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Research Article
Modulation of Diabetes Mellitus-Induced Male Rat
Reproductive Dysfunction with Micro-Nanoencapsulated
Echinacea purpurea Ethanol Extract
Chien-Feng Mao ,Xiu-RuZhang , Athira Johnson ,
Jia-Ling He , and Zwe-Ling Kong
Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 20224, Taiwan
Correspondence should be addressed to Zwe-Ling Kong; kongzl@mail.ntou.edu.tw
Received 25 June 2018; Revised 6 August 2018; Accepted 9 August 2018; Published 30 August 2018
Academic Editor: Sanyog Jain
Copyright © Chien-Feng Mao et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Diabetes mellitus is a major health problem that aects a patient’s life quality throughout the world dueto its worst complications. It
was recognized that chronic hyperglycemia with oxidative stress was the major cause of male infertility.Echinacea purpurea ethanol
extract (EE) contains phenolic acid and isobutylamides had been proven to ameliorate diabetic complications. Chitosan/silica
nanoparticles are well-known in the medicinal eld because of its controlled release and drug delivery properties. is study
was aimed at investigating whether the EE encapsulated chitosan/silica nanoparticle (nano-EE) can enhance the amelioration
of male infertility. Our results indicated that the average size of nano-EE was ± nm with an encapsulation ecien cy of
.% and loading capacity of .%. e reduction in oxidative stress and antioxidant activity of nano-EE was observed in LC-
cells. In in vivo experiment, mg/kg of streptozotocin (STZ) was used to induce diabetes in male Sprague-Dawley rats. Diabetic
rats were treated with nano (mg/kg), nano-EE (mg/kg), nano-EE (mg/kg), nano-EE ( mg/kg), and metformin
(Met) ( mg/kg) for weeks. e results show that the nano-EE can improve hyperglycemia, insulin resistance, and plasma
broblast growth factor (FGF ) resistance.It was also conrmed that nano-EE signicantly improved the testis tissue structure,
increasing sperm quality and DNA integrity as well as reducing reactive oxygen species level.
1. Introduction
Typediabetesmellitusisacomplexdiseasecharacterizedby
the improper use of insulin by the pancreatic beta cells associ-
ated with hyperglycemia and insulin resistance []. Oxidative
stress is responsible for the onset of several complications of
diabetes such as vascular diseases, kidney damage, and repro-
ductive dysfunction []. Both diabetic patients and animals
commonly experience reproductive system deciencies like
testicular dysfunctions. Diabetes could also induce structural
changes in the testis and spermatozoa and promote germ cell
apoptosis, impairment of sperm parameters, and hormonal
changes that nally results in infertility [].
Echinacea purpurea is also known as purple coneower
originated in North America and was brought to Europe
inthelatethcentury.emostactivecompoundsofE.
purpurea are isobutylamides and polyphenols-caeic acid
derivatives such as caaric acid, chlorogenic acid, cynarin,
echinacoside, and cichoric acid []. Extracts and dietary sup-
plements from this plant exhibited anti-immunosuppressant
[], antioxidative [], anti-inammatory [], antibacterial
[], antiviral [], and anticancer [] properties. Despite all
these wide spectra of pharmacological properties, the use
of E. purpurea extract in the biomedical application eld is
limited due to its bitter taste and astringent and low aqueous
solubility as well as low oral bioavailability []. Polyphenols
areknownasthemostactivecompoundsofE. purpurea.ey
are the secondary metabolites possessing radical scavenging
activity towards reactive oxygen species (ROS). Because of
the fast release, destruction against environmental stress, low
bioavailability, low permeation, and low solubility, the direct
use of phenolic compounds was limited [].
One of the promising ways to circumvent these problems
is through nanoencapsulation by using nanocarriers. It acts
Hindawi
BioMed Research International
Volume 2018, Article ID 4237354, 17 pages
https://doi.org/10.1155/2018/4237354
BioMed Research International
as a controlled drug delivery system and can improve the
oral bioavailability of the lipophilic and hydrophobic drugs.
ere are several methods adopted for nanoencapsulating
phenolic compounds into a carrier system. e methods are
primarily categorized into physical methods (spray-drying,
uid bed coating, centrifugal extrusion, etc.), physiochemical
methods (spray-cooling, ionic gelation, solvent evaporation
extraction, etc.), and chemical methods (in situ polymer-
ization, interfacial polymerization, etc.) []. Several inves-
tigations have been carried out using polymers (natural and
synthetic) for the development of nanoparticles to improve
the oral bioavailability []. Nowadays, the studies based on
nanotechnology are expanded in almost all research elds.
Nanoparticles got a lot of attention in drug delivery research
due to its versatility in targeting tissues and enabling deep
molecular targets [].
Chitosan is an N-acetylated derivative of chitin widely
used as a building material for nanoparticles []. It has
excellent biodegradable, biocompatible, nontoxic, nonim-
munogenic properties and it prolongs drug release time in
the gastrointestinal tract []. Most importantly, chitosan is
digested by chitosanase enzymes secreted by microorgan-
isms of the intestine aer oral ingestion. Silica is another
material usually used for the building up of nanoparticles
and is obtained from diatom skeletons, sponge speckles,
and animal dietary silicon []. e potential applications
of silica in nanobiotechnology include the encapsulation of
biomolecules, drug delivery, and gene transfer [].
is study has investigated the preparation and detailed
characterization of chitosan/silica encapsulated EE and also
performed the in vitro study to nd out the eect of nano-
EE on H2O2-induced oxidative stress in LC- and further
performed the in vivo study to investigate the eect of the
above said particle on diabetes-induced male rat reproduc tive
dysfunction.
2. Materials and Methods
2.1. Reagents. Chitosan was purchased from Lytone Enter-
prise, Inc., Taipei, Taiwan [degree of deacetylation (DD) =
%, molecular weight (Mw) = kDa]. Echinacea purpurea
was purchased from Taiwan Direct Biotechnology Corp.,
(Hsinchu, Taiwan). Sodium acetate and acetic acid were pur-
chased from Zhenfang Company (Taipei, Taiwan). Sodium
silicate was purchased from Wako Chemical (Osaka, Japan).
Nitroblue tetrazolium (NBT), -(,-dimethylthiazol--yl)-
,-diphenyltetrazolium bromide (MTT), sodium nitrite,
streptozotocin (STZ), acridine orange (AO), and DCFH-
DA were purchased from Sigma (St. Louis, MO, USA).
