in angiotensin II type 1 receptor-mediated insulin resistance.
Hypertension 59(2):493–499
63. Marx N, Froehlich J, Siam L etal (2003) Antidiabetic PPAR
gamma-activator rosiglitazone reduces MMP-9 serum levels in
type 2 diabetic patients with coronary artery disease. Arterioscler
Thromb Vasc Biol 23(2):283–288
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.