Ameliorative effects of bee pollen and date palm pollen on the glycemic state and male sexual dysfunctions in streptozotocin-Induced diabetic wistar rats

This study aimed to assess the effects of bee pollen (BP) and/or date palm pollen (DPP) suspensions on the
glycemic state, testicular dysfunctions, oxidative stress and antioxidant defense system in streptozotocin (STZ)-
induced diabetic male Wistar rats. Diabetes mellitus was induced by single intraperitoneal injection of STZ to
overnight-fasted rats at dose of 40 mg/kg body weight. After 1 week of STZ injection

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Biomedicine & Pharmacotherapy
journal homepage:www.elsevier.com/locate/biopha
Ameliorative effects of bee pollen and date palm pollen on the glycemic state
and male sexual dysfunctions in streptozotocin-Induced diabetic wistar rats
Nema A. Mohamed
a
, Osama M. Ahmed
b,⁎
, Walaa G. Hozayen
c
, Marium A. Ahmed
b
a
Department of Zoology, Faculty of Science, Alexandria University, Egypt
b
Experimental obesity and Diabetes Research Lab, Physiology Division, Department of Zoology, Faculty of Science, Beni-Suef University, Egypt
c
Biochemistry Division, Department of Chemistry, Faculty of Science, Beni-Suef University, Egypt
ARTICLE INFO
Keywords:
Diabetes mellitus
Streptozotocin
Bee pollen
Palm pollen
Pancreas
Testis
Oxidative stress
Rats
ABSTRACT
This study aimed to assess the eff ects of bee pollen (BP) and/or date palm pollen (DPP) suspensions on the
glycemic state, testicular dysfunctions, oxidative stress and antioxidant defense system in streptozotocin (STZ)-
induced diabetic male Wistar rats. Diabetes mellitus was induced by single intraperitoneal injection of STZ to
overnight-fasted rats at dose of 40 mg/kg body weight. After 1 week of STZ injection, diabetic rats were treated
with BP and/or DPP suspensions at dose levels of 100 mg/kg body weight/day for 4 weeks. The STZ-induced
diabetes significantly increased blood glucose levels and testicular nitric oxide (NO) and malondialdehyde
(MDA) levels parallel with disrupted testicular and pancreatic histological architecture and integrity. On the
other hand, STZ-induced diabetes significantly decreased body weight, testis and pancreas weights, levels of
serum insulin, testosterone, luteinizing hormone (LH) & follicle stimulating hormone (FSH) as well as sperm
count, motility and viability. The administration of BP and DPP suspensions resulted in a significant recovery of
the above mentioned parameters as compared to the diabetic control group. These improvements were asso-
ciated with enhancement of the testicular antioxidant system manifested by an increase in the lowered glu-
tathione content (GSH) and glutathione-S-transferase (GST), glutathione peroxidase (GPx) and superoxide dis-
mutase (SOD) activities in diabetic rats as a result of treatments with BP and DPP suspensions. Thus, it can be
concluded that BP and/or DPP suspensions may have potential protective role against diabetes-induced pituitary
testicular axis dysfunction and testicular histological deleterious changes in association with antihyperglycemic
actionsviatheir antioxidant properties and their efficiency to improve blood insulin level and beta cell function.
1. Introduction
Diabetes mellitus (DM) is an epidemic disease that is considered as a
common health problem around the world. According to the
International Diabetes Federation in 2013, 381 million people suffered
from diabetes, and it is estimated that it will be almost doubled by 2030
[1].
Testicular dysfunction, impotence, decreased fertility characterized
by reduced spermatogenesis and production of dysfunctional sperm,
potential and retrograde ejaculations, and erectile dysfunction are
conditions that have been described in diabetic males[2]. Oxidative
stress mechanisms may be wholly or in part contribute towards the
development of testicular dysfunction and degeneration in diabetic
animal models[3]. Thus, the search for treatment of diabetes and its
associated sexual dysfunction in male with antioxidants of natural
sources, to avoid side effects of conventional chemical drugs, is an in-
terestingfield of study of many investigators[4–6].
Bee pollen (BP) ofApis melliferais rich in proteins, essential amino
acids, unsaturated fatty acids, many vitamins, minerals and trace ele-
ments that contribute to the health effects[7]. The main active con-
stituents of BP included isoflavones,flavonols and lignans otherwise
known as plant hormones since these compounds have well-docu-
mented hormonal benefits for both men and women[8]. In addition,
the phenolic components of BP were reported to exhibit high levels of
antioxidant and radical scavenging activity[9,10]. Furthermore, BP
grains have many pharmacological functions as an antimicrobial[11],
anti-inflammatory[12], hepatoprotective modulator[13], anti-
artherosclerosis and antitumor[14]properties.
Flower pollens, that formfine powder-like material produced by
seed plants, were considered to be the male reproductive cells of
flowering plants[15]. The date palm pollen (DPP) ofPhoenix dactylifera
(P. dactylifera) were used in traditional medicine for the treatment of
various disorders which include memory disturbances, infl ammation,
fever, paralysis, loss of consciousness and many nervous disorders[16].
http://dx.doi.org/10.1016/j.biopha.2017.10.117
Received 3 September 2017; Received in revised form 21 October 2017; Accepted 21 October 2017
⁎
Corresponding author at: Experimental obesity and Diabetes Research Lab, Physiology Division, Department of Zoology, Faculty of Science, Beni-Suef University, Egypt.
E-mail addresses:[email protected],[email protected](O.M. Ahmed).
Biomedicine & Pharmacotherapy 97 (2018) 9–18
0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
T

2. Suspension of DPP has been widely used as a folk remedy for curing
male infertility in traditional medicine[17]. The high concentrations of
total phenolic content,flavonoids and anthocyanins as well as the
presence of significant quantity of selenoproteins inP. dactyliferamakes
it an excellent candidate for antioxidant processes[18]. DPP grains
contain triterpenoids, saponins, a crude gonadotropic substance, a D-
glucan, heteroxylon, galactomannans, estrone and cholesterol[19–21].
