Effects of Hydro-alcoholic Extract of Pumpkin Seeds on Oogenesis Pathway, Liver, and Kidney of Female Rats
Samaneh Motamed Jahromi1,* and Sadegh Niami Jahromi2
- Expert, Molecular Medicine Research Center, Hormozgan Health Institute , Hormozgan University of Medical Sciences, Bandar Abbas, Iran
- Technical Expert in charge of Electricity Department, Shipping Reservoir Unit, Qeshm Shipping, Bandar Abbas, Iran
* Corresponding author: Samaneh Motamed Jahromi, Expert, Molecular Medicine Research Center, Hormozgan Health Institute , Hormozgan University of Medical Sciences, Bandar Abbas, Iran. Tel: 09177921046; Email: Sanammorsal400500@yahoo.com
Received 2020 August 23; Revised 2020 August 30; Accepted 2020 September 08.
Background: Pumpkin seed extract can be a good alternative to hormone replacement therapy since it is rich in phytoestrogens.
Objectives: The present research aimed to investigate the effect of hydro-alcoholic extract of pumpkin seeds on the oogenesis pathway, liver, and kidney of female rats.
Methods: This experimental study was performed on 64 Wistar female rats (including 32 adults and 32 immature rats). The adult rats were randomly divided into three experimental and one control groups (n=8 per group). Moreover, the immature rats were allocated to groups in a similar manner. The experimental groups 1, 2, and 3 received a hydro-alcoholic extract of pumpkin seed in doses of 20, 50, 100 mg/kg, respectively, via intraperitoneal injection for 21 consecutive days. For the purposes of the study, blood samples were taken one day after the last injection to determine the serum levels of female hormones as well as renal and hepatic factors. The ovaries, livers, and kidneys of the rats were also separated for histological tests.
Results: Based on the results, significant increases were observed in the bodyweight of all immature rats; estrogen levels in the adult experimental group 3 and immature experimental groups 2 and 3; progesterone and creatinine levels in the immature experimental group 3; aspartate aminotransferase, total protein, uric acid(UA), and the renal diameter in the immature experimental groups 1 and 2; follicle-stimulating hormone in the adult experimental group 3 and the immature experimental groups 1 and 2; luteinizing hormone and Graafian follicles in the adult experimental group 3; and atretic follicles in the immature experimental group 1 and 3 (P<0.05). Moreover, significant decreases were observed in the alkaline phosphatase in the adult experimental group 3; total protein, UA, and renal diameter in the immature experimental group 3; diameters of proximal and distal tubule as well as Henle’sloop in all immature rats; diameter of glomerular in the immature experimental groups 1 and 2; diameter of the renal cortex, glomerular, and Bowman's capsule in the adult experimental groups 2 and 3; secondary follicles in the adult experimental group 1, immature experimental groups 1 and 3; and primitive and early follicles in all the adult rats, compared with the control group (P<0.05).
Conclusion: Based on the findings, it can be concluded thatthe pumpkin seeds provide the nutritional needs of the body at the onset of sexual maturity, prepare the body for sexual maturity, and regulate female sex hormones without having adverse effects on the hepatic tissues. However, it must be noted that its consumption at the onset of sexual maturity requires dosage determination and further studies.
Keywords: Estrogen, Extract, Female, Hormone, Kidney, Liver, Rat
Chemical medicines used for disease treatment can have various side effects. For example, hormone
replacement therapy used for the treatment of sexual maturation disorders or genital anomalies is
associated with hyperplasia, endometrial cancer, breast cancer, or thromboembolism that can also
affect renal and hepatic functions (1). In general, due
to the side effects of chemical medicines, researchers have decided to focus on the use of herbal
products in a variety of medical fields (2). It should be
noted that herbal medications, which have been used in traditional medicine, are made from a group
of herbs with activeingredients used to treat or prevent various diseases (3).
According to the review of the related literature, pumpkin is also known as Cucurbita maxima and it is originally
from South America. It belongs to the family Cucurbitaceae and has been widely cultivated around the world since
its discovery. Pumpkin seeds are rarely used by people around the world and may even be considered as a waste.
