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ORIGINAL RESEARCH
Impact of Ginkgo biloba extract and magnetized
water on the survival rate and functional capabilities
of pancreatic β-cells in type 2 diabetic rat model
This article was published in the following Dove Press journal:
Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy
Ahmed Saleh
1,2
Mamdouh M Anwar
3,4
Ahmed E Zayed
5,6
Manal El Sayed Ezz Eldeen
7
Gamal Afifi
1,8
Hassien M Alnashiri
5
Asmaa MS Gomaa
4
Mahmoud Abd-Elkareem
6
Alaa Sayed Abou-Elhamd
6,9
Emad S Shaheen
10
Ghada A Mohamed
7
Helal F Hetta
11,12
Ahmed M Kotb
6
1
Department of Physics, Faculty of Science,
Jazan University, Jazan, KSA;
2
Exploratory
Center of Science and Technology, Cairo,
Egypt;
3
Department of Pharmacology, Faculty
of Pharmacy, Jazan University, Jazan, KSA;
4
Medical Physiology Department, Faculty of
Medicine, Assiut University, Assiut, Egypt;
5
Department of Biology, Faculty of Science,
Jazan University, Jazan, KSA;
6
Department of
Anatomy and Histology, Faculty of Veterinary
Medicine, Assiut University, Egypt;
7
Endocrine Unit, Department of Internal
Medicine, Faculty of Medicine, Assiut
University, Assiut, Egypt;
8
National Institute
for Laser Enhanced Sciences, Cairo
University, Giza, Egypt;
9
Department of
Medical Laboratory Technology, Faculty of
Applied Medical Sciences, Jazan University,
Jazan, Saudi Arabia;
10
Medical Research
Centre, Jazan University, Jazan, KSA;
11
Department of Medical Microbiology and
Immunology, Faculty of Medicine, Assiut
University, Assiut, Egypt;
12
Department of
Internal Medicine, University of Cincinnati
College of Medicine, Cincinnati, OH, USA
Introduction: Type 2 diabetes (T2D) is a widely distributed disease that affects large
population worldwide. This study aimed to verify the role of Ginkgo biloba (GB) extract
and magnetized water (MW) on the survival rate and functional capabilities of pancreatic β-
cells in type 2 diabetic rats.
Materials and methods: T2D was induced by feeding the rats on a high-fat diet (20% fat,
45% carbohydrate, 22% protein) for eight weeks followed by intra-peritoneal injection of a
single low dose of streptozotocin (25mg/Kg). Forty rats were randomly assigned to four
groups (n=10 rats) as follows: non treated control and three diabetic groups. One diabetic
group served as a positive control (diabetic), while the other two groups were orally
administered with water extract of GB leaves (0.11 g/kg/day) and MW (600 gauss) for
four weeks, respectively.
Results: The β-cell mass and insulin expression in these cells increased markedly after both
treatments, particularly in GB treated group. In addition, the immune-expression of the two
antioxidant enzymes; glutathione and superoxide dismutase 2 (SOD2) in the pancreatic tissue
demonstrated a down-regulation in GB and MW treated groups as compared with the
diabetic group.
Conclusion: A four-week treatment of GB and MW protected pancreatic β-cell cells and
improved their insulin expression and antioxidant status in type 2 diabetic rats.
Keywords: type 2 diabetes, β-cell, Ginkgo biloba extract, magnetized water, glutathione, SOD2
Introduction
Diabetes mellitus (DM) is classified into two main subtypes: 1 and 2. Type 1 DM
results from the destruction of the pancreatic β-cells and lack of insulin secretion; it
is accompanied by high blood glucose concentrations and ketoacidosis.
1,2
However,
Type 2 DM (T2D) is more common and is frequently linked to obesity.
3,4
It has been previously shown that T2D could affect the pancreatic endocrine
(islets of Langerhans) and exocrine systems (pancreatic acini). T2D in many cases is
accompanied by a decrease in body weight and many digestive disturbances, which
may rely on the enzymatic functional defect of the pancreatic exocrine system.
5–7
Even more, T2D is accompanied by high blood insulin levels and
hyperlipidemia.
