Potential of phloroglucinol to improve erectile dysfunction associated with streptozotocin-induced diabetes in rats

Abstract

Objective: Diabetes is a common metabolic disease with several complications in its patients. Often,
people living with diabetes develop erectile dysfunction (ED). The primary aim of this work was to
investigate the effect of phloroglucinol in diabetes-induced ED in rats.
Methods: Male Wistar rats were given 52 mg/kg of streptozotocin, by intraperitoneal injection, to induce
diabetes and ED. Subsequently, animals were grouped into three groups: group 1, diabetic control; group
2, low-dose phloroglucinol (150 mg/kg body weight); and group 3, high-dose phloroglucinol (250 mg/kg
body weight). A group of six normal rats served as a normal control. The rats were treated with
phloroglucinol for six weeks and then were assessed for treatment effects. Sexual behavior, glycosylated
hemoglobin A1c (HbA1c) values, serum testosterone, serum nitric oxide (NO), blood pressure and sperm
count were measured after the end of treatment.
Results: After a 6-week treatment period, the high dose of phloroglucinol significantly decreased HbA1c
values in diabetic rats. Rats treated with phloroglucinol had increased serum testosterone, NO and sperm
count. Animals treated with 250 mg/kg phloroglucinol performed similar to normal rats in the sexual
behavioral study, suggesting the reversal of complications of ED. Conversely, a decrease in the blood
pressure in treated groups was observed.
Conclusion: The results highlight the protective effect of phloroglucinol in diabetes-induced ED in rats
warranting further studies.

Full text

Original Research Article
Potential of phloroglucinol to improve erectile dysfunction associated
with streptozotocin-induced diabetes in rats
Syed Kashif
a
, Rema Razdan
a
, Ramanaiah Illuri
b,
⇑
a
Department of Pharmacology, Al-Ameen College of Pharmacy, Bengaluru, Karnataka 560027, India
b
PG and Research Centre in Biotechnology, MGR College, Hosur, Tamil Nadu 635109, India
article info
Article history:
Received 12 October 2018
Accepted 20 December 2018
Available online 5 April 2019
Keywords:
Diabetes
Streptozotocin
Erectile dysfunction
Phloroglucinol
Glycated hemoglobin A
abstract
Objective: Diabetes is a common metabolic disease with several complications in its patients. Often,
people living with diabetes develop erectile dysfunction (ED). The primary aim of this work was to
investigate the effect of phloroglucinol in diabetes-induced ED in rats.
Methods: Male Wistar rats were given 52 mg/kg of streptozotocin, by intraperitoneal injection, to induce
diabetes and ED. Subsequently, animals were grouped into three groups: group 1, diabetic control; group
2, low-dose phloroglucinol (150 mg/kg body weight); and group 3, high-dose phloroglucinol (250 mg/kg
body weight). A group of six normal rats served as a normal control. The rats were treated with
phloroglucinol for six weeks and then were assessed for treatment effects. Sexual behavior, glycosylated
hemoglobin A1c (HbA1c) values, serum testosterone, serum nitric oxide (NO), blood pressure and sperm
count were measured after the end of treatment.
Results: After a 6-week treatment period, the high dose of phloroglucinol significantly decreased HbA1c
values in diabetic rats. Rats treated with phloroglucinol had increased serum testosterone, NO and sperm
count. Animals treated with 250 mg/kg phloroglucinol performed similar to normal rats in the sexual
behavioral study, suggesting the reversal of complications of ED. Conversely, a decrease in the blood
pressure in treated groups was observed.
Conclusion: The results highlight the protective effect of phloroglucinol in diabetes-induced ED in rats
warranting further studies.
Please cite this article as: Kashif S, Razdan R, Illuri R. Potential of phloroglucinol to improve erectile dys-
function associated with streptozotocin-induced diabetes in rats. J Integr Med. 2019; 17(4): 282–287.
Ó2019 Shanghai Changhai Hospital. Published by Elsevier B.V. All rights reserved.
1. Introduction
Diabetes is now becoming a common condition all over the
world. This debilitating metabolic disease is associated with
increased blood glucose. Primary reasons for the disease are dam-
age to pancreatic b-cells, which leads to failure in insulin produc-
tion, and resistance of cells to the insulin that is produced [1].
Over 382 million people were estimated to have diabetes in
2013, and the prevalence is expected to increase to more than
592 million by 2035 [2].
