الفهرس 
Review of literatureOnly 14 pages are availabe for public view
Abstract
Polycystic ovary syndrome (PCOS) is the most common
endocrine disorder among women of fertile age, which causes
anovulation in animal and women. The prevalence of PCOS varies
between 13.5 and 26.5% of population. Its hyperandrogenic
manifestations include menstrual irregularity, acne, hirsutism and
oligo-ovulation/anovulation. Metabolic abnormalities such as
dyslipidemia and insulin resistance that persist in PCOS are
responsible for making the patient more prone to diabetes, obesity,
cancer, infertility and coronary heart diseases. PCOS is contributed
to abnormal follicles, hyperglycemia, oxidative stress, and altered
sex hormones (luteinizing hormone (LH), follicle stimulating
hormone (FSH), estrogen (E2), progesterone (PROG), and
testosterone) levels.
Letrozole, a non-steroidal aromatase inhibitor produces a
PCOS model which in numerous ways depicts human PCOS. It
blocks conversion of testosterone and androstenedione to estradiol
and estrone respectively and simulates PCOS like condition by
causing hormonal imbalance, circulating hyperandrogenism and
intra ovarian androgen excess leading to appearance of polycystic
ovary. Follicular atresia and abnormal follicular development is
observed due to induced elevation of androgen levels inside the
ovary.
Licorice roots (Glycyrrhizae glabra) are known as medicinal
herb that considered as a medicine and a flavoring agent to disguise
the unpleasant flavor of other medications. Besides its main active
component, glycyrrhizic acid, licorice also consists of high amount
of flavonoids, saponins, triterpenes, isoflavonoids and chalcones.
Glycyrrhizic acid is used for herbal treatment of various diseases due to its anti-inflammatory, neuro-protective, anti-carcinogenic
and antiviral features. Isoliquiritigenin (ISL), chalcone and
liquiritigenins (LTG’s) are the flavonoids in the structure of
licorice. While ISL has anti-inflammatory, antioxidant, antitumor
activities and liver protective effect against oxidative stress, LTG
has estrogenic effect.
Marjoram (Origanum majorana L.) is a member of the mint
family, Lamiaceae. Typically, products identified as marjoram are
the dried leaves and flower tops of marjoram, which is found
throughout the world. It contains phenolic terpenoids, flavonoids,
tannins, phenolic glycosides, sitosterol, protein and steroids. The
antiviral, bactericidal, antiseptic and antifungal effects of marjoram
are attributed to ursolic acid and essential oil.
Date Palm pollen (DPP) (Phoenix dactylifera L. Palmae) is a
useful traditional medicinal plant. Fresh pollen consists of water (5-
36%) and solids (64-95%). Palm pollen ingredients contain various
vitamins (e.g. vitamin A, E and C) as well as elements (e.g. zinc,
copper, selenium, cobalt, iron, nickel and manganese); it also
contains essential and non-essential amino acids, fatty acids,
flavonoids, estradiol, beta sitosterol, estrone, tri-terpenoidal,
saponins, and a crude gonadotrophic substance. Palm pollen in
traditional medicine has been recommended to treat infertility and it
was used in ancient Egypt to treat infertility in women.
The present study was conducted to investigate the effect of
oral administration of aqueous extracts of licorice, marjoram, date
palm pollen and their mixture against the effect of metformin on
letrozole-induced PCOS in rats.
Eighty five healthy adult virgin female white albino rats
weighing 160±10gram were maintained on standard commercial pellets diet and tap water ad libitum, and kept individually in
stainless cages in constant condition for one week. After
acclimation period the two normal estrous cycles of eighty five
female rats have been determined to ensure their regular
menstruation cycle.
After two weeks of estrous cycle follow up, seventy rats were
induced PCOS by administration of letrozole (1mg/kg body weight)
dissolved in 1% CMC and given orally for 21 days. The remaining
fifteen rats were administered CMC orally and considered as
healthy control group. On the other hand, the length of phases of
estrous cycle was determined for all rats along the periods of
letrozole administration. At the end of 21days, blood samples were
collected to determine testosterone, LH, FSH, progesterone,
estrogen (E2) and insulin hormones in addition to perform
microscopic examination of ovaries to ensure the prevalence of
PCOS.
After prevalence of PCOS seventy rats (60 PCOS rats+ 5
healthy rats) were used in the experimental animals and divided into
seven groups each group consisted of 10 rats as follow:
group 1, Healthy control (-ve control): Healthy rats administered
saline solution by gastric tube daily.
group 2, PCOS control (+ve control): Rats induced PCOS and
received saline solution orally.
group 3, PCOS + Licorice roots: Rats induced PCOS and
administered licorice extract (300mg/kg b.wt./day) orally.
group 4, PCOS + Marjoram: Rats induced PCOS and
administered marjoram extract (300mg/kg b.wt./day) orally. group 5, PCOS +Date palm pollen: Rats induced PCOS and
administered date palm pollen extract (300mg/kg b.wt./day) orally.
group 6, PCOS + Mixture of herbs: Rats induced PCOS and
administered mixture of licorice, marjoram and date palm pollen
extracts by a ratio (1:1:1) at a dose (300 mg/kg b.wt.) given orally.
Group7, PCOS +Metformin: Rats induced PCOS and
administered metformin (200mg/kg b.wt./day) dissolved in distal
water and given orally.