Metformin HCl was purchased from TCM Biotech Interna-
tional Corp. (Taipei, Taiwan). Alanine transaminase (ALT),
aspartate transaminase (AST), blood urea nitrogen (BUN),
creatinine, and glucose ELISA kits were purchased from
Randox (Colorato, USA). Interleukin- beta (IL-𝛽), inam-
matory cytokines tumor necrosis factor-alpha (TNF-𝛼),
FGF , and Testosterone ELISA kits were purchased from
Abcam (Connecticut, US). Insulin ELISA kit was purchased
fromMercodia(Uppsala,Sweden).PlasmaAGEwithAGEs
determine ELISA kit. Dulbecco’s modied eagle’s medium,
nutrient mixture F (DMEM-F), and Trypsin, RPMI
, were purchased from Gibco (Carlsbad, California,
USA). Fetal bovine serum (FBS) was purchased from PAA
(Pasching, Austria). Dulbecco’s phosphate-buered saline
was purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo,
Japan). LC- (rat testicular Leydig cell lines) came from
the Food Industry Research and Development Institute
(Hsinchu, Taiwan).
2.2. Preparation of Echinacea purpurea Extract (EE). Exper-
iment sample of Echinacea purpurea was provided by Tai-
wan Direct Biotechnology Corp. Echinacea purpurea were
extracted with % ethanol and later freeze-dried (Freeze
drying system FD . XL, Kingmech Co., Ltd., Taipei,
Taiwan). e acquired EE powder yield approximately %.
2.2.1. High-Performance Liquid Chromatography (HPLC)
Analysis for the Determination of Alkylamides and Total Phe-
nolic Acid in EE. Series III LC pump (Scientic Soware, Inc.,
Pleasanton, CA, USA) was used as a system for performing
HPLC analysis. 𝜇l of EE was injected onto a Phenomenex
C column ( mm ×. mm, 𝜇m) (Torrance, CA, USA)
at ∘C. Mobile phase A was acetonitrile: water: phosphoric
acid (::) and mobile phase B was acetonitrile: water:
phosphoric acid (::). A gradient solvent delivery was
used with A maintained at % (∘C) for minutes. e
proportion of B decreased to % and the column was washed
with % A for min until the next injection. e ow
rate was . mL/min, and the wavelength of the UV-visible
detector was set ( nm for total phenolic acids and nm
for alkalamides). e data were collected (HyperQuan Inc.,
VUV-, Colorado Springs, Colorado, USA) and detected
(Showa Denko, Japan).
2.3. EE Loaded Chitosan/Silica Nanoparticle Preparation.
According to the previously reported method [], chi-
tosan/silica nanoparticles were prepared by mixing silicate
solution and a chitosan solution. e silicate solution was
prepared by dissolving sodium silicate in . M sodium
acetate solution (.% w/w). Chitosan solution was prepared
by dissolving chitosan in . M acetic acid solution (.%
w/w). Chitosan and silica mixing ratio were : and the
acidity of both solutions was adjusted to pH .. EE was added
to the chitosan/silica solution. e ratio of solution and EE
was : and stirred homogeneously at room temperature for
minutes. Aer stirring, the mixture was centrifuged (high
speed centrifuge, Hettich CR-, Germany) at ×gfor
minutes and the supernatant was freeze-dried (freeze drying
system FD . XL, Kingmech Co., Ltd., Taipei, Taiwan). e
acquired freeze-dried product was nano-EE [].
2.4. Particle Size, Zeta Potential, and Morphology Analysis.
eshapeandsizeoftheparticleweredeterminedby
scanning electron microscope (SEM) imaging []. e con-
ductive double-sided tape was attached on the surface of a
test plate. e sample was dissolved in % of alcohol for
minutes. Later, the sample was dropped on the tape and
was dried in the convection oven for hours. e sample
was sputter-coated with a thin layer of gold and observed
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under SEM (Olympus Hitachi, Tokyo, Japan). Zeta potential
andparticlesize(forconrmation)weredeterminedby
using Dynamic Light Scattering (DLS) (Zetasizer Nano ZS,
Malvern Instruments, United Kingdom) method []. e
sample was diluted to a thousand times with deionized water
and was shaken for minute in an ultrasonic water bath
for getting homogenous d ispersion. 𝜇Lofsamplewas
injected into the disposable capillary sample cell (DTS)
for the analysis.
2.5. Preparation of Encapsulation Eciency and Loading
Capacity. e encapsulation eciency and loading capacity
were determined by HPLC (Series II LC pump, SSi, USA,
HyperQuan Inc. VUV-, Shodex RI- detector, Showa
Denko, Japan) analysis. e analysis was conducted by
detecting the content of caaric acid of nano-EE and EE.
e encapsulation eciency and loading capacity of nano-EE
were calculated by the following formulas []:
Encapsulation eciency =[(A−B)
A×100%]()
A is total amount of caaric acid and B is free caaric acid
in the supernatant.
Loading capacity =[(A−B)
C×100%]()
A is total amount of caaric acid, B is free caaric acid in
the supernatant, and C is weight of the nanoparticles.
2.6. In Vitro Cell Experiments of Nano-EE and EE
2.6.1. Cell Culture. e LC- were cultured in Dulbecco’s
modied eagle medium: nutrient mixture F- (DMEM-F)
medium supplemented with fetal bovine serum (FBS) and
maintained at ∘C in a humidied incubator containing %
CO2[].
2.6.2. Cell Viability Assay. e cell viability assay was per-
formed by using the -(, -dimethylthiazol--yl)-, -
diphenyltetrazolium bromide (MTT) reagent. e samples
were dissolved in dimethyl sulfoxide (DMSO) and diluted
in cell culture medium. e nal concentration of cells in
DMSO was .%. e dierent doses were determined by
weighing method. In control experiments, this concentration
did not have any eects on the measured parameters. e
cells were cultured in well plate at a concentration of
×5cells/well and stimulated with hydrogen peroxide
(H2O2). Aer hours of preconditioning, the culture
medium was aspirated and cells were treated with a variety
of concentrations of the sample for hours. Subsequently,
𝜇lofMTTdye(mg/ml)wasaddedtothecultureand
further incubated for hours at ∘C. e cell viability was
calculated by measuring the absorbance at nm (ELISA
Reader, ermo Fisher , Germany) [].
2.6.3. Eect of EE on Nitric Oxide (NO) Production in H2O2-
Induced LC-540 Cells Oxidative Stress Model. Nitrite as the
end product of NO assay was measured by using the Griess
reagent. Briey, culture supernatants ( 𝜇l) were mixed with
𝜇l of the Griess reagent and the nitrite concentration
in the culture supernatant was obtained by measuring the
absorbance at nm. e nitrite concentration was deter-
mined with reference to the standard curve using sodium
nitrite [].
2.7. Animal Model Design. -week-old Sprague-Dawley (SD)
rats has purchased form BioLASCo Taiwan. Each rat was
housed individually in cages under controlled temperature
( ±∘C) and humidity ( ±%) with lighting on a h
light/ h dark cycle. Feed and water were provided as ad
libitum. Each rat was domesticated in the rst week and the
laboratory Rodent Diet was given as the main diet. Aer
domestication, rats were divided into two groups such as
control group (Con) that were fed with Lab Diet and
diabetic group that were fed with high-fat diet (HFD) (%
fat calories) for the entire experiment. Aer feeding with
HFD for weeks, the diabetic group rats were injected with a
low dose of streptozotocin (STZ) to induce diabetes mellitus
(DM). Aer week, oral glucose tolerance test (OGTT) was
conducted to determine the successful induction of DM.