However, the extract of these seeds have numerous benefits for human health and can be used as nutrient
enrichment of food products or an alternative to oil (4-6).
Pumpkin seed is an excellent source of minerals, including phosphorus, potassium, and selenium, which help the
body fight various diseases, such as arthritis, inflammation, and prostate cancer. In addition, it is said to be
rich in various elements, namely calcium, manganese, and zinc (6). According to the previous research, pumpkin seeds
extract contains chemical compounds, such as linoleic acid and oleic acid, which reduce blood cholesterol (7-9). It is also a good source of unsaturated fatty acids
and phytosterols (β-sitosterol) that can prevent chronic diseases.
Pumpkin seeds can also effectively reduce the risk of bladder-stone disease and treat lower urinary tract
symptoms and benign prostatic hyperplasia (10). It should be noted that β-Sitosterol can
reduce cholesterol, affect estrogen, and have anticancer activities (11). Based on the findings of previous studies, pumpkin
has potential antioxidant activity.
Reactive oxidants in the follicles are necessary for the ovulatory response; therefore, the ablation of the
ovarian oxygen specieshinders the ovulation and the whole repertoire of essential preovulatory responses (12
13). It should be noted that more changes were observed
in sex hormones at the onset of sexual maturation in comparison to after the completion of sexual
development. Abnormal levels of sex hormones, especially estrogen and progesterone, can affect the kidney and
Renal and hepatic failure is common among women who take steroid-containing contraceptives (14)
.According toprevious studies, the use of certain amounts of synthetic estrogen and progesterone for the treatment of female hormone disorders may affect renal
and hepatic functions in certain circumstances (13-15). For example, tamoxifen is a selective estrogen
receptor modulator that can lead to cholestasis (16). Moreover, ethinyl estradiol, which is used for
treating ovarian insufficiency and menopausal symptoms, can have side effects, such as changes in hepatic
function and jaundice associated with biliary obstruction. In addition, HD pills, used for menstrual regulation,
can increase the risk of biliary disease and gallstoneformation (17).
It is noteworthy that peliosis hepatitis is a form of hepatic disease that can be associated with the use of
anabolic-androgenic steroids, oral contraceptives, azathioprine, tamoxifen, and danazol. This disease can be
asymptomatic or manifest itself as hepatomegaly, hepatic failure, and intraperitoneal hemorrhage or even cause
death (16-18). Infection, inflammation, and removal of the
gallbladder have been reported in women taking oral estrogen, which may be due to the hepatic effects of oral
administration of estrogen. Estrogens increase biliary cholesterol secretion and saturation which is associated
with decreased gallbladder motility and increased crystallization of cholesterol in bile
Given the side effects of chemical medications, pumpkin seed extract can be used as an alternative sex hormone
regulator. However, it can replace chemical medications only after it is proved that this extract does not have
any adverse effects on the kidney and liver at the onset or end of sexual maturation (21).
The present research aimed to investigate the effect of hydro-alcoholic extract of pumpkin seeds on the oogenesis pathway, liver, and kidney of female rats.
3.1. Subjects and group allocation
This study was performed on immature and adult female Wistarrats. All ethical issues regarding how to
work with laboratory animals and the principles of laboratory animal care were considered in the present
research (ethics code for immature and adult rats were IR.HUMS.REC.1398.426 and jums.REC.
1393.071, respectively). All the selected immature rats were at the onset of puberty. The adult and
immature rats were kept in an animal breeding room under similar conditions for a week to adapt. They
were maintained in a 12/12 h light/dark cycle and the humidity was about 50-55% (22-25).
For the purposes of the study, 32 adult Wistar female rats that were 60 days old and weighted
180±10 g were randomly divided into three experimental and one control groups (n=8 per group.
Moreover, 32 immature Wistar female rats that were 30-35 days old and weighted 100±80 g were
randomly divided into three experimental and one control groups (n=8 per group.