Erectile dysfunction (ED) is defined as the inability of the male
to maintain a reasonable penile erection during sexual intercourse
[3]. A healthy sexual function involves a healthy vascular and hor-
monal system. ED is commonly reported among diabetic patients,
leading to a reduced quality of life [4,5]. Men with diabetes have an
elevated risk for developing ED [6,7]. Several drugs are available for
managing of ED, but these medications cause several side effects in
humans [8–10]. This has led researchers to look for alternative
treatment options which are safe and effective for ED [11–14].
The rise of herbal and traditional medicine in management of
ED has been well documented over last few decades [15]. Circula-
tory stimulants like ginger and turmeric, and adaptogens like
American ginseng and ashwagandha have been explored for
management of ED in human [16]. Phloroglucinol is a flavonoid
derivative with antioxidant and anti-inflammatory activities [17].
In a 10-year follow-up study, higher flavonoid intake reduced ED
in human subjects [18]. Gut microbiota plays a critical role in the
biotransformation of xenobiotics and phytocompounds in the
human gastrointestinal tract [19,20].
Phloroglucinol and its derivatives are reported to have antioxi-
dant effects and are also known as nitric oxide (NO) donors [21,22].
https://doi.org/10.1016/j.joim.2019.04.002
2095-4964/Ó2019 Shanghai Changhai Hospital. Published by Elsevier B.V. All rights reserved.
⇑
Corresponding author.
E-mail address: illuri.ramanareddy@gmail.com (R. Illuri).
Journal of Integrative Medicine 17 (2019) 282–287
Contents lists available at ScienceDirect
Journal of Integrative Medicine
journal homepage: www.jcimjournal.com/jim
www.journals.elsevier.com/journal-of-integrative-medicine

In this work, we investigated the impact of phloroglucinol in ED
associated with diabetes. It will be interesting to know whether
these effects can be further explored to improve the ED in people
with diabetes. Streptozotocin (STZ) is commonly used by
researchers to induce diabetes in rats and is administered by
intraperitoneal injection [23]. Rats with fasting blood glucose
(FBG) levels above 250 mg/dL were considered to be diabetic and
used in the study. STZ-induced diabetic rats in treatment groups
were given different doses of phloroglucinol (150 mg/kg and
250 mg/kg body weight) for six weeks, and sexual function was
evaluated using several common parameters.
2. Materials and methods
2.1. Chemicals and reagents
STZ was purchased from MP Biomedicals and phloroglucinol
from HiMedia, India. Glucose test kits were purchased from Span
Diagnostics Limited, India. Diethylstilbestrol and progesterone
were purchased from Penta Pharmaceuticals, India and Sun Phar-
maceutical India Limited, India, respectively. Standard rat feed
was procured from Pranav Agro Industries Limited, India.
2.2. Study animals
Ethical clearance from the Institutional Animal Ethics
Committee of Al-Ameen College of Pharmacy, Bangalore, India
was obtained before the start of the study. All the experiments
were performed following the Guide for the Care and Use of Labo-
ratory Animals of the National Institutes of Health and guidelines
set by the Committee for the Purpose of Control and Supervision
of Experiments on Animals, India. Albino Wistar rats (12-week
old) between 250 and 300 g were purchased from the Animal
House of Al-Ameen College of Pharmacy, Bangalore, India. Rats
were housed in polypropylene cages with paddy husk bedding, at
(25 ± 2) °C and exposed to 24 h light/dark cycle. All animals were
allowed fresh food and water ad libitum. All animal dissection
experiments were performed under anesthesia as per institutional
ethics guideline.
2.3. Induction of diabetes and treatment groups
Healthy rats were injected with STZ (52 mg/kg body weight,
intraperitoneal injection) prepared in 100 mmol/L sodium citrate
buffer (pH 4.5), to induce diabetes. STZ was freshly prepared before
dosing and immediately injected intraperitoneally, as described
previously [24,25]. A 5% glucose solution was provided ad libitum
to rats for one day, starting 24 h after STZ. FBG was measured
72 h after STZ injection, using a glucose kit to check the status of
diabetes [25]. The animals with FBG >2.5 g/mL were considered
diabetic and were divided into groups for the diabetic control
(n= 6), low-dose phloroglucinol (150 mg/kg body weight) and
high-dose phloroglucinol (250 mg/kg body weight). A nondiabetic
group of 6 rats was kept as a normal control. Phloroglucinol was
prepared freshly before each administration in distilled water
and administered orally, once a day for 6 weeks.