8
Maintaining optimal blood glucose levels can delay further DM
progression. Whereas, a steady rise in plasma glucose levels occurs regardless of
the degree of control or type of treatment. Therefore, β-cell function declines
linearly with time, and it was reported that after 10 years more than 50% of patients
Correspondence: Helal F Hetta
Department of Internal Medicine, University
of Cincinnati College of Medicine,
Cincinnati, 231 Albert B. Sabin Way, PO Box
670595, OH 45267-0595, USA
Email hettahf@ucmail.uc.edu
Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy Dovepress
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Open Access Full Text Article
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http://doi.org/10.2147/DMSO.S209856
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require insulin therapy.
9
The underlying changes in β-cell
function have been well described,
10,11
and β-cell mass
decreases steadily during the course of T2D.
12,13
It is
strongly accepted that T2D is a worldwide chronic pro-
gressive syndrome. Therefore, a high likelihood demand
for insulin therapy is important to maintain at optimal
glycemic status.
14
Free radicals play a major role in the pathogenesis of T2D
and most likely its further complications. Nevertheless, the
formation of reactive oxygen species (ROS) is a direct con-
sequence of hyperglycemia.
15
Increased ROS and decreased
antioxidant systems induce a critical oxidative stress in dia-
betic patients. Substances having ROS scavenging ability can
have potential effectiveness in diabetic animals with high
oxidative stress level.
16
Modulation of the levels of common
antioxidants including vitamins A, C, and E, glutathione, and
the enzymes superoxide dismutase, catalase, glutathione per-
oxidase, and glutathione reductase can be applied to counter-
act this oxidative stress condition.
17–19
It has been suggested that dietary antioxidants may
play a role in reducing the risk of T2D as well as its
complications.
20
The extracts derived from Ginkgo biloba
(GB) have been frequently used in traditional medicine
and has been shown to exhibit antioxidant potency.
21
GB
extract leads to significant alterations in antioxidant
enzymes (superoxide dismutase, catalase, and glutathione
peroxidase) and total antioxidant status.
22
The magnetized water (MW), however, has been also
reported to reduce blood glucose, improve antioxidant status,
and lipid profiles in streptozotocin-induced diabetes in rats.
21,23
This protective effect of MW is induced by elevating the
concentration of glutathione peroxidase (GSH-Px) in serum
after one or two months of exposure.
24
The role of natural antioxidants ie GB and MW in
protecting β-cells is so far not mentioned in the available
literature. This novel study was designed to verify the
protective role of administration of GB and MW on pan-
creatic β-cells.
Materials and methods
Animals and experimental design
This experiment was performed on 40 adult males Wister
rats weighing 200±20 g. The experiment was performed in
the animal house, Jazan University, KSA, and was
approved by the ethical committee of Jazan University.
We followed our previously published protocol for design-
ing the current experiments.
21
Shortly, animals were
housed in separate cages under normal day and night
cycles. The animals were divided into two main groups:
a control group (n=10) and a diabetic group (n=30). The
control group was fed standard laboratory ration and
allowed free access to water. The diabetic group was
further subdivided into three groups (10 rats each).
Group I was kept as non-treated control, diabetic group.
Group II was orally administered with water extraction of
GB leaves (0.11 mg/kg/day/four weeks) purchased from
Novo Mesto Company, Slovenia, Diabetic+ GB. Group III
was orally administered magnetic treated (magnetized)
water for four weeks, Diabetic+ MW.
Ethical statement
All experiments were carried out in accordance with Jazan
University, KSA laws and University guidelines for the
care of experimental animals. The data used to support the
findings of this study are included within the article.
Induction of T2D
T2D was induced by feeding the rats on a high-fat diet
(20% fat, 45% carbohydrate, 22% protein) for eight weeks
At the beginning of the ninth week, animals fasted for
12 hrs then injected intraperitoneally by a single dose (25
mg/kg) of streptozotocin (STZ) purchased from Sigma
Chemical Co, St. Louis, MO, USA. After injection, rats
were given 10% glucose for the next 24hours to avoid fatal
hypoglycemia that may result from the massive pancreatic
insulin release following STZ injection.
25
After three days,
the development of diabetes was confirmed by measuring
glucose levels in blood samples obtained from the tail
vein. Rats with blood glucose level over 200 mg/dl were
considered diabetic.