The sample size was calculated using the information extracted from a study performed by Solomon et al.
and based on the formula N= in which α=0.05 and 1-β=0.9. Accordingly, the formula N= 8
was considered in each experimental group (26). Both the experimental and control
groups received the right doses of solutions via insulin syringe through intraperitoneal injection for
21 successive days on a daily basis, at 10 a.m. and once a day (27). The details are described
Adult rat groups:
Group 1: received 20 mg/kg bw of hydro-alcoholic extract of pumpkin seeds (0.2 ml in each daily
intraperitoneal injection) (27).
Group 2: received 50 mg/kg bw of hydro-alcoholic extract of pumpkin seeds (0.2 ml in each daily
intraperitoneal injection) (27).
Group 3: received 100 mg/kg bw of hydro-alcoholic extract of pumpkin seeds (0.2 ml in each daily
intraperitoneal injection) (27).
Group 4 (Control): received no medicine.
Immature rat groups:
Group 1: received 20 mg/kg bw of hydro-alcoholic extract of pumpkin seeds (0.1 ml in each daily
intraperitoneal injection) (27).
Group 2: received 50 mg/kg bw of hydro-alcoholic extract of pumpkin seeds (0.1 ml in each daily
intraperitoneal injection) (27).
Group 3: received 100 mg/kg bw of hydro-alcoholic extract of pumpkin seeds (0.1 ml in each daily
intraperitoneal injection) (27).
Group 4 (Control): received no medicine.
On day 22, after weighing the rats, blood samples were taken directly from their hearts using a 5 cc
syringe (under anesthesia induced by Ketamine hydrochloride and Xylazine). Thereafter, their abdomens
were cut and their livers, kidneys, and ovaries were removed from the surrounding adipose tissue by
scalpel and pence for histological studies.
3.2. Preparation and extraction of plant materials
Extraction was performed using the Soxelle method (28). Dry pumpkin seeds (100 g),
(Cucurbita maxima genus, “Cucurbitaceae” family, and C. maxima [danhobak] species)
(4, 5) were comminuted and combined
with 500 mLof 80% ethanol and stored in a percolator. Finally, the extract drops were collected after
three days. When all of the drops were collected, 80% ethanol was poured into the machine to clear the
color of extract. Moreover, in order to thicken it, the resulting mixture was passed through a filter
and evaporated in a rotary evaporator at 40°C. In addition, the remaining material was under vacuum
in a desiccator for 24 h to lose its moisture. Afterward, the obtained dry material was weighed and
evaluated its efficiency which indicated that 16 g of dry extract was obtained from 100 grams of pumpkin
seeds powder and the remaining was waste. At last, a 16% pumpkin seed extract was acquired through this
procedure. Finally, the researchers determined lethal, maximum, moderate, and minimum doses of this
3.3. Dose determination
Several various concentrations of pumpkin seed extract were randomly selected and injected in rats (four
experimental groups of 8). The lethal dose 50 (LD50) of a group was found to be 400 mg/kg and the
maximum, moderate, and minimum doses were specified as well. There was 400 mg of pure extract in each
liter and for LD50 determination, 1 ml of it was injected into 5 rats that weighed 200 g. To obtain a
dose of 100 mg/kg bw, the basic extract was diluted four times using normal saline (group 1),
and to provide a dose of 50 and 20 mg/kg bw, the extract was diluted twice and five times using
normal saline, respectively (groups 2 and 3) (27).
3.4. Measurement of plasma biochemical parameters
Bloodsamples were used in order to measure the serum levels of blood urea nitrogen (BUN), creatinine,
uric acid(UA), alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase
(ALP), total protein (TP), bilirubin, albumin (Alb), estrogen, and progesterone, follicle-stimulating
hormone (FSH), and luteinizing hormone (LH). The blood biochemical factors were measured based on the
colorimetric method using biochemical test kits, and Selectra XL
fully-automatedanalyzer (manufactured in Netherlands).