Later, DM group were divided into groups DM group
without treatment, Met group (DM treated with metformin),
nano group (DM with chitosan/silica nanoparticle), and
three experimental nano-EE groups (DM with low, medium
and high dose of nano-EE; , and mg/kg B.W,
respectively). Treatments were given until the end of the
experiment. During the experiment, the body weight and
food and water intake were monitored and recorded between
every three days. All experimental rats were sacriced at the
age of weeks. Blood samples were collected in heparinized
tubes by hepatic portal vein sampling. Blood plasma was
separated by centrifugation at rpm at ∘Cformin.
Body organs were weighed and frozen for further analysis.
2.7.1. Postmortem Analyses/Analyses aer the Sacrice
(1) Sperm Analysis. e swim-up method was used to collect
sperm from the epididymis. Epididymis was cut into
pieces, immersed in Roswell Park Memorial Institute (RPMI)
medium and shaken in an orbital shaker at rpm for
min. Epididymis with RPMI medium was centrifuged
at ×g for min and incubated at ∘Cin%CO
2
incubator for minutes. Finally, the sperms were collected
from the supernatant and observed under the microscope
for determining the sperm count, motility, and abnormality.
Reactive oxygen species (ROS) damage was determined by
using uorescent dyes dichloro-dihydro-uorescein diacetate
(DCFH-DA) and ow cytometry []. e DNA fragmen-
tation of sperm was noticed by using acridine orange (AO)
staining method under the uorescent microscope [].
Nitric oxide (NO) and nitroblue tetrazolium (NBT) assay
were conducted by using chemical assay methods [, ].
(2) Testis Histology Analysis. Aer sacrice, the testicles were
collected and were immersed in % formaldehyde. e testis
was cut into mm from the middle part with a scalpel. e
group was marked with pencil on the lid and soaked back
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into formalin. Specimens were later sent to Rapid Science
Co., Ltd. at room temperature for paran-embedding and
hematoxylin & eosin (H&E) staining.
(3) Preparation of Testis Homogenates. mg of testicular
tissue was cut, rinsed with PBS, centrifuged for min,
and homogenized (Tissue homogenizer, Hitachi SCR BA,
Tokyo, Ja p a n ) in 𝜇L of homogenizing solution. 𝜇Lof
PBSwasaddedandthemixtureswerechilledto−∘Cfor
hours, followed by −∘C thawing, frozen again to −∘C,
then thawed, and centrifuged at ∘C ( ×grpm)for
minutes []. Supernatants were collected and were stored at
−∘C(−∘C Freezer, Nuaire, United Kingdom). e total
protein content of homogenates was determined by Bradford
assay.
(4) Analysis of Testis Homogenates. Inammation levels in
testis were determined by measuring tumor necrosis factor-𝛼
(TNF-𝛼)andIL-𝛽content in homogenates [].
2.8. Statistical Analysis. e data were analyzed using SPSS
. soware. All data were expressed as a mean ±standard
error of measurement (SEM) (N= ). Comparison among
groups was made by one-way analysis of variance (ANOVA).
Multiple comparisons of dierent groups were analyzed by
Duncan’s test at the value of p<. as signicant level.
3. Results and Discussion
3.1. e Chemical Composition of Phytopharmacological
Preparations and Encapsulation Eciency of Nano-EE. Echi-
nacea purpurea contained many active compounds such
as polysaccharides, lipid compounds, isobutylamides, total
polyphenols, avonoids, and inorganic salts. e content of
total polyphenols and alkylamides in EE depended upon
the extracted parts, species, and the methods of extraction.
Alkylamides are commonly found in the roots of Echi-
nacea and are related to the eects of immunomodula-
tion, anti-inammation, and antidepression. e polyphe-
nols are largely obtained from the oral part (also seen
in stem and leaves) of Echinacea and are well-known to
produce antioxidant activity. Previous studies show that the
isobutylamides and total polyphenols compounds of EE had
many pharmacological activities []. Increased resistance
to oxidation stress was related to the number of hydroxyl
groups on the phenol. e number of hydroxyl groups and
scavenging activities is directly proportional [ ]. e ecacy
order of EE components was in the following manner: -
echinacoside >cichoric acid >cynarin >chlorogenic acid >
caeic acid >caaric acid []. e content of isobutylamides
and total polyphenols of nano-EE and EE were evaluated
by HPLC analysis. Dodecatetraenoic acid isobutylamide was
identied from HPLC analysis (Figure ). e total phenolic
compounds which were detected from the HPLC analysis are
caaric acid, chlorogenic acid, echinacoside, and cichloric
acid (Figure (a)) and the standard curve of caaric acid
was given in Figure (b). e isobutylamides of freeze-dried
nano-EE and EE were . mg/g and . mg/g, respectively,
while the total polyphenols of freeze-dried nano-EE and
T : e contents of the extraction yield, alkylamide, total
phenolic acids, encapsulation rate, and loading capacity in freeze-
dried EE and nano-EE were examined by HPLC analysis.
Quality Characteristics Content
Nano-EE EE
(1)Extraction yield (%) -
(2)Total phenolic acids (mg/g freeze dried extract) . .
() Isobutylamides (mg/g freeze dried extract) . .
() Encapsulation eciency (%) . -
() Loading capacity (%) . -
–: not detected.
T : Particle size (PS), polydispersity index (PDI), and zeta
potential (ZP) of formulated nano-EE by DLS.
PS (nm) PDI ZP (mV)
Nano-EE ± . ±. -. ±.
0.020
0.010
AU
0.016
0.014
0.012
0.018
0.008
0.004
0.002
0.006
0.000
dodecatetraenoic acid isobutylamides
45.00 50.00 55.00 60.0010.005.00 15.00 20.00 25.00 30.00 35.00
Minutes
40.00
F : e HPLC chromatograms of the alkylamides in EE. e
chemical prole of EE was detected at UV nm.
EE were .mg/g and . mg/g respectively (Table ). In
addition to this, the extraction yield of EE obtained was
%. e loading capacity and encapsulation eciency of
nano-EE were about .% and .%, respectively. So the
results conrm that nano-EE has good loading capacity and
encapsulation eciency (Table ).
3.2. Characterization of Nano-EE. Chitosan/silica nanopar-
ticle has a small particle size, controlled release, and good
stability and it can be used as potential drug delivery systems
for biomedical applications []. e size and shape of the
particle were determined by SEM analysis. e average
particle size (conrmation), dispersion, and zeta potential of
the nano-EE were measured by DLS method. e average
particle size of nano-EE obtained from DLS method was
± nm (Table ) (Figure ). From the size distribution
data, it was evident that a minor portion of the nanoparticle
population has higher particles size.
e nanoparticles showed high polydispersity index
(PDI) value in the range of .±. (Table ). PDI is a kind
of particle size dispersion index indicating the homogeneity
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AU
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
0.090
0.100
0.110
0.000
2.000.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
Minutes
1.