In the same regard, Hassan et al.[22]and Hassan[23]revealed the
presence of estrone, estradiol and estriol, besidesflavonoid compounds
(β-sitosterol, quercetin and rutin).
Therefore, this study was designed to explore the effect of STZ-in-
duced DM on male sexual functions and the probable curative effects of
BP and/or DPP suspensions in improving the glycemic state and in
preventing the male reproductive dysfunctions.
2. Materials and methods
2.1. Experimental animals
Male Wistar rats, weighing 150–170 g, were obtained from the
Animal House of the Faculty of Medicine, Alexandria University,
Alexandria, Egypt. They were housed under controlled conditions
(25 ± 1 °C constant temperature, 55% relative humidity, 12-h lighting
cycle), kept under laboratory conditions for two weeks prior to ex-
periment for acclimation and received standard diet and waterad li-
bitum.All animal procedures and the experimental protocols were ap-
proved by the local Animal Ethics Committee and were in accordance
with the recommendations of the Canadian Council on Animal Care
(CCAC) (1998)[24]for the proper care and use of laboratory animals
[25].
2.2. Chemicals
Streptozotocin (STZ) was purchased from Sigma Chemical
Company, St. Louis, MO, USA. All other used chemicals were of the
highest analytical grades and were commercially available. BP ofApis
melliferaand DPP ofP. dactyliferawere purchased from an authentic
source at local herbal market (Alexandria, Egypt).
2.3. Preparation of BP suspension
The powder of BP grains (2 g) was suspended in distilled water
(100 ml) at concentration 2% w/v and mixed vigorously. The prepared
suspension was administered by oral gavage at volume of 5 ml/ kg b. w.
that is equivalent to dose level of 100 mg/kg b. w.[26].
2.4. Preparation of DPP suspension
The DPP suspension was freshly prepared by adding 2 g powdered
DPP grains ofP. dactyliferato 100 ml of distilled water at concentration
2% w/v. The suspension was mixed thoroughly till complete dispersion.
Five ml of the prepared suspension was administered by oral gavage to
rats at a dose level of 100 mg/kg b. w.[27].
2.5. Induction of diabetes
Experimental diabetes was induced in overnight fasted rats by a
single intraperitoneal dose of STZ (40 mg/kg b. w.)[28]dissolved in
ice-cold citrate buffer (pH 4.5). One week post-STZ injection, the blood
glucose level was measured in rats deprived of food and water over-
night and after 2 h of oral glucose administration (3 g/kg b. w.) using
reagent strips (Accu-ChekW, Roche)with a drop of blood obtained by
lateral
tail-vein puncture. Animals were considered diabetic if blood
glucose levels after 2 h of oral glucose loading were higher than
200 mg/dl.
2.6. Experimental design
Thirty adult male Wistar rats were selected and divided intofive
groups (n = 6) as follows:
Group I:healthy rats receiving the equivalent volume of the vehicle
(distilled water) daily by oral gavage for 4 weeks (Normal group);
Group II:diabetic rats receiving the equivalent volume of the ve-
hicle (distilled water) daily by oral gavage for 4 weeks (Diabetic control
group);
Group III:diabetic rats treated with the aqueous suspension of BP
ofApis mellifera(100 mg/kg b.w./day)[26]by oral gavage for 4 weeks.
Group IV:diabetic rats treated with the aqueous suspension of DPP
ofP. dactylifera(100 mg/kg b.w./day)[27]by oral gavage for 4 weeks.
Group V:diabetic rats treated with both aqueous suspensions of BP
and DPP by oral gavage at dose levels of 100 mg/kg b.w./day for 4
weeks.
At the end of the experiment, fasting and 2 h post-glucose loading
blood glucose levels were measured. At the following day, the rats were
overnight-fasted, weighed and blood samples, testes and pancreases
were obtained.
2.7. Blood, semen and tissue sampling
For the purpose of studying various biochemical parameters, blood
samples were collected from carotid artery of normal, diabetic control
and diabetic treated rats after mild diethyl ether anesthesia. Serum was
separated from blood cells by centrifugation at 8000 rpm for 5 min. The
serum was then stored at−20 °C pending determination of serum in-
sulin, testosterone, follicle-stimulating hormone (FSH) and luteinizing
hormone (LH) levels.
After decapitation and dissection, pancreas and two testes of each
rat were rapidly excised and weighed. One testis and pancreas of each
rat werefixed in neutral buffered formalin (10%) for histological in-
vestigation. The other testis of each rat was homogenized in 0.9% NaCl.
The homogenates were centrifuged and the homogenate supernatants
were aspirated and kept at−20 °C until used for detection of oxidative
stress and antioxidant defense system markers.
Semen was collected the cauda epididymis and was diluted with
physiological solution for measurement of sperm count, motility, ab-
normality percent and viability percent.
2.8. Determination of serum insulin, testosterone, LH and FSH levels
Serum insulin was assayed using kits obtained from RayBio
®
ELISA
kits obtained from RayBiotech, 3607 Parkway Lane, Suite 100,
Norcross, GA 30092 according manufacturer's instruction. Serum tes-
tosterone, FSH and LH levels were estimated by using Cobas electro-
chemiluminescence immunoassay kits obtained from Roche Diagnostics
GmbH, Sandhofer Strasse 116, D-68305 Mannheim according to
Wheeler[29]Johnson et al.[30]and Beastall et al.[31]respectively.
2.9. Determination of testis oxidative stress and antioxidant defense system
markers
Colorimetric determination of nitric oxide (NO) level was performed
according to the method of Montgomery and Dymock[32]. Testis lipid
peroxidation (LPx) end product, malondialdehyde (MDA), was mea-
sured according to the method of Ohkawa et al.[33]. Also, the level of
reduced glutathione (GSH)[34]and the activities of the antioxidant
enzymes including GSH-S-transferase (GST)[35], glutathione perox-
idase (GPx)[36]and superoxide dismutase (SOD) activities[37]were
assayed in the testis homogenate supernatant.