Preparation of MW
The MW was prepared by passing drinking water through
our hand-made electro-magnet unit.
21
A transistor-con-
trolled DC current is flowing in two coils connected in
series. A potentiometer was used to control magnetic field
strength. Water was pumped through a flexible tube by a
water pump installed inside the unit. The distance between
the magnetic coils was about 15 mm. The produced mag-
netic strength was 600 G (measured by WT10A
Teslameter), it was uniform and perpendicular to the
water flow. Water flow was at a relatively low speed
(0.34 L/min) to avoid overflow. The 600G is an average
strength that has been tested to cause no pathological
lesions in experimental rats.
26
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Semithin sectioning
Small tissue samples (2 mm thick) of the pancreas were
processed for semithin sectioning and stained with 1%
Toluidine Blue according to our published protocol.
27
Sections were examined and photographed with a light
microscope.
Histology and immunohistochemistry
Tissue samples from the pancreas were fixed in 4% parafor-
maldehyde solution, dehydrated in ascending graded ethanol,
embedded in paraplast. Thin sections (3-5 μm thick) were
sectioned by a Leica RM 2125RT microtome. For H&E
(Roche) and immunostaining, paraffinsections(3-5µm)
from the pancreas were used. We detected insulin in pancrea-
tic β-cells, according to our previous protocol
28
by using the
specific primary antibodies (polyclonal anti-insulin) obtained
from Chongqing Biospes Co., Ltd, China. Power-Stain
TM
1.0
Poly HRP DAB Kit for Mouse + Rabbit was obtained from
Genemed Biotechnologies, Inc, San Francisco, CA USA.
Image J software was used for histological sections
analysis as well as measuring of protein expression
intensities.
Statistical analysis
The data were analyzed by means of one-way analysis of
variance (ANOVA) and presented as mean ± standard
error. Statistical analysis was done following Student’st-
test. A difference was considered significant when P<0.05.
Results
GB and MW protect the pancreatic
structure against T2D
To investigate the effect of T2D on the rat pancreatic
structure, we used paraffin sections stained with H&E. We
found that diabetes-induced structural changes in the
Endocrine portion (islets of Langerhans) and Exocrine
(Acinus) portion as well when compared to control (Ctrl).
The % size of islets of Langerhans and its cellular contents
reflects the healthy condition of the endocrine pancreatic
system. We observed that the size of islets of Langerhans
was markedly decreased in the diabetic animals compared
to Ctrl (Figure 1A a,b). Furthermore, we found a marked
decrease in the cellular contents of islets of Langerhans in
diabetic rats compared to Ctrl (Figure 2A a,b,e,f). Using
Image J software, we measured the correlation of the whole
islets of Langerhans size in Ctrl and other rat groups. In
addition, with image J, we counted the cellular content of
islets of Langerhans. The size of islets of Langerhans was
significantly decreased in diabetic pancreas compared to
Ctrl. Furthermore, the cell number of diabetic islets of
Langerhans was markedly decreased compare to control
(Figure 1C). Furthermore, we have noticed that the acidic
staining of the pancreatic acini in diabetic rats was mark-
edly decreased compared to Ctrl (Figure 1A a,b). With
Image J, we measured the intensity of the acinar acidic
staining and we noticed a significant decrease in the diabetic
rats compared to Ctrl (Figure 1A e,f).
To study the protective effect of GB and MW on the
pancreas, we treated diabetic rats with GB and MW. We
observed that the pancreatic phenotype was partially rescued
in GB and MW treated rats compare to control (Figure 1A c,
d,g,h). Furthermore, the islets of Langerhans (size and
cellular contents) in GB and MW treated groups were nearly
comparable to control (Figure 1A a,c,d). In addition, with
Image J we were able to confirm the comparable changes in
the size of islets of Langerhans and the cellular contents in
GB and MW groups compared to Ctrl (Figure 1C).
Furthermore, we found that the pancreatic acinar staining
intensity after GB and MW treatment was significantly
increased compared with the diabetic rats (Figure 1B).