2.
3.
4.
Caaric acid
Chlorogenic acid
Echinacoside
Cichloric acid
(a)
5000000
4000000
3000000
2000000
1000000
0
Caaric acid concentration (mg/ml)
Peak area
y=1000000x-19987
0.8
1.6
2.4
3.2
4.0
R2=0.9994
(b)
F : (a) e HPLC chromatograms of the total phenolic acids in EE (the chemical prole of EE was detected at UV nm); (b) standard
curve of caaric acid.
0.1
15
5
0110
10
20
100 1000 10000
Size (d.nm)
Intensity (Percent)
F : Dynamic light scattering (DLS) of nano-EE particle size.
of the carrier system []. e better PDI value was obtained
for nano-EE group.
Zeta potential is an important parameter for interpreting
the electrostatic potential near the surface of nanoparticles
and predicting their degree of stability. It is a measure of the
magnitude of charges on nanoparticles and also denoting the
stability of the particles. e obtained zeta potential of the
nanoparticles was −. ±. mV (Table ). e negative
charge of zeta potential indicated that the nano-EE has a
negative charge and is stable in water. ese charged particles
can interact more favorably with the cell membrane and
thereby enhance the uptake of the particle.
Furthermore, with SEM analysis, the size and shape of
the prepared nano-EE (Figure ) were analyzed. e SEM
investigation of the nano-EE showed that the average particle
size of the nano-EE was ± nm in diameter. Particles
with a size between and nm belong to the micro-
nanograde particles. However, From SEM (x) analysis,
it was found that the particles were clustered in nano-EE
group. At , times magnication, the results indicated
that nanoparticles were appeared similar to small beads.
Finally, it was founded that the particle size of the DLS
was greater than that of the SEM. e reduction in the size
of the particle was due to particle shrinkage during SEM
analysis. It could be assumed that the size of the nanoparticle
was reduced due to the loss of moisture content from the
samples and the nanoparticles to get closer to each other.
It was understood that the chitosan-silica nanoparticle was
formed with poor dispersion and has spherical clusters with
agglomeration.
3.3. Cell Experiments
3.3.1. H2O2-Induced LC540 Oxidative Stress Model
(1) Protective Eect of Nano-EE on H2O2-Induced Cytotoxicity.
e cytotoxicity of nano-EE and EE in LC- cells was
evaluated by MTT assay (Figure ). e results show that
there is not any signicant dierence in the cytotoxicity
of nano-EE and EE groups at concentrations ranging from
𝜇g/mL to 𝜇g/mL. us, the highest concentrations of
nano-EE available for LC- cell line were 𝜇g/mL. Cell
viability of LC- cells with 𝜇g/mL dose of nano-EE and
EE has a signicant dierence. e result indicated that nano-
EE has a slow and sustained release of the EE solution.
e cytoprotective eect of nano-EE (, ., ., ., .,
., and 𝜇g/mL) in H2O2-induced LC- cells was exam-
ined in Figure . Cell viability of LC- cells exposed to
𝜇MH
2O2decreased below % and increased signicantly to
% in the nano-EE-treated group at 𝜇g/mL (Figure ).
(2) Eect of Nano-EE on H2O2-Induced NO Production.
Oxidative stress plays an important role in male infertility.
e imbalance between ROS production and antioxidants
activity might lead to a rise in ROS levels in the semen.
High level of ROS aects the unique ability of the male
germ cells to move forward and also upsets their ability to
fertilize to the oocyte. e fertilizing ability of the human
spermatozoa is inversely proportional to the sperm ROS
production. H2O2is one of the major ROS associated withthe
oxidative stress. It readily penetrates into cells and reacts with
intracellular metal ions such as iron or copper to generate
highly reactive O2- and nitric oxide (NO-) radicals []. It
was identied that a strong inhibition of cell growth by H2O2
and the protective eect of nano-EE against H2O2-induced
oxidative stress in LC- cells. It was further observed
that nano-EE and EE could protect the LC- from H2O2-
induced oxidative stress and reduced NO production. e
results indicated that nan o-EE was c apable of reducing H2O2-
induced cytotoxicity. e NO release of nano-EE and EE (,
., ., and 𝜇g/mL) in H2O2-treated LC- cells was
evaluated aer hours (Figure ). e results show that the
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(a) (b)
(c)
F : Scanning electron micrographs (SEM) of EE and nano-EE structures EE at times magnication (a), nano-EE at , times
magnication (b), and , times magnication (c).
production of NO was signicantly reduced with an increase
in the concentration of nano-EE and EE. As compared
to nano-EE, EE has a signicant dierence in inhibiting
NO production. e hig hest concentration ( 𝜇g/mL) of
nano-EE and EE exhibited more NO reduction than other
concentrations. erefore, nano-EE and EE can signicantly
reduce NO production and thereby lower the oxidative stress.
3.4. Animal Experiments
3.4.1. Evaluation of the Body Weight, Food Intake, and
Water Intake in Diabetic Rats Fed with Nano-EE. e STZ-
induced experimental diabetic animal model was character-
ized by hyperglycemia, increased food, and water intake, and
decreased body weight has been chosen for the present study
[]. From this study, it could be understood that the weight
loss has happened when fed with nano-EE and nano-EE
may be caused due to an increase in the metabolism of the
body.
Metformin (Met) which is an important antihypergly-
caemic agent commonly used for the treatment of type
diabetes mellitus has the ability to decrease the hepatic
glucose production via a mild and transient inhibition of the
mitochondrial respiratory chain complex I. In addition, it
results in the reduction of hepatic energy status and activates
AMP-activated protein kinase (AMPK) a cellular metabolic
sensor, providing a generally accepted mechanism for the
action of metformin on hepatic gluconeogenesis [].
We recorded the body weight of each rat from weeks
old to weeks old (Figure ). Rats were induced to diabetes
at the age of weeks by using STZ. e results show
that the body weight of control (Con) group was increased
in every week while the body weight of diabetic-induced
groups was signicantly lower than that of the control group.
e metformin (Met) and dierent dose of nano-EE were
administered at the age of weeks. e result showed that
there was a signicant reduction in the body weight of Met,
nano-EE, and nano-EE in comparison with DM group and
nano groups, though no signicant dierences were observed
within these three groups.
efoodandwaterintakeofConwaslowerthanthatof
diabetes-induced groups (Figure ). At the age of weeks,
the calories and water intake of DM group were relatively
higher than the Con group. At the age of weeks, there were
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0
6.25
12.5
25
50
100
200
400
800
EE
Nano-EE
∗∗
Concentration (
g/mL)
0
20
40
60
80
100
120
cell viability (%)
F : Cell viability of LC-540 was treated with nano-EE and EE
for 24 hours. LC- cells were adjusted to ×5cells/ml and
treated with nano-EE and EE for hours. e cells viability was
analyzed by MTT assay. Results were shown by mean ±SEM (n =
). P<. (∗∗) indicates signicant dierences between EE and
nano-EE.