2.4. Animal body weight
The weight of each animal was taken in triplicate at the end of
every week during treatment, to monitor the general health of the
animal. The weight was taken before the induction of diabetes and
was continued at the end of every week until the end of the study.
2.5. Blood pressure and diabetes status
The tail-cuff method was used to measure blood pressure in rats
[26]. The method includes noninvasive systolic blood pressure
measurement using LabChart v7 (ADInstruments South Asia, New
Delhi, India). Subsequently, rat blood was collected in ethylenedi-
aminetetraacetic acid-coated tubes, and percentage of glycosylated
hemoglobin A1c (HbA1c) was determined using a glycosylated
hemoglobin kit (Coral Clinical Systems Goa, India), according to
the manufacturer-suggested protocol.
2.6. Sexual behavioral studies
The male rats were trained for sexual activity before the induc-
tion of diabetes. Sexual training was provided in the presence of
estrous ovariectomized female rats [27]. For the sexual behavioral
study, the female rats were ovariectomized and treated with
diethylstilbestrol (1 mg/kg, per os, administered 48 h before sexual
behavior study). Estrous phase was induced by giving progesterone
(5 mg/kg, subcutaneous injection, administered 4 h before the sex-
ual behavior study). A male rat was first introduced in the cage and
allowed to acclimatize for 5–10 min before introducing the female
rat. Following sexual behavior, parameters were evaluated [28,29].
Mount frequency represents the number of the mounts in a sexual
cycle before ejaculation; intromission frequency represents the
number of intromissions in a sexual cycle before ejaculation;
mount latency represents the time from the introduction of the
female into the cage of the male up to the first mount; intromission
latency represents the time from the introduction of the female up
to the first intromission by the male; ejaculation latency represents
the time from the first intromission to the ejaculation; postejacu-
latory interval represents the time from the end of the first ejacu-
lation to start of next intromission.
2.7. Serum NO and testosterone levels
Serum NO was measured by mixing 100 mL of the sample with
100 mL of the Greiss reagent in a 96-well culture plate. Absorption
of the mixture was measured at 570 nm in an ultraviolet–visible
spectrophotometer, after 30 min of incubation at room tempera-
ture. The amount of nitrite formed was determined by comparing
the sample values with a NaNO
2
standard curve [30]. A testos-
terone enzyme-linked immunosorbent assay kit from Abcam
Ò
,
USA was used to measure the serum testosterone.
2.8. Sperm count and mobility
In the postsexual behavioral study, the epididymal tail was
excised and placed in a Petri dish containing 1 mL of 100 mmol/L
phosphate buffer (pH 7.4). The tail was further swirled gently for
homogeneity and to allow sperm diffusion in the solution for
10 min under 37 °C. The resulting solution containing sperm cells
was assessed for sperm motility, number, and gross morphology.
Aliquots of 10 mL were placed in a hemocytometer chamber, and
motility was observed under a light microscope. Further,
100 sperms were studied for each animal and characterized based
on motility (motile and immotile) as described previously [31].
Number of sperms per caudal epididymis
¼mean count 50 ðtotal volumeÞ
0:01 0:01 ðvolume of counting chamberÞ
2.9. Statistical analysis
One-way analysis of variance followed by Tukey’s multiple
comparison test was used to evaluate the statistical significance
S. Kashif et al. / Journal of Integrative Medicine 17 (2019) 282–287 283

of treatment. P< 0.05 was considered statistically significant. All
the values are reported as mean ± standard deviation or standard
error of the mean. GraphPad Prism 5 (GraphPad Inc., La Jolla, CA,
USA) was used for graphing and statistical testing.
3. Results
3.1. Effects of phloroglucinol on animal weight
The changes in body weight of rats treated with phloroglucinol
were significantly different from that of the untreated diabetic rats
(Fig. 1). Normal control rats on vehicle gained significant weight,
compared to diabetic control. Interestingly, the diabetic control
group showed a reduction in body weight. The animals treated
with phloroglucinol showed a < 10% decrease in body weight.
3.2. Effects of phloroglucinol on blood pressure
The systolic blood pressure of diabetic rats was significantly
higher than that of normal control rats (Fig. 2). Treatment with
phloroglucinol significantly lowered rat blood pressure and was
active at both doses (P< 0.001).
3.3. Sexual behavioral studies
The results for different parameters of the sexual behavioral
study are presented in Table 1. Compared to normal control, all
the diabetic rats showed diminished sexual activity. Phloroglucinol
treatment at a higher dose (250 mg/kg) was effective to improve
sexual activity when compared to the diabetic control except
mount frequency.