2.10. Sperm analysis
The epididymal sperm count (million/ml) was determined with a
N.A. Mohamed et al. Biomedicine & Pharmacotherapy 97 (2018) 9–18
10

3. haemocytometer (Improved Neubauer slide, LABART, Darmstadt,
Germany) using a method described by Turk et al.[38]and Sonmez
et al.[39]. Percent sperm motility was evaluated according to the
method of Sonmez et al.[40]. The percent of the morphologically ab-
normal spermatozoa as well as spermatozoa viability percent were
evaluated by using eosin-nigrosin (1.67 g eosin, 10 g nigrosin, and 2.9 g
sodium citrate per 100 ml distilled water) stain according to the
methods of Kose et al.[41]and Chalah and Brillard[42].
2.11. Histopathological investigation of the testis and pancreas
Thefixed testes and pancreases were dehydrated by upgrading from
30 to 100% series of ethyl alcohol and then to xylene each for 1 h,
embedded in paraffin wax. Paraffin blocks were sectioned at 5μm
thicknesses. The sections were then stained with hematoxylin and eosin
according to the method of Bancroft et al.[43]. Microscopicfields were
selected randomly for the evaluation of histopathological alterations of
seminiferous tubules of testis and islets of Langerhans of pancreas. The
stained sections were examined with a light microscope. Permanent
preparations of testicular and pancreatic tissues were histologically
examined and photographed using a digital camera (Canon Virginia
Inc., Newport News, VA, USA) mounted on a light microscope (Carl
Zeiss Inc., Jena, Germany).
2.12. Statistical analysis
The data were analyzed using the one-way analysis of variance
(ANOVA) (PC-STAT, University of Georgia, 1985) followed by LSD test
to compare various groups with each other. Results were expressed as
mean ± standard error (SE) and values of P > 0.05 were considered
non-significantly different, while those of P < 0.05 and P < 0.01 were
considered significantly and highly significantly different, respectively.
F-probability expressed the effect between groups.
3. Results
3.1. Effect of BP and/or DPP suspensions on body weight and body weight
gain
The derived data inTable 1showed a highly significant (P < 0.01)
decrease in thefinal body weight and body weight gain of the diabetic
control rats in comparison with the normal rats; the recorded percen-
tage changes were−44.91% and−290.73% respectively. Moreover,
the treatment of diabetic rats with BP and/or DPP suspensions induced
a highly significant (P < 0.01) increase in thefinal body weight and
body weight gain as compared to the diabetic control rats. It was ob-
vious that the administration of DPP suspension alone to diabetic rats
had the most potent effect on body weight decrease.
3.2. Effect of BP and/or DPP suspensions on testis and pancreas weights
and their relative weights
As shown inTable 2, the diabetic rats exhibited a highly significant
(P < 0.01) decrease in testis and pancreas weights recording percen-
tage
changes of−56.222% and−31.481% respectively as compared
with normal rats. Moreover, the treatment of diabetic rats with BP, DPP
and their mixture induced a highly significant increase (P < 0.01) in
testis weight as compared to untreated diabetic rats; the recorded
percentage changes were 67.149%, 71.064% and 42.468% respectively.
The pancreas weight, on the other hand, was significantly increased as
result of treatment of diabetic rats with BP and its combination with
DPP recording percentage increases of 32.432% and 24.865% respec-
tively while it was significantly decreased as a result of treatment with
DPP recording percentage change of−24.324%. The relative testis
weight was significantly increased (p < 0.05; 11.681%), non-sig-
nificantly increased (p > 0.05; 8.141%) and significantly decreased
(p < 0.05;−9.469%) as a result of treatments with suspensions of BP,
DPP and their mixture respectively. The relative pancreas weight was
highly significantly increased (P < 0.01) in diabetic rats as compared
with normal rats and it was significantly decreased as a result of
treatments of diabetic rats with suspensions of BP, DPP and their
mixture recording percentage decreases of 8.383%, 49.700% and
16.167% respectively.
3.3. Effect of BP and/or DPP suspensions on fasting and post-prandial
blood glucose concentrations
The diabetic rats showed a highly significant (P < 0.01) increase in
the fasting and post-prandial blood glucose levels recording percentage
changes of +253.98% and +283.02% respectively. They also exhibited
a highly significant decrease (P < 0.01) in serum insulin level re-
cording percentage change of−57.30% in comparison with normal
rats. The treatment of diabetic rats with BP and DPP suspensions in-
duced a detectable decrease in the elevated fasting and post-prandial
serum glucose concentrations. The effect of BP and its co-administra-
tion with DPP to diabetic rats induced a significant decrease
(P < 0.05) of the elevated postprandial blood glucose level; the re-
corded percentage changes were−25.18% and−20.34% respectively.
On the other hand, the lowered serum insulin concentration in diabetic
rats was significantly ameliorated (P < 0.01) as a result of treatment
with BP and DPP; the bee pollen suspension seemed to be most potent
since it produced percentage increase of 90.85% as compared with the
diabetic control (Table 3).
3.4. Effect of BP and/or DPP suspensions on testosterone, FSH and LH
levels
As illustrated inTable 4, the diabetic rats exhibited a detectable
Table 1
Effect of BP and/or DPP suspensions on body weight and body weight gain in diabetic rats.
Parameters Groups Initial body weight (g) % Change Final body weight (g) % Change Body weight gain (g) % Change
Normal 155.16 ± 2.89
a
– 183.29 ± 4.40
a
– 28.15 ± 2.26
b
–
Diabetic control 154.70 ± 3.42
a
−0.30 100.98 ± 4.36
c
−44.91 - 53.69 ± 1.23
a
−290.73
Diabetic treated with
BP
150.16 ± 2.18
a
−2.93 154.63 ± 1.07
b
+53.13 4.50 ± 1.27
d
+108.38
Diabetic treated with
DPP
149.22 ± 3.29
a
−3.54 159.72 ± 3.44
b
+58.17 10.55 ± 2.08
c
+119.65
Diabetic treated with BP and DPP 151.17 ± 1.33
a
−2.28 159.12 ± 1.72
b
+57.57 8.01 ± 0.56
cd
+114.92
F-probability P < 0.05 P < 0.001 P < 0.001
LSD at the 5% level – 9.598 4.673
LSD at the 1% level – 12.986 6.3224
- Data are expressed as Mean ± SE. Number of animals in each group is 6.