GB and MW maintain the pancreatic
structure against T2D
Histologically, the pancreas of the normal control group,
which appears formed of an exocrine portion (pancreatic
acini and ducts) and an endocrine portion (islets of
Langerhans).The islets were randomly distributed amidst
the pancreatic acini and were frequently neighboring the
pancreatic ducts. The islets were formed of clusters or cords
of cells of varying size and staining intensities. We found
that, in the diabetic group, the frequency of occurrence and
areas occupied by the islets of Langerhans were drastically
reduced. Each islet contained few β-cells demonstrating a
degenerative alteration as compared to Ctrl (Figure 2A a,b).
The degenerative changes were represented by a form of
nuclear pyknosis and cytoplasmic vacuolization (Figure 2A
b arrow). The GB and MW treated groups (Figure 2A c,d),
however, showed more or less restoration of the normal
morphology of the β- cells seen in the normal control with
a comparatively better picture in GB treated group.
Furthermore, the GB and MW treated groups showed less
autophagy cytoplasmic vacuoles compared to the diabetic
group (Figure 2A f-h). Image analysis showed that the %
size of islets of Langerhans and the number of ß-cells were
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1.5 150
100
50
0
% Size of Islets of Langerhans
**
** ***
*** **
**
** **
*
% Cell number
Ctrl
Diabetic
Diabetic+GB
Diabetic+MW
% Acinar secretion intensity
BC
Ctrl
OverviewAcini
A
a
IL
IL
IL
IL
ef gh
bcd
Diabetic Diabetic+GB Diabetic+MW
1.0
0.5
0.0
Ctrl
Diabetic
Diabetic+GB
Diabetic+MW
Figure 1 GB and MW protective effects against the diabetic nephrotoxic effect. (A)Paraffin sections stained against H&E. (A-D) islets of Langerhans (IL) (green bordered) was
decreased in size in the diabetic pancreas and comparable to control in Diabetic+GB and Diabetic+MW. Scale bar 200 µm. Magnification of pancreatic acini (E-H). Image J
measurements of acinar staining intensity (B), islets of Langerhans size and its cellular content in relation to Ctrl (C). *P<0.05, **P<0.01 and ***P<0.001 vs control group.
Abbreviations: GB, Ginkgo biloba; MW, magnetized water ; Ctrl, control.
Ctrl
ADiabetic Diabetic+GB Diabetic+MW
Islets of LangerhansMagnification
a
IL
bcd
ef gh
IL IL IL
Figure 2 In the diabetic pancreas, cellular disturbances in islets of Langerhans rescued after GB and MW treatment. (Aa-h) Semithin sections stained with Toluidine Blue. In
diabetic rats, islets of Langerhans were smaller than Ctrl (A,B). The size of islets of Langerhans was comparable to Ctrl in GB and MW treated rats (C,D). The cells of
islets of Langerhans were few in number and showed autophagy cytoplasmic vacuoles (asterisk). Scale bar 50 µm (A-D), 20 µm (E-H).
Abbreviations: GB, Ginkgo biloba; MW, magnetized water ; Ctrl, control.
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significantly decreased in diabetic islets compared to Ctrl
(Figure 1C). The GB and MW treated groups (Figure 2A c,
d), showed a marked protective effect which included an
increase in the cell number and the % size of islets of
Langerhans (Figure 1C).
GB and MW are able to rescue low levels
of pancreatic insulin caused by T2D in
rats
In order to investigate the pancreatic insulin after T2D
induction, we stained against insulin in the pancreatic
β-Cell. We found a marked decrease in insulin
expression in diabetic pancreas compared to control
(Figure 3A a-b).
Image analysis using Image J showed that the intensity
of the insulin expression was significantly decreased up to
10 folds in diabetic pancreas compared to Ctrl (Figure 3B).
Interestingly, insulin expression was almost comparable
to control after GB and MW treatment (Figure 3A c,d).
Image J analysis confirmed the induction in the intensity of
the insulin after GB and MW treatment (Figure 3B).
Altogether, GB and MW treatments were able to
induce insulin expression in diabetic pancreas.
GB and MW treatment decreased
diabetic effect on the pancreatic
glutathione reductase and SOD2 protein
expression
In order to investigate the pancreatic oxidative stress after
T2D induction, we stained against glutathione reductase
and SOD2 antibodies.