3.5. Microscopic examination of renal, hepatic, and ovarian tissues
After the bleeding ended, the abdomens of the rats were cut, and their kidneys, livers, and ovaries were
separated from their surrounding tissues. Afterward, they were weighed, washed with normal
saline, and stored in a 10% formalin solution for 14 days. For the preparationof microscope slides,
the kidneys, ovaries, and livers were sent to the histology laboratory. The slides were used to study
the tissue since they were separately prepared from different parts of renal and ovarian tissues.
In each kidney, the diameters of the cortex, medulla, glomerular, Bowman capsules, urinary space,
proximal tubule, distal tubule, and Henleloop (in millimeters) were determined. Moreover, each liver was
evaluated regarding congestion, hyperemia, atresia, and mean value of liver tissue destruction which
included disruption of the polarized arrangement of hepatocytes,vasodilation, hyperemia, hepatic
necrosis, Kupffer cell aggregation, inflammatory cell infiltration, and portal space changes.
Furthermore, in each ovary, hyperemia, vacuolization of the cells of ovarian tissue and follicular
atresia, as well as the mean value of primitive, early, secondary, Graafian, atretic,and yellow
follicles were measured by an optical microscope with a magnification of 400X in 10 fields of view (a
total of 50 fields of view for each animal). Thereafter, their mean values were calculated and
the groups were compared (27).
3.6. Statistical analysis
The collected data were analyzed in SPSS software (version 23) using descriptive statistical tests (e.g.,
mean and standard deviation), one-way ANOVA, and Chi-squared test. It should be noted that a p-value of
0.05 was considered statistically significant. Healthy adult and immature female Wistar rats were
included, while the dead rats were excluded from the study.
Effect of hydro-alcoholic extract of pumpkin seeds on:
Comparison of the bodyweight of the rats on the first and last days of pumpkin seed extract injection revealed a
significant increase in the three immature experimental groups, compared to the control group (P<0.001).
However, no significant alterations were observed in the bodyweight of the adult experimental groups
4.2. Serum concentrations of BUN, Creatinine, and UA
There were a significant increase and decrease in the level of creatinine (P<0.001) and UA (P=0.04),
respectively, in the three immature experimental groups, compared to the control group. Moreover, a significant
increase was observed in the UA in the immature experimental groups 1 and 2 in comparison to the control group
(P=0.04) (Figure 1). However, no other significant
changes were found in
the blood test results of the adult (P=0.016) and immature groups (P>0.05) in this regard.
A significant increase was observed in the UA in the immature experimental groups receiving doses of 20 and 50
mg/kg in comparison to the control group (P<0.05). No significant difference was observed between the columns
with at least one letter in common (P>0.05).
4.3. Serum concentrations of ALT, AST, ALP, OT, PT, T.pr.t , and Alb
The ALP in the adult experimental group 3 underwent a significant decline in comparison to the control group
(P=0.023). In addition, there was a significant increase in AST in the immature groups 1 and 2, compared to the
control and the immature group 3 (P=0.04). Furthermore, a significant increase was observed in the total protein
level in the immature groups 1 and 2 in comparison with the control group, while the immature group 3
experienced a significant decline in this regard, compared to the control and other immature groups (P=0.023).
No significant alterations were observed in other blood test results of the adult and immature groups
4.4. Serum concentrations of female sex hormones
According to the results, there was a significant increase in the level of FSH in the adult experimental group 3
in comparison to other adult experimental groups and the control group (P=0.002). Moreover, FSH underwent a
significant increase in the immature experimental groups 1 and 2 in comparison to the control group
(P<0.001). In addition, there was a significant increase in LH in the adult experimental group 3, compared to
the control group (P=0.046).