- Values, which have the same superscript symbol(s), are not significantly different.
- Percentage changes (%) were calculated by comparing the diabetic control group with normal and the diabetic treated groups with diabetic control.
N.A. Mohamed et al.
Biomedicine & Pharmacotherapy 97 (2018) 9–18
11

4. decrease in testosterone (P < 0.01), FSH (P > 0.05) and LH
(P < 0.01) levels recording percentage decreases of 26.51, 14.78 and
36.44 respectively in comparison with normal group. The treatment of
diabetic rats with BP, DPP and their mixture induced a significant
(P < 0.01) increase in serum testosterone level as compared to dia-
betic control rats; the recorded percentage changes were +30.10,
+49.25 and +32.83 respectively. DPP was the most potent in in-
creasing the serum testosterone level. The administration of BP and the
co-administration of BP and DPP induced a significant improvement of
the lowered serum FSH level; the co-administration of BP and DPP was
the most effective. Similar to the effect on testosterone, the serum LH
level was highly significantly increased as a result of treatments with
BP, DPP and their mixture recording percentage increases of 55.94%,
67.83% and 67.83% respectively.
3.5. Effect of BP and/or DPP suspensions on various sperm parameters in
diabetic rats
As demonstrated inTable 5, the sperm count, sperm motility per-
cent and viability percent exhibited a significant (P < 0.01) decrease
in the diabetic rats recording percentage changes of−87.13%,
−36.11% and−30.67% as compared with normal rats. In contrast, the
abnormal sperm percent was significantly (P < 0.01) increased in the
diabetic rats recording percentage increase of 196.30%. On the other
hand, the treatment of diabetic rats with BP and/or DPP suspensions
induced a significant (P < 0.01) alleviation of the deteriorated sperm
count, sperm motility percent, abnormal sperm percent and viability
percent in the diabetic rats. The BP appeared to be the most potent in
improving the decreased sperm count, sperm motility percent and
viability percent recording percentage increases of 160.90%, 31.30%
and 21.45% respectively. On the other hand, the most potent ameli-
orative effect on abnormal sperm percent was attained as a result of co-
administration of BP and DPP suspensions; the recorded percentage
change was−41.25% as compared with the diabetic control rats.
3.6. Effect of BP and/or DPP suspensions on antioxidant defense system in
testis
Data showing the effect of BP and DPP on testis GSH content and
GST, GPx and SOD activities of diabetic rats were represented in
Table 6. The testis GSH content was significantly decreased in diabetic
rats recording percentage changes of−26.88%. The depleted GSH
content was detectably increased as a result of treatment of diabetic rats
with BP and DPP. However, while the effect of treatment with BP was
significant (p < 0.01), the effect of treatment with DPP suspension or
its co-administration with BP induced a non-significant increase
(p > 0.05). The antioxidant enzymes GST, GPx and SOD activities
were significantly decreased in diabetic rats recording percentage
changes of−28.13,−19.20 and−22.63% respectively. The treatment
of diabetic rats with BP significantly (p < 0.01) increased the lowered
GST
activity while the treatment with DPP or the co-administration of
BP and DPP did not (p > 0.05). The three treatments BP, DPP and
their combination significantly improved the lowered GPx activity of
diabetic rats recording percentage increases of 14.21%, 25.01% and
18.26% respectively. Testis SOD activity was significantly increased in
diabetic rats as a result of treatment with DPP (43.66%) or co-admin-
istration of BP and DPP (25.60%).
3.7. Effect of BP and/or DPP suspensions on testis NO and MDA levels
The results inTable 7depicted that testis NO and MDA levels had
obviously increased in diabetic rats as compared to control; the re-
corded percentage increases were 25.95% and 96.01% respectively.
The treatment of diabetic rats with suspensions of BP, DPP and their
combination induced a significant increase (P < 0.01) in testis NO
level as compared to the diabetic control rats; the recorded percentage
Table 2
Effect of BP and/or DPP suspensions on absolute and relative testis and pancreas weights in diabetic rats.
Parameters Groups Testis weight (g) % Change Relative testis weight % Change Pancreas weight (g) % Change Relative pancreas weight % Change
Normal 2.684 ± 0.009
a
– 1.366 ± 0.008
a
– 0.270 ± 0.007
a
– 0.138 ± 0.005
c
–
Diabetic control 1.175 ± 0.043
d
−56.222 1.130 ± 0.076
c
−17.277 0.185 ± 0.019
c
−31.481 0.167 ± 0.003
a
+21.014
Diabetic treated with
BP
1.964 ± 0.017
b
+67.149 1.262 ± 0.004
b
+11.681 0.245 ± 0.005
b
+32.432 0.153 ± 0.003
b
−8.383
Diabetic treated with DPP 2.010 ± 0.011
b
+71.064 1.222 ± 0.094
bc
+8.141 0.140 ± 0.007
d
−24.324 0.084 ± 0.006
d
−49.700
Diabetic treated with BP and DPP 1.674 ± 0.020
c
+42.468 1.023 ± 0.006
d
−9.469 0.231 ± 0.004
b
+24.865 0.140 ± 0.001
bc
−16.167
F-probability P < 0.001 P < 0.001 P < 0.001 P < 0.001
LSD at the 5% level 0.069 0.100 0.016 0.010
LSD at the 1% level 0.092 0.134 0.022 0.014
- Data are expressed as Mean ± SE. Number of animals in each group is 6.