We found a marked increase in glutathione reductase
and SOD2 expressions in diabetic pancreas compared to
control (Figure 4A a-b,e-f, respectively).
Image analysis using Image J showed that the intensity
of the glutathione reductase expression was significantly
increased up to 10 folds in STZ-treated pancreas compared
to Ctrl (Figure 4B). Furthermore, we noticed a significant
increase in SOD2 intensity up to 6.6 folds in diabetic
pancreas compared to control (Figure 3B).
Interestingly, glutathione reductase (Figure 4A c,d)
and SOD2 (Figure 4A g,h) expressions were almost
1.5
% Insulin intensity
**
*
***
1.0
0.5
0.0
Ctrl
Diabetic
Diabetic+GB
Diabetic+MW
Ctrl
ADiabetic
Insulin
Diabetic+GB Diabetic+MW
B
abcd
Figure 3 GB and MW treatment rescue insulin expression in islets of Langerhans. (Aa-d) Paraffin sections stained with anti-insulin antibody. The expression of insulin was
decreased in diabetic pancreas and back to almost normal after the use of GB and MW compare to Ctrl. Scale bar 100 µm. Image J analysis displayed a significant decrease of
insulin expression intensity in diabetic islets of Langerhans compared to Ctrl. In Diabetic+GB and Diabetic+MW pancreas, insulin protein expression intensity was increased
to be comparable with Ctrl (B). *P<0.05, **P<0.01 and ***P<0.001 vs control group.
Abbreviations: GB, Ginkgo biloba; MW, magnetized water ; Ctrl, control.
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comparable to control after GB and MW treatment. Image
J analysis confirmed the reduction in the intensity of the
glutathione reductase and SOD2 after GB and MW treat-
ment (Figure 4B).
Altogether, GB and MW treatments were able to
reduce the oxidative stress in the diabetic pancreas.
Discussion
In this study, we investigated the expected effects of the
extract of GB and MW in the restoration of β-cell mass and
amelioration of their functions after induction of T2D in rats.
Our results verified a dramatic pancreatic (β-cells and acinar
cells) failure in non-treated diabetic (positive control) rats. β-
cells showed a marked pyknosis with signs of autophagy and
apoptosis. These findings coincide with the data has been
published previously,
29
it was reported that β-cell failure in
T2D occurs when islets were unable to sustain β-cells
compensation as a result of insulin resistance. Furthermore,
the failure is progressive, particularly after hyperglycemia
was established, where β-cells become poorly functioning,
de-differentiated and apoptotic.
29
In the current study, diabetic rats showed a drastic
decrease in acinar staining intensity reflecting the defect in
the pancreatic digestive effect which could explain the
decreased body weight of diabetic rats. Furthermore, diabetic
rats showed a drastic decrease of β-cells masses and staining
intensity of acinar cells. Moreover, insulin granules and their
intensity in β-cells, as shown by immunohistochemistry, were
downregulated in these animals. In this concern, it was
reported that T2D starts by increasing insulin resistance and
then β-cells will undergo apoptosis or necrosis.
14,29,30
In our
diabetic rat, β-cells succumbed necrotic changes (cytoplasmic
vacuolation and nuclear pyknosis) with a subsequent apop-
tosis and/or autophagy. Autophagy has been reported to play
10
B
9
8
7
6
5
4
3
2
1
0
Ctrl Diabetic Diabetic+GB Diabetic+MW Ctrl Diabetic Diabetic+GB Diabetic+MW
**
*** **
***
Glutathione reductase
SOD2
**
**
Ctrl
Glitathion reductaseSOD2
ADiabetic Diabetic+GB Diabetic+MW
a
IL
ef gh
bcd
IL
IL
IL
IL
IL
IL IL
Figure 4 GB and MW protective effect against oxidative stress induced by type 2 diabetes. (Aa-d) Paraffin sections stained with anti-glutathione reductase antibody. The
expression of glutathione reductase was increased in diabetic pancrease and back to almost normal after the use of GB and MW. Furthermore, (Ae-h) Paraffin tissue sections
stained withanti SOD2 antibody. SOD2 expression was intensively increasedin diabetic pancreaswhile with the use of GB and MW wascomparable with control. Scale bar 100µm.