Based on the results of the estrogen test in different adult and immature groups, there was a significant
increase in the estrogen level of the adult group 3 in comparison to the control group (P=0.003). Similarly, the
estrogen level of immature experimental groups 1 and 2 underwent an increase in comparison to the control group
(P=0.02) (Table 1). Moreover, a significant increase was
observed in the progesterone level in the immature experimental group 3 in comparison to the control group
(P<0.001). However, there was no other significant alteration regarding the other ovarian serum data
4.5. Renal tissues
Based on the results, the pumpkin seed extract significantly decreased the diameters of the renal cortex
(P=0.04), glomerular capsule (P<0.001), and Bowman’s capsules (P=0.023) in the adult experimental
groups 2 and 3 in comparison to the control group. Moreover, a significant decrease and increase were observed
in Bowman’s capsule and urinary space dimensions in the immature experimental groups 2 and 1,
respectively, in comparison to the control group (P<0.001).
There was also a significant increase in the renal diameter in the immature groups 1 and 2, compared to the
control group, while there was a significant diminution in this regard in the immature group 3 in comparison to
the control and other immature groups (P=0.023). Furthermore, the diameters of the proximal tubule (P<0.001),
distal tubule (P=0.008), and Henleloop (P<0.001) underwent a significant diminution in all the immature
experimental groups in comparison to the control group. Additionally, there was a significant diminution in the
diameter of glomerular in the immature groups 1 and 2 in
Figure 1. Comparison of uric acid in the immature rats after the
injection of pumpkin seed extract
Table 1. Comparison of estrogen levels in the adult and immature
groups after the injection of hydro-alcoholic extract of pumpkin seeds
Estrogen levels in the adult group (g/ml)
Estrogen levels in the immature group (g/ml)
* Group 1 (receiving a dose of 20 mg/kg); Group 2 (receiving a dose of 50 mg/kg); Group 3 (receiving a dose of
100 mg/kg); Group 4 (control) (P<0.05).
** No significant difference was observed in the mean values of the rows with at least one letter in common
according to Duncan's test (P>0.05).
comparison to the control (P=0.008). No more significant alterations were observed in other renal tissue results
in the adult and immature groups (P>0.05).
4.6. Hepatic Tissues
Based on the results, the pumpkin seed extract had no significant effect (including hyperemia, inflammatory
cell infiltration, necrosis, vacuolation, clearance of cytoplasm, and portal space changes) on the hepatic
tissues in the adult and immature experimental groups (P>0.05) (Figure 2).
4.7. Ovarian Tissues
According to the findings, pumpkin seed extract was able to significantly increase and decrease the atretic and
secondary follicles, respectively, in the immature groups 1 and 3 (P=0.008), compared to the control group and
immature group 2 (P=0.023). Moreover, a significant increase was observed in the Graafian follicles in the adult
experimental group 3. Besides, there was a significant diminution in the early (P=0.01) and primitive follicles
in the three adult experimental groups in comparison with the control group (P=0.013).
Furthermore, the secondary follicles underwent a significant decrease
Figure 2. Normal structure of liver tissue in the mature experimental group 2
(receiving a dose of 50 mg/kg body weight) in comparison to the control group (hematoxylin-eosin,
in adult group 1, compared to adult groups 2 and 3 (P=0.038) (Table 2). Meanwhile, there was no significant
difference between the mean values of the rows with at least one common letter according to Duncan’s test.
It is noteworthy that no change was observed regarding the other variables (P>0.05).
Table 2. Changes in ovarian tissue variables in immature female rats
after the injection of hydro-alcoholic extract of pumpkin seeds
Changes in ovarian tissue variablesIn
different groups compared to the control group
Group 4 (control)
Ovarian tissue variables
Number of secondary follicles (percent)
Number of atretic follicles
*Group 1 (receiving a dose of 20 mg/kg body weight); Group 2 (receiving a dose of 50 mg/kg body
weight); Group 3 (receiving a dose of 100 mg/kg body weight); Group 4 ( control)
** No significant difference was observed in the means between the rows with at least one common letter according
to Duncan's test (P<0.05).