- Values, which have the same superscript symbol(s), are not significantly different.
- Percentage changes (%) were calculated by comparing the diabetic control group with normal and the diabetic treated groups with diabetic control.
Table 3
Effect of BP and/or DPP suspensions on fasting and post-prandial blood glucose and serum insulin concentrations in diabetic rats.
Parameters Groups Fasting glucose (mg/dl) % Change Postprandial blood glucose (mg/dl) % Change Insulin ( μIU/ml) % Change
Normal 102.83 ± 3.30
c
– 117.67 ± 5.67
c
– 22.27 ± 0.95
a
–
Diabetic control 364.01 ± 47.94
ab
+253.98 450.67 ± 30.07
a
+283.02 9.51 ± 1.29
d
−57.30
Diabetic treated with
BP
302.67 ± 44.05
b
−16.85 337.17 ± 37.07
b
−25.18 18.15 ± 0.52
b
+90.85
Diabetic treated with
DPP
275.00 ± 12.56
b
−24.45 399.33 ± 13.54
ab
−11.39 16.96 ± 0.66
c
+78.34
Diabetic treated with BP and DPP 393.02 ± 17.89
a
+7.97 359.00 ± 41.19
b
−20.34 15.71 ± 0.37
c
+65.19
F-probability P < 0.001 P < 0.001 P < 0.001
LSD at the 5% level 89.60 87.13 2.408
LSD at the 1% level 121.22 117.88 3.257
- Data are expressed as Mean ± SE. Number of animals in each group is 6.
- Values, which have the same superscript symbol(s), are not significantly different.
- Percentage changes (%) were calculated by comparing the diabetic control group with normal and the diabetic treated groups with diabetic control.
N.A. Mohamed et al.
Biomedicine & Pharmacotherapy 97 (2018) 9–18
12

5. increases were 17.01%, 87.13% and 60.48% respectively. In contrast,
the treatment of diabetic rats with suspensions of BP, DPP and their
combination induced a significant decrease (P < 0.01) in testis MDA
level as compared to the diabetic control rats; the recorded percentage
decreases were 49.00%, 55.97% and 54.73% respectively.
3.8. Histopathological studies
The histological photomicrographs of pancreas sections of normal,
diabetic and treated diabetic rats were depicted inFig. 1.
Histopathological observations of pancreatic tissues of normal
group (Photomicrograph 1A) showed intact pancreatic architecture.
The closely packed pancreatic acini are composed of pyramidal shaped
cells with rounded nuclei while the pale stained normal islets of
Langerhans are scattered in between acini with well-preserved cyto-
plasm and nucleus and intact interlobular connective tissue and inter-
lobular duct appearance. The islets of Langerhans are composed of
alpha, beta and delta cells.
The pancreatic islet cells of diabetic rats, showed vacuolated cyto-
plasm, necrotic cells; the tissue of pancreatic Langerhans and the beta
cells of diabetic rats have been degenerated (Photomicrograph 1B). In
addition, the pancreatic islets of diabetic rats exhibited smaller size
with less number of islet cells than those of normal rats.
On the other hand, BP and/or DPP treated rats showed recovery
signs including few degenerative and necrotic changes and increased
number of pancreatic cells which appeared more intact and granulated
(Photomicrographs 1C, D and E). While the pancreatic islets of DPP
treated diabetic rats were completely normalized, the pancreatic islets
of diabetic rats treated with BP and its combination with DPP still
showed few necrotic foci and vacuolations.
The histological photomicrographs of testis sections of normal,
diabetic and treated diabetic rats were depicted inFig. 2.
Histological sections of control testis showed that the seminiferous
tubules appeared normal with numerous active and intact germ cells.
Each seminiferous tubule has a normal spermatogenic cell lineage
formed of a number of spermatogonia, spermatocytes, spermatids and
spermatozoa arranged around central lumen. Alternating with the
spermatogonia, there are nutritive cells known as Sertoli cells
(Photomicrograph 2A).
The most consistentfindings in the testicular sections of diabetic
rats were loss of germ cells, abnormality of germinative epithelium, and
sperm with abnormal concentration (Photomicrograph 2B). The testis
section of diabetic rats exhibited remnants of Leydig cells, degenerated
and atrophic seminiferous tubules, severe destruction of spermatids and
spermatozoa, with scanty to absent germ cells.
The previous changes in testis of diabetic rats were noticeably im-
proved with oral administration of BP and/or DPP suspensions re-
vealing a marked repairing of testicular abnormalities, as shown in
Figs. 2C–E, demonstrating maximum healing effects against STZ-in-
duced testicular damage, showing sperm with a normal concentration
near to the normal group. The treatment with BP seemed to be more
effective in improving the testis histological architecture than DPP or
BP and DPP in combination.
4. Discussion
Weight loss is one of the symptoms of diabetes mellitus occurring
especially when glycemic control is poor. The present reduction in body
weight of diabetic rats was in agreement with a previously published
report of Yu et al.[44]. It was known that glycosuria causes a sig-
nificant loss of calories for every gram of glucose excreted and most
likely; this loss results in severe weight loss in spite of increased ap-
petite, particularly when it is coupled with loss of muscle and adipose
tissue due to excessive breakdown of protein[45–47]. The body weight
loss may be due to decrease of secretion of insulin which is considered
to be one of the most anabolic hormones in the body[48].
Table 4
Effect of BP and/or DPP suspensions on serum testosterone, FSH and LH levels in diabetic rats.