-induced LC-540 cell oxidative stress
∗∗ ∗∗∗
∗∗∗∗
∗∗∗∗
∗∗∗∗
0
25
50
75
100
125
cell viability (%)
46.9 93.8 187.5 375 750 1500 30000
HO
HO
(M)
）＃50 = 1100 －
F : Cell viability of various H2O2-induced LC-540 cell stress
for 24 hours. Cells were adjusted to ×5cells/ml and treated with
H2O2for hours. e cells viability was analyzed by MTT assay.
Results were shown by mean ±SEM (n = ). P <. (∗∗∗∗), P
<. (∗∗∗), and P <. (∗∗)versusng/mlH
2O2.
not any signicant dierences observed in the calories and
water intake of DM, nano, and nano-EE groups. e Met,
nano-EE, and nano-EE groups show relatively lower intake
of calories and water. In the nal week, lowered intake of
calories and water were observed in each group by OGTT.
3.4.2. Eect of Plasma Biochemical Parameters in Di abetic Rats
(1) Glucose Tolerance Test (OGTT) and the Area under the
Curve (AUC) in Diabetic Rats aer 7 Weeks. OGTT is known
as a marker for diagnosing diabetes. It was usually carried out
0
0.8
1.6
3.1
6.3
12.5
25
g/ml
0
10
20
30
40
50
60
70
80
90
100
cell viability (%)
HO
(1100 M) + EE
HO
(1100 M) + Nano-EE
F : LC-540 was treated with dierent EE, nano-EE, and H2O2
for 24 hours. Cells were adjusted to ×5cells/ml were treated for
hours with EE, nano-EE, and H2O2( 𝜇M). e cells viability
was analyzed by MTT assay. It can decide the amount of the damage
of the H2O2. Results were shown by mean ±SEM (n = ).
aer overnight fasting or glucose loading. e plasma glucose
level was determined aer days of STZ administration
(Figure (a)). When compared to the Con group, the fasting
glucose level of other groups was elevated to above mg/dl.
It was observed that, aer hours, the blood glucose level of
STZ-induced groups was greater than mg/dl. e blood
glucose level of STZ-induced groups was not signicantly
lowered to normal values and the AUC of blood glucose
was also increased (Figure (c)). So, the results indicated
that rats were successfully induced with diabetics. e blood
glucose level of DM and nano groups has no any signicant
reductionatmin(Figure(b)).But,inthecaseofMet
and nano-EE treatments, the blood glucose levels were
lowered when compared to the DM group (Figure (a)).
Furthermore, we found that the AUC of blood glucose in the
nano-EE group was lowered to the AUC of the Met group in
a dose-dependent manner (Figure (d)).
(2) Eects of Plasma Insulin Level, Homeostatic Model
Assessment-Insulin Resistance (HOMA-IR), Advanced Glyca-
tion End Products (AGEs), and Plasma Fibroblast Growth
Factor 21 (FGF 21) in Diabetic Rats Fed with Dierent Doses
of Nano-EE aer 7 Weeks. Insulin is a peptide hormone
produced by the beta cells of the pancreas. It is functionalized
to maintain the blood sugar level and protect the body from
hyperglycemia (sugar level high) and hypoglycemia (low
sugar level) []. e insulin resistance is dened by HOMA-
IR. e pathogenesis of diabetes is mediated by AGEs [].
Diabetes can be caused by the binding of plasma glucose to
the protein in the blood. Aer a series of chemical reactions,
AGEs were produced as irreducible substances and can aect
thenormalfunctionsoftheDNAandgeneexpressionofthe
cells. Accumulation of AGEs will generate a large amount of
ROS and eventually cause an increase in oxidative stress [].
BioMed Research International
NO assay
Control
0
6.3
12.5
25
+ EE (g/ml)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Nitric oxide release (
M)
∗∗∗
∗∗ ∗∗ ∗∗
HO
(a)
NO assay
Control
0
6.3
12.5
25
+ Nano-EE (g/ml)
0.00
0.02
0.04
0.06
0.08
0.10
Nitric oxide release (
M)
∗∗ ∗∗
∗∗
HO
(b)
F : Eects of EE and nano-EE on H2O2induced nitric oxide release for 24 hours by NO assay in LC-540. Cells were adjusted to ×5
cells/ml were treated for hours with EE (a), nano-EE (b), and H2O2( 𝜇M). e cells viability was analyzed by NO assays. It can decide
the amount of the damage of the H2O2. Results were shown by mean ±SEM (n = ). P<. (∗∗); P<. (∗)versusng/mlH
2O2+EEor
nano-EE.
Con
STZ
OGTT
Gavage
OGTT
Sacrificed
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
∗∗
∗∗
∗∗
∗∗ ∗∗∗
∗∗∗
∗∗∗ ∗∗∗
∗∗ ∗∗∗
150
175
200
225
250
275
300
325
350
375
400
425
450
475
500
Body weight (g)
7 8 9 10 11 12 13 14 15 16 17 186
Age (weeks)
∗∗∗
∗∗∗
∗
∗∗
∗∗
F : Body weight of rats during experiment. Data are shown as
the mean ±SEM (n = rats/group). Con: control; DM: diabetes;
Met: diabetes + mg/kg p er day metformin; Nano: diabetes
+ mg/kg per day chitosan and silica; Nano-EE: diabetes +
mg/kg per day EE; Nano-EE: diabetes + mg/kg per day EE;
Nano-EE: diabetes + mg/kg per day EE. P <. (∗∗∗), p <
. (∗∗), and p <. (∗)versusDM.
FGF is an endocrine hormone predominantly seen in liver,
pancreas, and adipose tissue and is relatively less seen in
other organs such as testis. e FGF are functionalized to
increase the glucose uptake in the adipose tissue, augment
lipolysis, enhance production of ketone bodies in the liver,
and regulate energy balance and physical stress responsive-
ness in humans. Serum FGF is also a superior biomarker
to other adipokines in predicting incident diabetes []. e
level of plasma FGF is increased in insulin-resistant states
and correlates with hepatic and whole-body (muscle) insulin
resistance in type diabetes []. Diabetes and its related
reproductive impairments have been widely studied, but the
exact mechanisms for the reproductive dysfunction in males
are not completely understood.
Aer the administration of dierent doses of nano-EE for
weeks, the levels of plasma insulin, HOMA-IR, advanced
glycation end products (AGEs), and plasma FGF were
determined (Table ). e results showed that DM group
possesses insulin over secretion. No signicant dierence
was observed between nano and DM group. But, nano-EE
exhibited a reduction in insulin secretion. e signicant
reduction of insulin secretion was observed in the Met group.
Furthermore, DM group and nano-EE had signicantly
increased the level of HOMA-IR and AGEs compared to
Con group. Treatment with nano-EE eectively improved
theHOMA-IRandAGEslevelstoaslowastheMetgroup.
Finally, the FGF level in DM group signicantly increased.