Cucurbita maxima are cultivated worldwide for both food and medicinal purposes. It is one of the most
popular vegetables that are also affordable and easy to grow (29); in addition, its seeds
have significant quantities. According to the previous research, the highest mean weight of its seed
massperfruit is 110.7 g (30, 31). Pumpkin seeds contain
water (94 g), protein (1.1 g), fat (0.1 g), starch (6.3 g), calcium (28 mg), phosphorus (30 mg), iron
(0.4 mg), potassium (2.2 mg), and vitamins (i.e., A: 400 units, B1: 0.05 mg, B2: 0.09 mg, B3: 1 mg, and
C: 22 mg). They are also good sources of zinc, unsaturated fatty acids, phytosterols
(β-sitosterol), antioxidants, carotenoids, tocopherols, and fatty acids (i.e., palmitic, stearic,
oleic, and linoleic acids) which have positive effects on body tissues.
However, bezoarsare one of the disadvantages of pumpkin seeds. Bezoars are
thedigestive tract.Nevertheless,based on previous research,pumpkin seed extract has had no
adverse effects on the liver and kidney so far (32, 33).
In this study, there was asignificant increase in the body weight in all of the immature
experimental groups due to the nutrient composition of pumpkin seed extract asit fulfilled the
nutritional requirements of the rats at the onset of their sexual maturation when
their body underwent potential changes.
According to previous studies, pumpkin seeds contain phytoestrogens which are
estrogen-like molecules that can have complex effects on non-reproductive behaviors,
such as anxiety and movement. This means that they can exert anxiolytic effects that, in turn, increase
motor activity and exploratory behaviors. These effects are associated with hormone action and sex
steroids. Increased mobility, which can lead to weight loss (33, 34), was observed in the groups
that received moderate and maximum doses of pumpkin seed extract. Moreover, no weight gain was observed
in the adult experimental groups which may be due to the increased mobility; however, this finding
requires further investigation.
The phytoestrogens present inthe humanfoodstuffs are isoflavones that are classified as
flavonoids. Isoflavones are phytoestrogens that are able to bind to estrogen receptors in different organs (35).
Today, synthetic estrogens are used for the treatment of female hormone disorders and infertility. According to the findings of a
study performed by Siano (2016), only certain amounts of synthetic estrogens in special circumstances
can affect the liver (36). According to the findings of another study conducted by Sangiovanni et al. (2020), hepatic dysfunction was observed in women using
estrogen-containing steroids for contraceptive purposes (37).
In the present study, the hydro-alcoholic extract of pumpkin seeds had no harmful effects on the
hepatic tissues in the adult and immature experimental groups, while the estrogen levels significantly
increased in the adult and immature experimental groups 3 and in the immature experimental group 2 in
comparison with the control group. Hence, this extract can be used at the onset of puberty and in the
menstruation period. However, there is a need for further analysis and subsequent tests.
A significant increase was also observed in progesterone in the immature group 3 in comparison with the
control group. However, the adult and immature groups underwent no significant alterations regarding the
albumin level. It should be noted that estrogens and progesterone are transported in the blood by
binding to albumin and specific sex hormone-bindingglobulins. The attachment of the hormones to
this protein are so weak that they are released to the tissues over a period of approximately
30 min (38). Hence, it can be concluded that in the present study, liver
functionwas normal in terms of albumin pathway.
The estrogen found in the contraceptive pills can lead to cholestasis; moreover, this effect can be
exacerbated by the progestin. Furthermore, the consumption of anabolic steroids and androgens
has been associated with the development of hepatocellular adenoma, hepatocellular
carcinoma, and hepatic angiosarcoma (39). The sexhormone
disordersthatreduce testosterone levels and elevate
estradiol levels are symptoms of hepatic cirrhosis (40). Important effects
of estrogens ontheliverinclude cholestasis, liver tumors,
Budd–Chiari syndrome, and dose-dependent jaundice. It should be noted
that medication-induced cholestasisis completely reversible after medication withdrawal
A significant diminution was observed in ALP in the adult group 3 in comparison with the control group.