Parameters Groups Testosterone (ng/ml) % Change FSH (mIU/ml) % Change LH (mIU/ml) % Change
Normal 5.47 ± 0.21
b
2.03 ± 0.06
c
2.25 ± 0.09
a
Diabetic control 4.02 ± 0.12
c
−26.51 1.73 ± 0.20
c
−14.78 1.43 ± 0.08
b
−36.44
Diabetic treated with
BP
5.23 ± 0.10
b
+30.10 2.95 ± 0.20
b
+70.52 2.23 ± 0.18
a
+55.94
Diabetic treated with
DPP
6.00 ± 0.04
a
+49.95 1.68 ± 0.17
c
−2.89 2.40 ± 0.29
a
+67.83
Diabetic treated with BP and DPP 5.34 ± 0.11
b
+32.83 3.65 ± 0.07
a
+111.0 2.40 ± 0.07
a
+67.83
F-probability P < 0.001 P < 0.001 P < 0.001
LSD at the 5% level 0.371 0.449 0.481
LSD at the 1% level 0.501 0.608 0.650
- Data are expressed as Mean ± SE. Number of animals in each group is 6.
- Values, which have the same superscript symbol(s), are not significantly different.
- Percentage changes (%) were calculated by comparing the diabetic control group with normal and the diabetic treated groups with diabetic control.
Table 5
Effect of BP and/or DPP suspensions on sperm count ( × 10
6
) and sperm motility, abnormality and viability percents in diabetic rats.
Parameters Groups Sperm count ( × 10
6
) % Change Sperm motility (%) % Change Sperm abnormality (%) % Change Viability (%) % Change
Normal 76.91 ± 1.72
a
– 64.25 ± 0.56
a
– 13.5 ± 0.39
e
– 75.0 ± 0.45
a
–
Diabetic control 9.90 ± 0.40
d
−87.13 41.05 ± 0.47
e
−36.11 40.0 ± 0.45
a
+196.30 52.0 ± 0.45
e
−30.67
Diabetic treated with
BP
25.83 ± 0.96
b
+160.90 53.90 ± 0.40
b
+31.30 27.5 ± 0.22
c
−31.25 63.0 ± 0.45
b
+21.15
Diabetic treated with DPP 18.10 ± 0.47
c
+82.83 48.80 ± 0.36
d
+18.88 31.00 ± 0.45
b
−22.50 55.0 ± 0.45
d
+5.77
Diabetic treated with BP and DPP 19.45 ± 0.53
c
+96.46 50.80 ± 0.36
c
+23.75 23.50 ± 0.22
d
−41.25 58.0 ± 0.45
c
+11.54
F-probability P < 0.001 P < 0.001 P < 0.001 P < 0.001
LSD at the 5% level 2.78 1.271 0.966 1.303
LSD at the 1% level 3.76 1.719 1.307 1.763
- Data are expressed as Mean ± SE. Number of animals in each group is 6.
- Values, which have the same superscript symbol(s), are not significantly different.
- Percentage changes (%) were calculated by comparing the diabetic control group with normal and the diabetic treated groups with diabetic control.
N.A. Mohamed et al.
Biomedicine & Pharmacotherapy 97 (2018) 9–18
13

6. The decrease in the weight of pancreas could be attributed to the
disruption and the disappearance of pancreatic islets and selective de-
struction of insulin-producing cells[49]. Although the pancreas abso-
lute weight was significantly decreased in diabetic rats, the relative
pancreas weight was increased. This increase in relative body weight
may be attributed to the severe body weight loss. In contrast to the
present study, Mohammadi and Naik[50]reported that the relative
weights of pancreas were reduced in STZ treated rats.
In the present study, a significant decrease in the weight of the testis
was observed, suggesting that the weight of testis and the accessory sex
organs are sensitive end points that can be used in evaluation of ne-
gative effect on male reproduction[51]. Thisfinding was consistent
with Navarro-Casado et al.[52]and Jordaan et al.[53]who demon-
strated a decrease in testicular weight in STZ-induced diabetes.
The attenuation of body weight decline by the administration of BP
and/or DPP suggested that the BP and/or DPP could possess some
protective constituents that prevented the loss of body weight in dia-
betic treated rats. Accordingly, the significant increase in the re-
productive organ weights of diabetic rats that were treated with DPP
may be attributed to the improvement in testosterone levels or re-
sorption effects of estradiol which come from phytoestrogen component
in BP or DPP. This effect can be also explained by the antioxidant
property of BP and DPP that prevents cellular damage occurring as a
result of oxidative stress in spermatogenic cells of the seminiferous
tubules and Leydig cells of the testes[54]. This attribution was sup-
ported by the results of the present study which evidenced the decrease
of the testis oxidative stress markers including NO and LPx levels and
the increase in the testis antioxidant defense markers including GSH
content and GST, GPx and SOD activities. Moreover, this effect might be
due to the presence of gonadotropin-like substances or steroidal com-
ponent present in the BP and DPP[55].
In the current study, the biochemical investigations of glucose and
insulin levels after using STZ, ensured induction of DM in rats. DM was
induced by a single intraperitoneal injection of STZ which could da-
mage the insulin secreting cells of the pancreas and results in high
blood glucose levels that last for a long period[56]. Hyperglycemia,
hypoinsulinemia, polyphagia, polyuria and polydipsia accompanied by
weight loss were seen in adult rats within three days of streptozotocin
treatment[57].
In the current study, the treatment with BP and DPP caused a sig-
nificant improvement in the elevated blood glucose levels. The anti-
hyperglycemic effects of BP and DPP are due to their potencies to
ameliorate the lowered serum insulin levels secondary to the amend-
ment of pancreatic islets histological architecture and integrity and
increase in the number of insulin secretory beta-cells as indicated in the
present study. In another way, the hypoglycemic effect of BP and DPP
may be attributed to their minerals, phenolics and phytoestrogens
constituents. These minerals such as magnesium, zinc, chromium and
selenium play a key role in regulation of insulin action and insulin-
mediated-glucose uptake. Phenolics present inP. dactyliferawere con-
sidered to be potent inhibitors of alpha- glycosidase and alpha amylase,
leading to reduction of carbohydrates digestion and absorption that
may counteract the hyperglycemia present in DM[58].
DM is a chronic disease affecting many tissues and systems of the
body. Some of these manifestations were spermatogenic and steroido-
genic. In the present investigation, the STZ-induced DM resulted in a
reduction of testosterone, FSH and LH levels and impaired sperms count
and quality sperm and these results came in accordance with Khaneshi
et
al.[59].