Nano-EE and Met group can be reduced the FGF level in a
dose-dependent manner, but no signicant dierence among
the groups was observed.
(3) Eects of Plasma ALT, AST, Urea, and Creatinine in
Diabetic Rats Fed with Dierent Doses of Nano-EE aer 7
Weeks. Elevation in the concentration of aspartate amino-
transferase (AST) and alanine aminotransferase (ALT) are
seen in diabetic condition. e level of urea and creatinine is
higher in diabetes condition and it is known as a signicant
marker for renal dysfunction []. Aer the administration
of dierent doses of nano-EE for weeks, the activities
of plasma enzymes AST and ALT were signicantly higher
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80
100
120
140
160
180
Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
Food intake (kcal)
12 13 14 15 16 1711
Age (week)
∗∗∗∗
∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗
∗∗∗∗
∗∗∗∗
∗∗∗∗
∗∗∗∗
∗∗∗∗
∗∗∗∗
∗∗
∗∗∗
∗
∗∗
∗
∗∗∗∗
∗∗
(a)
Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
12 13 14 15 16 1711
Age (week)
0
20
40
60
80
100
120
140
Water intake (g)
∗∗ ∗∗ ∗∗ ∗∗
∗∗ ∗∗
∗
(b)
F : Eects of nano-EE on food intake (a) and water intake (b) during 6 weeks in diabetic rats. Data were shown as the mean ±SEM (n =
rats/group). Con: control; DM: diabetes; Met: diabetes + mg/kg per day metformin; Nano: diabetes + mg/kg per day chitosan and
silica; Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes + mg/kg per day EE.
P<. (∗∗∗∗), P <. (∗∗∗), p <. (∗∗), and p <. (∗)versusDM.
T : Plasma insulin level, homeostasis model assessment equation (HOMA-IR), advanced glycation end products (AGEs), and plasma
FGF in diabetic rats fed dierent doses of nano-EE aer weeks.
Con DM Met Nano Nano-EE Nano-EE Nano-EE
Insulin (𝜇U/mL) . ±.5c. ±.3ab . ±.6bc . ±.7abc . ±.6a. ±.4ab . ±.2bc
HOMA-IR . ±.6d. ±.9a. ±.0bc . ±.4ab . ±.38a. ±.4abc . ±.8c
AGEs (𝜇g/ml) . ±.6c. ±.7a. ±.4c. ±.3a ±.2ab . ±.78abc . ±.1bc
Plasma FGF (ng/mg protein) . ±.1b. ±.1a. ±.1b. ±.1ab . ±.4ab . ±.2ab . ±.1b
Data were shown as the mean ±SEM(n=rats/group).Con:control;DM:diabetes;Met:diabetes+mg/kgperdaymetformin;Nano:diabetes+
mg/kg per day chitosan and silica; Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes + mg/kg
per day EE. e values with dierent letters (a-b) represent signicantly dierent (p<.) as analyzed by Duncan’s multiple range test.
HOMA-IR = fasting plasma glucose (mg/dL) ×fasting plasma insulin (𝜇U/mL)/.
T : Eects of plasma ALT, AST, BUN, and creatinine in diabetic rats fed dierent doses of nano-EE aer weeks.
Con DM Met Nano Nano-EE Nano-EE Nano-EE
AST (U/L) . ±.8d. ±.0a. ±.2bc . ±.4ab . ±.1cd . ±.4cd . ±.0d
ALT (U/L) . ±.9b. ±.9a. ±.8b. ±.5ab . ±.5b. ±.3b. ±.0b
BUN (mg/dL) . ±.7b. ±.6ab . ±.8ab . ±.0a. ±.8ab . ±.8b. ±.1b
Creatinine (mg/dL) . ±.6bc . ±.0a. ±.2abc . ±.6ab . ±.1ab . ±.6cd . ±.6d
Data were shown as the mean ±SEM(n=rats/group).Con:control;DM:diabetes;Met:diabetes+mg/kgperdaymetformin;Nano:diabetes+
mg/kg per day per chitosan and silica; Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes +
mg/kg per day EE. e values with dierent letters (a-b) represent signicantly dierent (p<.) as analyzed by Duncan’s multiple range test.
(i) Alanine aminotransferase (ALT).
(ii) Aspartate aminotransferase (AST).
in the diabetic group when compared to the Con group
(Table ). e treatment with the dierent dose of nano-
EE and Met signicantly prevented the rise of AST and
ALT activities under diabetic condition. Levels of urea and
creatinine are also increased signicantly in diabetic group
when compared to Con group. However, the administration
of nano-EE and Met prevented the increased urea and
creatinine levels.
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Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
Before
30 60 1200
Time (min)
0
50
100
150
200
250
300
Blood glucose (mg/dl)
∗
∗
∗
(a)
0
30
60
120
Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
Aer
Time (min)
0
50
100
150
200
250
300
350
Blood glucose (mg/dl)
∗
∗
∗
∗
∗
∗
∗
∗
(b)
Before
Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
A
AAAAAA
0
2500
5000
7500
10000
12500
15000
17500
AUC (mg/dL
∗
min)
(c)
Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
A
B
B
AB
B
AA
Aer
0
5000
10000
15000
20000
AUC (mg/dL
∗
min)
(d)
F : Eects of nano-EE on oral glucose tolerance test (OGTT) (aer oral glucose load of 2 g/kg) (a-b) and the area under curve (AUC)
(c-d) in diabetic rats and before and aer 7 weeks. Data were shown as the mean ±SEM (n = rats/group). Con: control; DM: diabetes; Met:
diabetes + mg/kg per day metformin; Nano:diabetes + mg/kg per day chitosan and silica; Nano-EE: diabetes + mg/kg per day EE;
Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes + mg/kg per day EE. p<. (∗) versus DM. e values with dierent
letters (A-B) are signicantly dierent (p<.) as analyzed by Duncan’s multiple range test.
3.4.3. Evaluation of Nano-EE Reproductive Function in
Diabetic Rats
(1) Eects of Parameters on Sperm Count, Motility, Abnormal-
ity, and DNA Integrity by Acridine Orange (AO) Staining in
Diabetic Rats. Increase in the incidence of diabetes mellitus
in worldwide in men is associated with subfertility and
infertility. Long-term diabetes mellitus with uncontrolled
hyperglycemia is responsible for the testicular dysfunction
and resulting in the reduction of fertility potential. Later, it
was described that STZ-induced diabetes mellitus in the male
rats had altered sex behavior and diminished reproductive
organ weight, testicular sperm content, and epididymal
sperm content, as well as sperm motility. Increased sperm
DNA damage was also reported in diabetic men at repro-
ductive age associated with high oxidative stress resulting
from sperm over exposure to glucose []. e mechanisms
of sperm nuclear DNA damage in diabetic men promoted
by ROS are suggested to be mediated through the activity of
AGEs. ese AGEs accumulate in the reproductive tract of
diabetic men. Moreover, increased oxidative stress and higher
DNA fragmentation are interconnected with apoptosis [].