Since this hepatic serum change was observed only in adult groups, it can be said that pumpkin seed
extract can be the best choice for hormone replacement therapy in adult groups. Moreover, there was a
significant increase in the AST level in immature groups 1 and 2 in comparison with the control group
and the immature group 3. In addition, there was a significant increase in the total protein level in
the immature group 1 and 2in comparison with the control group. Moreover, there was a significant
diminution in the total protein level in the immature group 3 in comparison with the control and the
other immature groups; however, this finding requires further studies.
According to previous studies, the route of estrogen administration may affect its function. More
specifically, the effect of exogenous estrogens on the liver depends on the type and dose of estrogen as
well as the route of administration. Based on previous studies, oral consumption of synthetic estrogen
has a great effect on proteins (plasma and coagulation), lipoproteins, and triglycerides,
whileestradioltopicalhas minimal effect on the liver function.
Clinically, oral steroids are effective for the treatment of prostate cancer; however, they are
associated with severe cardiovascular side effects and changes in livermetabolism (41). Infection, inflammation,
and removal of gallbladder are more common in women taking oral estrogen, probably due to its hepatic effects. Estrogens enhance biliary
cholesterol secretionand saturation, increase cholesterolin bile,
and decrease gallbladder movement through the increase of crystallization in the bile (41
Transdermal (e.g., gels) or subcutaneous (implants) administration of estrogens maintains blood estrogen
as they are transported in the blood without leaving a high concentration of conjugated estrogens in the
liver(44).In this study, the intraperitoneal injection was performeddue to better absorptioncaused by the
high number of arteries and veins in the peritoneal cavity (45).
Results of previous studies have indicated that pumpkin seed drops can be used to treat prostate
to the findings of a study performed by Sun et al. (2018), there are only three types of estrogen in
human plasma (i.e., β-estradiol, estriol, and estrone) whileβ-estradiol is the major
estrogen produced in the ovaries.Moreover, they found that the secretion of estrone is
limited and it ismainlyderived from the conversion ofandrogens secreted by ovarian theca cells
andadrenal gland in peripheral tissues (47).
Witorsch et al. (2016) in their study found that estradiol is metabolized in the liver and converted to
estrone and estriol which are excreted in the urine as glucuronide and sulfate conjugates (48).
Therefore, in addition to the hepatic pathway, its metabolism has a renal pathway. In the present study, renal serum
results indicated a significant increase in the creatinine level and a significant diminution in UA in
the immature group 3 in comparison with the control group. Moreover, there was a significant increase in
UA in immature groups 1 and 2 in comparison with the control group.
Furthermore, Bowman’s capsule and urinary space dimensions underwent a significant decrease and
increase in the immature experimental groups 2 and 1, respectively, in comparison with the control
group. Besides, a significant increase was observed in the renal diameter in the immature experimental
groups 1 and 2 in comparison with the control group. However, the renal diameter underwent a significant
decrease in the immature experimental group 3 in comparison with the control and other immature
According to the results, the diameters of the proximal tubule, distal tubule, and Henleloop underwent a
significant diminution in all the immature experimental groups in comparison with the control group.
Additionally, there was a significant decrease in the diameter of the glomerulus in the immature groups
1 and 2, compared to the control group. No other significant alterations were observed in other
resultsaboutkidney tissues in all of the adult and immature experimental groups (P<0.05).
Based on the results, most of the changes occurred in immature experimental groups. Nevertheless, there
was also a significant diminution in the adult groups regarding the diameters of the renal cortex,
glomerular capsule, and Bowman’s capsule in groups 2 and 3 in comparison with the control group.
According to the results of this study and the review of the literature, it can be said that there is a
need for caution in the use of this extract in immature groups. This may be due to the younger age range
of immature rats in comparison with the adult ones.
The sex hormone activity and body weight were strongly affected by steroids. Moreover, successful
differentiation of follicles pertains to the steroids and follicular growth factors. Growth and
differentiation of ovarian follicles take place via proliferation and differentiation processes of the
granulosa cells (49). Adequate amounts of both FSH and LH are required for oocyte
growthand development and meiotic division (50).