Image J analysis displayed a significant increase of glutathione reductase and SOD2 intensities in diabetic pancreas compared to Ctrl. In Diabetic+GB and Diabetic+MW pancreas,
glutathione reductase and SOD2 protein expression intensities were decreased to be comparable with Ctrl (B). *P<0.05, **P<0.01 and ***P<0.001 vs control group.
Abbreviations: GB, Ginkgo biloba; MW, magnetized water ; Ctrl, control.
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an important role in pancreatic β-cell dysfunction and insulin
resistance in T2D.
31
Autophagy was stimulated at the begin-
ning of T2D as a protective mechanism for β-cell
32
and then
the accumulation of autophagosomes in β-cells will lead to
cellular damage and apoptosis.
33,34
The β-cell dysfunction and failure of insulin secretory
capacity in T2D could be attributed to glucotoxicity, lipo-
toxicity and/or oxidative stress.
29,35,36
Our observation of
hyperglycemia in diabetic rats,
21
could be attributed to
exhaustion and failure of β-cells. A similar explanation
has been suggested by Wang J & Wang H (2017), who
mentioned that hyperglycemia leads to glucotoxicity to β-
cells and induction of their apoptosis or necrosis. Reduction
of serum glucose levels, however, has been supposed to
increase survival of β-cells,
37,38
a suggestion that agrees
with our findings where GB and MW treatments reduced
blood glucose
21
and increased β-cells survivals.
Our suggestion that the damage of β-cells results from
dyslipidemia and lipotoxicity was concomitant with what
has been mentioned before.
38–40
The latter authors added
that elevation of triglycerides leads to elevation of free
fatty acids which causes lipotoxicity that impairs the sur-
vivals of β-cells, insulin secretion and subsequently
damages β-cells.
Even more, our previous observation of the marked
improvement of dyslipidemic and high glucose status in
diabetic rat,
21
could be attributed to our current findings
of the ameliorating effects of GB and MW on the pancreatic
β-cells. Similarly, it was reported that GB reduced
hyperlipidemia
41
that has been suggested to be due to its
content of flavonoid components.
42
The MW has been also
reported to have a powerful hypolipidemic action in T2D.
43
However, in our present study the improvement of both
morphological picture of β-cells as well as serum glucose
level and the lipid profile as revealed in our previous,
21
were comparatively better in GB treated rats than those
treatedwithMW.
A possible factor for destroying β-cell function is the
increasing free radical or oxidative stress that accompanies
T2D.
39
Immunohistochemically, our findings demonstrated
an overexpression of antioxidant enzymes; glutathione per-
oxidase and SOD2 in pancreatic islets of diabetic rat. These
results coincide with Wang J & Wang H (2017), who
reported that hyperglycemia, hyperlipidemia, hypoxia, and
endoplasmic reticulum (ER) stress lead to ROS generation
in β-cells. Hyperglycemia, in particular, can be directly
associated with increased ROS generation.
39
In chronic
hyperglycemia, β-cells are exposed to high glucose
concentrations for long time, where the normal route of
glycolysis gets saturated and excess glucose is shifted
towards alternative ROS-forming pathways including
glycosylation,
44
glucose autoxidation,
45,46
and glucosamine
pathway,
47
all of them lead to the accumulation of ROS and
induction of oxidative stress. Furthermore, increase ROS
production has been reported to decreases β-cell mass.
48
According to Mancini et al (2018), the total antioxidant
capacity of the diet may play a role in reducing the risk of T2D
as well as its complication. Our results demonstrated that
treatment with GB or MW ameliorated antioxidant status in
β-cells and hence downregulated antioxidants enzymes; glu-
tathione and SOD2. We suggest that both treatments scavenge
ROS with a subsequent decrease in the expression of both
enzymes in pancreatic islets. In the same context, GB has
been suggested to scavenge free radicals in vivo.
22
The cur-
rently increased survival of β-cells could be also attributed to
the amelioration of the antioxidant status in these cells.
In conclusion, treatment with GB or MW protects
pancreatic exocrine and endocrine systems against the
damaging effect of T2D in rats.
Disclosure
The authors report no conflicts of interest in this work.
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