Aer the administration of dierent doses of nano-EE for
weeks, parameters suchas sperm count, motility, abnormal-
ity (Figure ), and DNA integrity were determined (Figures
and ). Results show that a signicant reduction in sperm
count and motility as well as an increased abnormality was
observed in the DM group when compared to the Con. But,
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T : Eects of nano-EE and EE on diameters seminiferous tubules (DST), germinal cell layer thickness (GCLT), area of seminiferous
tubules (AST), and area of seminiferous lumen (ASL) aer weeks of treatment.
Con DM Met Nano Nano-EE Nano-EE Nano-EE
(diameter Size)
DST (𝜇m) . ±.1a. ±.9c. ±. 7a ±.4c ±.6bc . ±.7ab . ±.0a
GCLT (𝜇m) . ±.8a. ±.9c. ±.2a ±.0c. ±.5c. ±.9b. ±.0a
(Area)
AST (%) . ±.0a. ±.2bc . ±.1a. ±.7bc . ±.6c. ±.6b. ±.7a
ASL (%) . ±.0c. ±.2ab . ±.1c. ±.7a. ±.6a. ±.6b. ±.7c
Data were shown as the mean ±SEM(n=rats/group).Con:control;DM:diabetes;Met:diabetes+mg/kgperdaymetformin;Nano:diabetes+
mg/kg per day per chitosan and silica; Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes +
mg/kg per day EE. e values with dierent letters (a-c) represent signicantly dierent (p<.)as analyzed by Duncan’s multiple range test. e area %was
calculated by the formula, where As is area covered by seminiferous tubules or lumen. T is total area of the eld. %As = As ×.
Met has signicantly increased the sperm count and motility
and reduced the sperm abnormality. Treatment with nano-
EE improved the sperm motility and abnormality but there
were not any improvements seen in nano group (without EE).
e appearance of the sperm was observed the under
microscope (Inverted Phase Contrast Microscope, Olympus
IX-, Tokyo, Japan) (Figure ). Results showed that signif-
icant abnormalities such as the coiled tail, detached head,
and aggregated sperm were spotted in DM and nano groups.
Treatment with the Met and nano-EE groups shows better
results as compared to the DM group and nano group.
Finally, based on the properties of the uorochrome
acridine orange (AO), which uoresces green when bound
to native deoxyribonucleic acid (DNA) (double-stranded and
normal) and red when bound to denature DNA (single-
stranded). Orange and red stained spermatozoa (orange and
red arrows) were the abnormal sperm (denatured DNA)
while green stained (green arrows) spermatozoa were consid-
ered to be normal (nondenatured DNA). Results showed that
when compared to the Con group, the signicant sperm DNA
damage was seen in DM and nano group. Treatment with
the Met and nano-EE groups were shown better improve-
ments (Figure ). e quantitative analysis of the sperm
DNA integrity in Con and various experimental animals by
acridine orange (AO) staining was shown in Figure . e
percentage of the apoptotic cell was decreased in nano-EE
group compared to nano, Met, and DM groups.
(2) Eects of Nano-EE on the Sperm Production of ROS,
NO, and NBT aer 7 Weeks of Treatment in Diabetic Rats.
ROS production is known as the major process for oxidative
stress during diabetes condition. Generation of ROS was
determined by using DCFH-DA as a probe (Figure (a)).
ROS levels were signicantly increased in DM group when
compared to the Con group. But, treatment with nano-EE
and nano-EE signicantly reduced ROS level in the sperm.
Nano group did not show any improvement in ROS level.
e production of NO and superoxide anion in sperm
was observed in diabetic rats (Figures (b) and (c)).
e production of NO and superoxide anion in the DM
group was signicantly increased when compared to the Con
group. Treatment with Met and nano-EE could reduce their
productions. e reduction of NO and superoxide anion in
Met was most signicant, followed by the nano-EE group.
ese results are suggesting the anti-inammatory potential
of Met and nano-EE groups.
(3) Eects of Nano-EE and EE on Diameters Seminiferous
Tubules (DST), Germinal Cell Layer ickness (GCLT), the
Area of Seminiferous Tubules (AST), and Area of the Seminif-
erous Lumen (ASL) aer 7 Weeks of Treatment. e interior of
the testis is composed of many seminiferous tubules. ese
seminiferous tubules are supported by the Sertoli cell and
the Leydig cell exists in the space between the seminiferous
tubules. Sertoli cell and Leydig cell are responsible for the
regulation of androgen concentration and spermatogenesis
along with sperm maturation. e immature sperm cells are
located in the basement membrane while the mature sperm
is located in the centre of the lumen [].
Histological assessment of the testicular tissue showed
atrophy of seminiferous tubule, loss of testicular cells (such as
Leydig and Sertoli cells), sloughing of centrally located sper-
matozoa, and disappearance of spermatids in the seminifer-
ous tubular lumen in both DM and nano groups (Figure ).
However, dose-dependent treatments of these animals with
nano-EE for weeks attenuated these changes and provided
optimal protection at a dose of nano-EE (Figure ). So,
it is suggesting the protective action of nano-EE against
oxidative stress-mediated testicular damage in diabetic rats.
e diameters of seminiferous tubules (DST), germinal
cell layer thickness (GCLT), and the area of seminiferous
tubules (AST) were signicantly reduced, and the area of
seminiferous lumen (ASL) was increased in DM group as
compared to the Con, nano-EE, and nano-EE (Table ).
3.4.4. Analysis of Anti-Inammation
(1) Eect of the Plasma TNF-𝛼,IL-1𝛽Inammation Markers
aer 7 Weeks Treated with Nano-EE in Diabetic Rats. Anum-
ber of experimental and clinical data have clearly established
that adipose tissue, liver, muscle, and pancreas are the sites
of inammation in the presence of type diabetes mellitus
[]. Inltration of macrophages into these tissues was seen
in animal models of diabetes as well as in obese human
individuals with metabolic syndrome or type diabetes
mellitus []. ese cells are crucial for the production of
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Con DM Met
Nano Nano-EE1 Nano-EE3 Nano-EE5
F : Eects of nano-EE on seminiferous tubules of testis in diabetic rats aer 7 weeks of treatment. Representative images of hematoxylin
and eosin (H&E) sect ions in the testis of eac h group (magnications = X). Arrow : Leydig c ells, arrow : Sertol i cell, arrow : spermatogonia,
arrow : spermatocyte, arrow : sperm, blue line: diameters of seminiferous tubules (DST), black line: germinal cell layer thickness (GCLT),
and star: tubular shrinkage and extensive intertubular area.