3.4. Effects of phloroglucinol on testosterone and serum NO
Serum NO level was significantly lower in the diabetic control
group than those in the normal control group (Fig. 3). The levels
of NO increased considerably after treatment with phloroglucinol
when compared with diabetic control rats.
As expected, diabetes significantly lowered serum testosterone
level (Fig. 4). The serum testosterone levels of rats treated with
phloroglucinol were increased relative to diabetic control.
However, the results were only significant for the 250 mg/kg body
weight group when compared to untreated diabetic control rats.
3.5. Effects of phloroglucinol on serum HbA1c
Serum HbA1c of diabetic rats was significantly higher than that
in the normal control (Fig. 5). The low-dose phloroglucinol group
and diabetic control had similar Hb1Ac. In contrast, the Hb1Ac
level of high-dose phloroglucinol-treated rats was significantly
lower than that of diabetic control (Fig. 5).
3.6. Effects of phloroglucinol on sperm count
Diabetic control rats had a significantly lower sperm count
compared to normal control animals (Fig. 6). Only rats treated with
high-dose phloroglucinol showed a significant increase in sperm
count, compared to the diabetic control rats, suggesting a dose-
dependent effect. The high dose of phloroglucinol (250 mg/kg body
weight) significantly improved the proportion of defective sperms
in the total sperm count compared to diabetic control (Fig. 7).
4. Discussion
ED is the failure of an individual to sustain an erection for sat-
isfactory sexual intercourse. Diabetes is rising sharply in the mod-
ern population due to a shift in lifestyle and nutrition [32].A
significant percentage (over 50%) of diabetic men are likely to
develop ED at some stage of their life [5]. The possible mechanisms
for the development of ED in this population include oxidative
stress, nitrosative stress, hyperglycemia and hypertension due to
diabetes or even hypogonadism [5].
Phloroglucinol and its derivatives are reported to have antioxi-
dant effects, and phloroglucinol is a known NO donor [21,22].In
this work, we investigated the impact of phloroglucinol on ED
associated with diabetes. Diabetic rats with FBG levels above
2.5 g/mL were considered diabetic and used in the study.
Phloroglucinol was administered orally once a day at doses of
150 mg/kg and 250 mg/kg body weight for 6 weeks.
Change in body weight (%)
Normal control
Diabetic control
PHL (150 mg/kg)
PHL (250 mg/kg)
Fig. 1. Effects of oral administration of PHL on body weight. All values are
expressed as mean ± standard error of the mean (n= 6).
***
P< 0.001, compared with
normal control;
###
P< 0.001, compared with diabetic control. PHL: phloroglucinol.
Blood pressure (mmHg)
Normal control
Diabetic control
PHL (150 mg/kg)
PHL (250 mg/kg)
Fig. 2. Effects of oral administration of PHL on blood pressure. All values are
expressed as mean ± standard error of the mean (n= 6).
***
P< 0.001, compared with
normal control;
###
P< 0.001, compared with diabetic control. PHL: phloroglucinol.
284 S. Kashif et al. / Journal of Integrative Medicine 17 (2019) 282–287

Flavonoids are reported to possess antihypertensive properties
by inhibiting triphosphopyridine nucleotide (NADPH) oxidase, an
enzyme responsible for reducing the bioavailability of endothelial
NO, which is a well-known vasodilator [33]. We think phlorogluci-
nol, a flavonoid, induces changes in blood pressure through NADPH
oxidase inhibition. The reported antispasmolytic property of
phloroglucinol may also add to the hypotensive effect of
phloroglucinol [34]. Hyperglycemia-derived endothelial dysfunc-
tion is implicated in the development of ED, and extracts that
control the blood glucose are promising candidates for manage-
ment of ED [35].
Table 1
Effects of oral administration of PHL on sexual behavior.