STZ-induced diabetes in rats caused testicular dysfunction, leading
to the dramatic changes in the testicular histological architecture and
the alteration of spermatogenic process. The damaging effects of STZ on
the testes is mainly due to the decrease in testosterone level associated
with the absence or diminution of serum insulin levels, since insulin
acts as an anti-apoptotic factor capable to regulate testicular apoptosis
and sexual dysfunction induced by DM[60]. These effects may due to
that DM decreases serum testosterone levels[61,62]which are asso-
ciated with a steroidogenetic defect in Leydig cells. Furthermore, STZ-
induced DM is also associated with an increased oxidative stress, which
damages sperm nuclear and mitochondrial DNA.
It appeared that the hypothalamus-pituitary-testicular axis was
dysfunctional in the diabetic groups and this dysfunction was amelio-
rated by BP and/or DPP suspensions. The improving effect of DPP on
the sperm parameters in diabetic male rats might be due to the presence
of estrogenic material as a gonad-stimulating compound that improves
male fertility[63]. In this regard, Abbas and Ateya[64]and El-New-
eshy et al.[54]indicated that DPP containsflavonoid components that
have a positive effect on the sperm quality and male reproductive
Table 6
Effect of BP and/or DPP suspensions on testis GSH content and GST, GP
X, SOD activities in diabetic rats.
Parameters Groups GSH (nmol/100 mg) % Change GST (U/100 mg) % Change GP
X(mU/100 mg) % Change SOD (U/g) % Change
Normal 29.46 ± 1.33
a
– 53.32 ± 2.31
a
– 154.77 ± 5.01
a
– 11.31 ± 0.67
a
–
Diabetic control 21.54 ± 1.03
cd
−26.88 38.32 ± 1.39
b
−28.13 125.05 ± 3.29
c
−19.20 8.75 ± 0.64
c
−22.63
Diabetic treated with BP 26.76 ± 0.81
ab
+24.23 51.15 ± 3.13
a
+33.48 142.82 ± 1.32
b
+14.21 9.44 ± 0.42
bc
+7.89
Diabetic treated with DPP 23.28 ± 1.27
c
+8.08 37.69 ± 1.45
b
−1.64 156.33 ± 5.86
a
+25.01 12.57 ± 0.48
a
+43.66
Diabetic treated with BP and DPP 24.24 ± 1.01
bc
+12.53 38.82 ± 3.33
b
+1.30 147.88 ± 1.93
ab
+18.26 10.99 ± 0.65
ab
+25.60
F-probability P < 0.001 P < 0.001 P < 0.001 P < 0.001
LSD at the 5% level 3.227 7.162 11.341 1.691
LSD at the 1% level 4.365 9.689 15.342 2.029
- Data are expressed as Mean ± SE. Number of animals in each group is 6.
- Values, which have the same superscript symbol(s), are not significantly different.
- Percentage changes (%) were calculated by comparing the diabetic control group with normal and the diabetic treated groups with diabetic control.
Table 7
Effect of BP and/or DPP suspensions on NO level and LPx in testis of diabetic rats.
Parameters
Groups
NO (
μmole/
L) % Change LPx (nmole MDA/
100 mg/h)
% Change
Normal 24.55 ± 1.24
e
– 25.55 ± 1.07
b
–
Diabetic control 30.92 ± 1.20
d
+25.95 50.08 ± 0.64
a
+96.01
Diabetic treated
with BP
36.18 ± 1.44
c
+17.01 25.54 ± 1.70
b
−49.00
Diabetic treated
with DPP
57.86 ± 1.14
a
+87.13 22.05 ± 0.47
b
−55.97
Diabetic treated
with BP and
DPP
49.62 ± 1.84
b
+60.48 22.67 ± 0.31
b
−54.75
F-probability P < 0.001 P < 0.001
LSD at the 5%
level
4.069 2.837
LSD at the 1%
level
5.505 3.839
- Data are expressed as Mean ± SE. Number of animals in each group is 6.
- Values, which have the same superscript symbol(s), are not significantly different.
- Percentage changes (%) were calculated by comparing the diabetic control group with
normal and the diabetic treated groups with diabetic control.
N.A. Mohamed et al.
Biomedicine & Pharmacotherapy 97 (2018) 9–18
14

7. activity. In the same way, Hassan[23]stated that DPP exert a sig-
nificant protective effect against testicular dysfunction induced by
cadmium chloride (CdCl
2) through increases in estradiol level as well as
normalization of testosterone levels and sperm parameters. Moreover,
Abedi et al.[65]revealed that the aqueous extract ofP. dactylifera
pollen can be used as a sex enhancer and seems to cure male infertility.
In the current study, testicular antioxidants data showed a sig-
nificant reduction in GSH level and antioxidant defense enzyme (GST,
GPx and SOD) activities in diabetic testis, which is in accordance with
the study of Zhao et al.[66]. Other previous studies stated that the
activities of antioxidant enzymes as well as content of GSH were
reduced in tissues of diabetic rats. This may result in a number of de-
leterious effects which may be due to the accumulation of ROS; the
present study revealed an elevation of oxidative stress markers NO and
LPx in STZ-induced diabetic rats. One possible mechanism for this re-
duction in GPx, SOD and GST activities and GSH content may be due to
the inactivation caused by the excess of free radicals and/or by non-
enzymatic glycation due to the persistent hyperglycemia, which has
been extensively reported to occur in DM[67,68].