Sperm count
A
A
BC B BC BC
C
0
50
100
150
Cell (×)
Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
(a)
Sperm mobility
A
AB ABABC
BC
CC
Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
0
5
10
15
20
Percent (%)
(b)
CD
Sperm abnormality
A
AB
BCD
BC
D
CD
Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
0
5
10
15
20
25
Percent (%)
(c)
F : Eects of sperm parameters, (a) count, (b) mobility, and (c) abnormality, in diabetic rats fed dierent doses of nano-EE in diabetic
rats aer 7 weeks. Data were shown as the mean ±SEM (n = rats/group). Con: control; DM: diabetes; Met: diabetes + mg/kg per
day metformin; Nano: diabetes + mg/kg per day chitosan and silica; Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes +
mg/kg per day EE; Nano-EE: diabetes + mg/kg per day EE. e values with dierent letters (A-D) are signicantly dierent (p <
.) as analyzed by Duncan’s multiple range test.
BioMed Research International
Con DM Met
Nano Nano-EE1 Nano-EE3 Nano-EE5
F : Eects of nano-EE on sperm morphology seen in diabetic rats epididymal aer fed dierent doses of nano-EE in diabetic rats aer 7
weeks. Dierent sperm abnormalities in dierent dose of nano-EE-treated diabetic rats. Purple arrow: coiled tail, blue arrow: detached head,
and red arrow: aggregated sperm.
Con DM Met
Nano Nano-EE1 Nano-EE3 Nano-EE5
F : Evaluation of the sperm DNA integrity in control and various experimental animals by acridine orange (AO) staining. Orange and
red stained spermatozoa (orange and red arrows) were abnormal sperm (denatured DNA) while green stained (green arrows) spermatozoa
were considered to be normal (nondenatured DNA). For interpretation of the references to color in this gure legend, the reader is referred
to the histogram of Photoshot.
proinammatory cytokines, including TNF-𝛼, IL-, and IL-
𝛽. ey act in an autocrine and paracrine manner to promote
the insulin resistance by interfering with insulin signaling
in peripheral tissues through the activation of the c-JUN
N-terminal kinase (JNK) and nuclear factor-kappa B (NF-
𝜅B) pathways []. ese pathways are activated in multiple
tissues in type diabetes mellitus and have a central role in
promoting tissue inammation. In the pancreas, activation
of NLRP (NACHT, LRR, and PYD domains-containing
protein ) inammasome by high levels of glucose and
fatty acids and subsequent release of IL-𝛽lead to 𝛽cells
dysfunction, apoptosis, insulin deciency, and progression to
type diabetes mellitus []. In this study, the levels of IL-𝛽
and TNF-𝛼were increased in DM group but the levels were
decreased in the Met and nano-EE groups.
Results showed that, as compared to the Con group,
TNF-𝛼and IL-𝛽levels in the DM group were increased.
A signicant reduction in the level of TNF-𝛼and IL- 𝛽
was observed in Met and nano-EE groups and reached
almost similar values to the Con group while no signicant
dierence was observed between the Con, Met, and nano-
EE groups (Figure ).
BioMed Research International
AO ass ay
ab
a
b
cccd
d
Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
0
2
4
6
8
10
12
14
Apoptotic cells (%)
F : Quantitative analysis of the sperm DNA integrity in control and various experimental animals by acridine orange (AO) staining. For
interpretation of the references to color in this gure legend, the reader is referred to the histogram of Photoshot. Data were shown as the
mean ±SEM (n = rats/group). Con: control; DM: diabetes; Met: diabetes + mg/kg per day metformin; Nano: diabetes + mg/kg
per day chitosan and silica; Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes +
mg/kg per day EE. e values with dierent letters (a-d) are signicantly dierent (p<.) as analyzed by Duncan’s multiple range test.
A
BC
B
BC
BCD CD
D
Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
0
50
100
150
DCF fluorescence
(% of control)
(a) ROS
B
A
B
AB AB
AB
A
Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
0.0
0.5
1.0
1.5
2.0
NO (M)
(b)
BC
AB
C
ABC
A
ABC
BC
Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
0.0
0.1
0.2
0.3
0.4
0.5
Superoxide anion (mM)
(c)
F : Eects of nano-E E on the sperm produc tion of ROS (a) and NO (b) superoxide anion (c) aer 7 weeks of treatment in diabetic rats. Data
were shown as the mean ±SEM (n = rats/group). Con: control; DM: diabetes; Met: diabetes + mg/kg per day metformin; Nano: diabetes
+ mg/kg per day chitosan and silica; Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes + mg/kg per day EE; Nano-EE:
diabetes + mg/kg per day EE. e values with dierent letters (A-B) are signicantly dierent (p<.) as analyzed by Duncan’s multiple
range test.
BioMed Research International
BC
A
C
AAB
ABC
C
Plasma
Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
0
5
10
15
20
25
TNF-a (ng/mg protein)
(a)
Plasma
CD
A
D
ABC
A
AB
BCD
Con
DM
Met
Nano
Nano-EE1
Nano-EE3
Nano-EE5
0
10
20
30
40
IL-1 (pg/mg protein)
(b)
F : Eect of the plasma TNF-𝛼and IL-1𝛽inammation markers aer 7 weeks of nano-EE treatment in diabetic rats. Data were shown as
the mean ±SEM (n = rats/group). Con: control; DM: diabetes; Met: diabetes + mg/kg per day metformin; Nano: diabetes + mg/kg
per day chitosan and silica; Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes + mg/kg per day EE; Nano-EE: diabetes +
mg/kg per day EE. e values with dierent letters (A-B) are signicantly dierent (p<.) as analyzed by Duncan’s multiple range test.
4. Conclusion
In the present work, the observations suggested that the
micro-nanoparticles prepared by an easy process without
using high temperature and harsh organic chemicals might
be employed to improve the potential oral delivery of EE. EE
loaded chitosan/silica core-shell micro-nanoparticles (nano-
EE) were characterized by DLS and SEM analysis and evalu-
ated their eect both in vitro and in vivo system. e results
indicated that the size of nano-EE was ± nm with
an almost .% encapsulation eciency and .% loading
capacity. is chitosan/silica micro-nanoparticles enabled
the oral delivery of EE with low toxicity.
Our in vivo study revealed that nano-EE treatment might
provide eective protection against the oxidative damage
in plasma and testis of STZ-induced type diabetic rats.
Since this compound was able to ameliorate the enzymatic
and nonenzymatic antioxidant defense system to prevent
the lipid peroxidation in the tissues, nano-EE improved
oxidative stress, insulin resistance and restored liver and
kidney function and mitochondrial membrane potential in
diabetic rats.
erefore, the micro-nanoencapsulated EE can be used as
a promising tool for improving the reproductive function in
diabetic rats and provide a smart way for the development of
healthy products applicable to the pharmaceutical eld.
Data Availability
No data were used to support this study. e statements that
supporting our ndings are cited in references.
Conflicts of Interest
ere are no conicts of interest regarding the publication of
this paper.
Acknowledgments
is work was nancially supported by the Center of Excel-
lence for the Oceans, National Taiwan Ocean University,
from the Featured Areas Research Centre Program within
the framework of Higher Education Sprout Project by the
Ministry of Education (MOE), Taiwan. is work was also
granted by the Ministry of Science and Technology (MOST
--B--), Taiwan.
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