Group MF IF ML (s) IL (s) EL (s) PEI (s)
Normal control 4.5 ± 0.2 6.3 ± 0.02 159.9 ± 0.6 200.2 ± 0.1 317.0 ± 0.5 470.1 ± 1.0
Diabetic control 1.9 ± 0.2
***
3.6 ± 0.02
***
256.2 ± 0.1
***
319.5 ± 0.6
***
237.0 ± 0.9
***
601.4 ± 0.5
***
PHL (150 mg/kg) 2.2 ± 0.1 3.9 ± 0.1
#
251.6 ± 0.2
###
316.6 ± 1.6 245.4 ± 1.0
###
590.4 ± 1.5
###
PHL (250 mg/kg) 2.3 ± 0.2 4.2 ± 0.1
###
257.9 ± 0.5
#
312.6 ± 1.0
###
241.3 ± 0.8
##
580.6 ± 1.5
###
PHL: phloroglucinol. MF: Mount frequency; IF: intromission frequency; ML: mount latency; IL: intromission latency; EL: ejaculation latency; PEI: postejaculatory interval. All
values are expressed as mean ± standard error of mean (n= 6).
***
P< 0.001, compared with normal control;
#
P< 0.05,
##
P< 0.01,
###
P< 0.001, compared with diabetic control.
Serum nitric oxide (μmol/mL)
Serum nitric oxide (μmol/mL)
Normal control
Diabetic control
P
HL (150 mg/kg)
P
HL (250 mg/kg)
Serum nitric oxide (μmol/mL)
N
ormal control
D
iabetic control
PH
L (150 mg/kg)
PH
L (250 mg/kg)
Serum nitric oxide (μmol/mL)
Normal control
Diabetic control
PHL (150 mg/kg)
PHL (250 mg/kg)
Fig. 3. Effects of oral administration of PHL on serum nitric oxide. All values are
expressed as mean ± standard error of the mean (n= 6).
***
P< 0.001, compared with
normal control;
#
P< 0.05,
###
P< 0.001, compared with diabetic control. PHL:
phloroglucinol.
Serum testosterone (ng/dL)
Normal control
Diabetic control
PHL (150 mg/kg)
PHL (250 mg/kg)
Fig. 4. Effects of oral administration of PHL on serum testosterone. All values are
expressed as mean ± standard error of the mean (n= 6).
***
P< 0.001, compared with
normal control;
###
P< 0.001, compared with diabetic control. PHL: phloroglucinol.
HbA1c (%)
Normal control
Diabetic control
PHL (150 mg/kg)
PHL (250 mg/kg)
Fig. 5. Effects of oral administration of PHL on glycosylated hemoglobin. All values
are expressed as mean ± standard error of the mean (n= 6).
***
P< 0.001, compared
with normal control;
###
P< 0.001, compared with diabetic control. PHL:
phloroglucinol.
Sperm count
per caudal epididymis × 106
Normal control
Diabetic control
PHL (150 mg/kg)
PHL (250 mg/kg)
Fig. 6. Effects of oral administration of PHL on sperm count. All values are
expressed as mean ± standard error of the mean (n= 6).
***
P< 0.001, compared with
normal control;
#
P< 0.05, compared with diabetic control. PHL: phloroglucinol.
S. Kashif et al. / Journal of Integrative Medicine 17 (2019) 282–287 285

Serum NO is a neurotransmitter that is a well-known vasodila-
tor and is essential in both the arousal and maintenance of penile
erection [36]. As reported earlier, phloroglucinol is an NO donor;
our study also supports the reported activity of phloroglucinol
[22]. Testosterone is a potent vasodilator that activates Ca
2+
influx
and stimulates NO production [37–39]. Our results showed that
the rats treated with phloroglucinol had no significant change in
serum testosterone levels.
5. Conclusion
Our results showed a dose-related response, wherein we saw
that the rats treated with higher dose of phloroglucinol performed
significantly better than untreated rats and those who received a
lower dose of the drug. Additionally, it has been reported that
phlorotannins have angiotensin-converting enzyme inhibitory
activity. Since phlorotannins are formed by the polymerization of
phloroglucinol, it would of great interest to see whether these
phenols can produce the same effect as other polyphenols. Further
studies are warranted in different animal models of ED to ascertain
the beneficial impact of phloroglucinol in the disease.
Acknowledgements
The authors would like to acknowledge funding from Al-Ameen
College of Pharmacy, Bangalore, India.
Conflict of interest
The authors declare no conflict of interest for this work.
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Defective sperms (%)
Defective sperms (%)
N
ormal control
Dia
betic control
HL
(150 mg/kg)
HL
(250 mg/kg)
Defective sperms (%)
Normal control
Diabetic control
PHL (150 mg/kg)
PHL (250 mg/kg)
Fig. 7. Proportion of defective sperms in the 4 groups. All values are expressed as
mean ± standard error of mean (n= 6).
***
P< 0.001, compared with normal control;
##
P< 0.01, compared with diabetic control. PHL: phloroglucinol.
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