The mechanism for protection of the BP extract may involve
scavenging potentially toxic and mutagenic electrophiles and free ra-
dicals and modification of antioxidant pathways as suggested by Ohta
Fig. 1.Photomicrographs of sections of pancreas of normal, diabetic and treated diabetic rats. Photomicrograph of normal rat (A) showed pancreatic lobules divided by septa (S),
pancreatic acini (PA) and intact and organized islets of Langerhans (IL) that consists of alpha cells (a) in the core of the islets, beta cells (b) at periphery and delta cells (d) of bigger size
adjacent to alpha cells. Photomicrograph of pancreas of diabetic rat (B) showed disrupted islets of Langerhans (IL) of small size and few numbers of islet cells that exhibited necrosis (Nc)
and vacuolations (v). Photomicrograph of pancreas of BP treated diabetic rat (C) showed large islet of Langerhans (IL) with increased number of alpha (a), beta (b) and delta (d) cells
attained its intact shape in spite of presence of some vacuolations (v) and necrotic areas (Nc). Photomicrograph of pancreas of DPP treated diabetic rat (D) showed normalized large islet
of Langerhans (IL) with increased number of islet cells. Alpha (a), beta (b) and delta (d) cells were noticed. Photomicrograph of pancreas of diabetic rats co-treated with BP and DPP (E)
showed stimulated recovery. The islets (IL) regain their normal architecture with fewer vacuolated (v) hydropic cells and the alpha (α); beta (β) cells appeared more intact and granulated
(H & E;×200).
N.A. Mohamed et al.
Biomedicine & Pharmacotherapy 97 (2018) 9–18
15

8. et al.[69]. Other investigators indicated that, BP extract contains sig-
nificant amounts of polyphenolic substances, mainlyflavonoids
[70,14]. These polyphenols are antioxidants with redox properties
which allow them to act as reducing agents, hydrogen donators, and
singlet oxygen quenchers[71].
Like BP, DPP contains active antioxidant substances includingfla-
vonoids, phytosterols and carotenoids that have an essential role in
eliminating free radicals causing the enhancement of fertility para-
meters; thereby it can provide an important protective role against
oxidative stress[72]. Theflavonoids were reported to have potential
antioxidant properties[73].
In the present study, the treatment of STZ-induced diabetic rats with
BP and DPP suspensions successfully resulted in a suppression of oxi-
dative stress as indicated by the decrease in the elevated testis NO level
Fig. 2.Photomicrographs of sections of testes of normal, diabetic and treated diabetic rats. Photomicrograph of normal rat (A) showed seminiferous tubule (ST), spermatogonia (Sg),
primary spermatocytes (PSc), secondary spermatocytes (SSc), spermatids (Sd), interstitial connective tissue (ICT), Leydig cells (LC) and seminiferous tubules with numerous active germ
cells (GC). The lumen of tubules wasfilled with normal spermatozoa (Sz). Photomicrograph of testis section of diabetic rat (B) showed remnants of Leydig cells (LC), degenerated and
atrophic seminiferous tubules (small arrow), severe destruction of spermatids (Sd) and spermatozoa (Sz), with scanty to absent germ cells (*). Photomicrograph of section of testis of BP
treated diabetic rat (C) showed spermatogonia (Sg), increased number of sperms (Sz) and spermatids (Sd). Photomicrograph of section of testis of DPP treated diabetic rat (D) showed
slight reduction in spermatogonia (Sg), spermatocyte (Sc), spermatids (Sd) and spermatozoa (Sz) degeneration of interstitial tissue (*). Photomicrograph of section of testis of BP and DPP
treated diabetic rat (E) showed exfoliation (Ef) of the degenerated spermatogenic cell lineages in the lumen of seminefrous tubules beside germ cells degeneration resulting in areeas
devoid of germ cells (arrow), seminiferous tubules surrounded by interstitial tissue (*) (H & E; ×200).
N.A. Mohamed et al.
Biomedicine & Pharmacotherapy 97 (2018) 9–18
16

9. and LPx, and an augmentation of antioxidant defense system as evi-
denced by the increase of testis GSH content and stimulation of anti-
oxidant enzyme (GST, GPx and SOD) activities.
Histopathological analysis of the testis depicted that the STZ-in-
duced diabetic rats exhibited frequent abnormal architectural changes
in the seminiferous tubule lined by spermatogenic cell series, Sertoli
cells and Leydig cells, which lead to a decrease in plasma testosterone
levels[74]. The BP and DPP treatments improved the spermatogenesis
as evident by significant increase in the number of spermatids, sper-
matogonia, spermatocytes, and Sertoli cells as compared to diabetic
rats. The improved spermatogenesis in testis due to BP and DPP treat-
ments was concomitant with the increase in serum FSH level. Moreover,
the increase in serum testosterone level as a result of treatments with BP
and DPP to be attributed to the increase in the number of interstitial
Leydig cells on one hand and stimulated secretion of LH on the other
hand.
Pancreatic histological investigation of normal and diabetic rats
confirmed that the islet cells were destroyed due to the effects of STZ in
diabetic rats. STZ induces DM by destroying pancreaticβ-cells, possibly
through generating excess reactive oxygen species (ROS)[75,76]. STZ-
induced LPx and DNA breaks in pancreatic islet cells have been de-
monstrated[77].
Degenerative changes of pancreatic beta cells in STZ treated rats
were minimized by administration of BP and/or DPP treatments as
evidenced by histopathological examination. The pancreatic islets of
DPP treated diabetic rats were completely normalized while the pan-
creatic islets of diabetic rats treated with BP and its combination with
DPP still showed few necrotic foci and vacuolations These results
clearly indicate that BP and/or DPP treatments exert a therapeutic
protective nature in DM by decreasing oxidative stress and pancreatic
beta cells’damage which may be attributed to their antioxidative po-
tentials.
5. Conclusion
The preventive effect of BP and/or DPP grains against sexual dis-
turbances in the diabetic male rats may be attributed to the improve-
ments in the glycemic state secondary to ameliorations in the insulin
secretion and beta cell function as well as to enhancement of the tes-
ticular antioxidant defense system. The study also supports the tradi-
tional use of these pollen grains as acclaimed aphrodisiacs. However,
further clinical studies are required to assess the efficacy of BP and DPP
on impaired sexual dysfunctions in diabetic men.
Conflict of interest
The authors declare that there is no conflict of interest.
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