In the present study, FSH significantly increased in the adult group 3 in comparison with the other adult
experimental and control groups. The FSH also increased in immature groups 1 and 2, compared to the
control group. There was also a significant increase in the LH levels in the adult group 3 in comparison
with the control group. It can be said that the extract had a significant positive relationship with
these two hormones (i.e., FSH and LH). Deiman and Chaola (2008) in their study also found that androgen
production increased with the increase of LH, which is in line with the results of the present
study (51, 52). In this research, the estrogen increased in the adult group 3, while the immature groups require more investigation in this
Follicles trigger ovulatory cascade and fatty acidscause the growth
ofGraafianfolliclesand increase their number and size.As previously
mentioned, pumpkin seeds are also a good source of unsaturated fatty acids and flavonoids
(phytoestrogens). It is also noteworthy that estrogen is a potent stimulus of follicular growth in
rodents (53, 54).
According to the results of the present study, there was a significant diminution in the secondary
follicles in adult group 1 and immature groups 1 and 3 in comparison with the control groups. Moreover,
the atretic follicles increased in immature groups 1 and 3; hence, caution is advised regarding the use
of the extract in immature groups. There was also a significant diminution in the early and primitive
follicles in all the three adult experimental groups, compared to the control group, which is
inconsistent with the results of the previous research. This inconsistency may be due to some agents,
such as breed, hormone, and treatment duration.
It has been found that pumpkin seeds also have antioxidant activities. It is necessary to remember that
reactive oxidants in the follicles areessential for ovulation and their ablation hinders proper
ovulation (55, 56). The increase in the
Graafian follicles in the adult experimental group 3 is consistent with the results of a previous study
conducted by Forouzan Fard et al. (57).
Pumpkin seeds also contain calcium (28 mg) and phosphorus (30 mg). Average level of phosphorus in the
blood is 3 to 4 mg per 100 ml. Since each 1 kg of pumpkin seeds contains 30 mg of phosphorus, it enters
the blood faster than calcium and replenishes the reserves of the body. Excessive phosphorus intake
reduces calcium absorption since these two elements compete to enter intestinal cells. Moreover, it
can causetheexcretion of excessiveserum phosphorusin the urine. It is
essential to know the amount of phosphorus and calcium intake to evaluate the effect of pumpkin seeds on
the kidney since phosphorous precipitation in the kidneys can cause renal dysfunction (43). Therefore, future
studiesshould investigate the effect of pumpkin seed extract on phosphorus and calcium intake and
compare the results in adult and immature groups.
In conclusion, it can be said that the nutrients found in pumpkin seeds provide the nutritional needs at
the onset of sexual maturity. They affect female sex hormones and provide conditions for sexual maturity
without any adverse effects on the hepatic tissues and also regulate female sex hormones after puberty.
However, the use of this extract at the onset of sexual maturity requires caution and accurate dosage
determination. Moreover, in adults, it requires caution in terms of renal factors and tissues.
Therefore, further molecular biology studies are suggested in this regard.
Authors’ Contribution: Study concept and design: Samaneh Motamed Jahromi and Sadegh Niami. Analysis and interpretation of data: Samaneh Motamed Jahromi. Drafting of the manuscript: Samaneh Motamed Jahromi. Critical revision of the manuscript for important intellectual content: Samaneh Motamed Jahromi. Statistical analysis: Sadegh Niami.
Conflict of Interests: The authors have no conflict of interest to declare.
Ethical Approval: All of the experiments were reviewed and approved by the Ethics Committee of Hormozgan and Jahrom Universities of Medical Sciences (code of ethics for immature rats groups: IR.HUMS.REC.1398.426 and adult rats groups: jums.REC.1393.071).
Funding/Support: The authors would like to thank the Council for their contribution to this research. This study was part of a research project (Grant No: 970390 and 50/93 ) which was supported and funded by Hormozgan and Jahrom Universities of Medical Sciences.
Informed Consent: Not applicable.
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