Food intake was recorded daily. The animals were weighted
weekly to monitor body weight change and FER. As well as the
length of phases of estrous cycle was determined along the period
of experiment. Rats were scarified after 12hours fasting with water
ad libitum. Blood samples were collected from hepatic portal vein.
The ovaries were separated immediately, and weighted, then part of
the ovary specimens were taken and fixed in formalin 10% for
microscopic examination.
At the end of experiment the following nutritional, biological
and biochemical parameter were measured:
Licorice roots, marjoram and date palm pollen were
chemically analyzed for their total phenolic, total flavonoids
and total antioxidant capacity contents.
Estrous cycle phases length.
Food intake, body weight gain, and feed efficiency ratio.
Relative ovaries weight.
Serum sex hormones (LH, FSH, LH/FSH ratio, progesterone,
estrogen and total testosterone) levels.
Serum glucose, insulin and HOMA-IR levels.
Erythrocyte and ovarian GSH levels. Serum MDA levels and catalase activity.
Ovarian MDA levels.
Ovarian caspase3-protein level.
Serum lipid profile (TC, TAG, HDL-C, LDL-C, and VLDLC)
levels.
In addition, microscopic examination of ovaries was
performed.
The results of the current study are summarized as follow:
(1)Total phenolic, total flavonoids and total antioxidant
capacity of licorice roots, marjoram, and date palm
pollen aqueous extracts:
Total phenolic content of licorice roots, marjoram, and date
palm pollen was 110.5, 151.2, and 40.1 mg GAE/g, respectively.
Total flavonoid content was 212, 190, and 67mg CE/g, respectively.
As well as, total antioxidant capacity was 85.88mg%, 90.48mg%,
and 85.51mg%, respectively.
Data showed that total phenols and total antioxidant contents
were higher in marjoram than licorice roots and date palm pollen;
on the other hand total flavonoids were higher in the licorice roots
as compared to marjoram and DPP.
(2) Estrous cycle and its phase’s detection:
The present data represented a marked increase in estrous
cycle and its phases (proestrus, metaestrus and diestrus) with
disappearance of estrus phase that indicated irregular estrus
cyclicity in PCOS rats due to the alteration of steroid hormones,
which regulate ovarian function. While, an administration of
licorice roots, marjoram, date palm pollen, mixture of herbs, and metformin caused significant decrement in estrous cycle and its
phase’s caused regulation of estrus cyclicity. Herbal extracts
contain estrogen-like compounds that aid in hormonal regulation
and controlling estrous cycle.
(3)Food intake, body weight gain, feed efficiency ratio
and relative ovaries weight:
Letrozole administration cause significant increase in daily
food intake, body weight gain, and relative ovaries weight.
Moreover administration of licorice roots, marjoram, date palm
pollen, and their mixture as well as metformin caused significant
reduction in daily food intake, body weight gain, and relative
ovaries weight. Herbal treatments were more effective than
metformin drug. Mixture of herbs has the best effect in reducing
food intake (787.96g) and relative ovaries weight (0.045%), while
DPP extract has the best effect in reducing body weight change
(70.7g). The reduction of body weight through licorice, marjoram,
DPP, and their mixture consumption may probably due to a
combination of enhanced lipolysis and inhibition of lipogenesis,
which also contributes to the reduction of hyper-insulinemia.
(4) Hormonal status measurements:
PCOS control group showed hormonal disturbance that was
manifested by marked elevation in serum LH, LH/FSH ratio and
testosterone levels. As well as there were marked depletion in
serum FSH, PROG, and E2 levels.
Current results proved the androgen-lowering effects of
licorice roots, marjoram, DPP, mix of these herbs and metformin
that were manifested by marked elevation of serum FSH, PROG
and E2 levels. Moreover, they caused marked depletion in serum LH, LH/FSH ratio, and testosterone levels. Herbal treatments were
more effective than metformin drug. Mixture of herbs has the best
effect in increasing serum levels of FSH)0.55mlu/ml),
E2(67.59pg/ml), and PROG(19.47ng/ml) and decreasing serum
level of total testosterone(1.53 ng/ml), while DPP has the best effect
in decreasing serum levels of LH(0.55mlu/ml) and LH/FSH
ratio(1.03). These herbs contain flavonoid and alkaloid compounds
that have estrogenic activity and could activate aromatase enzyme
and prevent the conversion of estrogen to testosterone hormone.
(5) Serum glucose hemostasis status:
It was noticed that there was significant elevation in serum
fasting glucose, insulin and HOMA-IR levels in PCOS control
group as compared to healthy group.
The present study demonstrated the hypoglycemic effect of
licorice roots, marjoram, DPP, mix of herbs and metformin as clear
by the reduction of glucose, insulin and HOMA-IR levels. Herbal
extracts were more effective than metformin in regulation of
glucose hemostasis. Licorice root has the best effect in reducing
serum levels of glucose (87.76mg/dl), insulin (9.21ldμIU/ml) and
HOMA-IR (1.99). Herbal extracts not only decreases glucose
production but also increases target tissue sensitivity to insulin. This
may be caused by inhibiting 11β- hydroxysteroid dehydrogenase
type 1 (11β-HSD 1), which in turn decreases the hexose-6-
phosphate dehydrogenase (H6PDH) activity and glucocorticoid
production resulted in decrease the conversion of oxaloacetate into
phosphoenolpyruvate and carbon dioxide in the gluconeogenesis
pathway. 6) Antioxidant status and lipid peroxidation
measurements:
Rats induced PCOS showed disturbance in antioxidant
enzymes including erythrocyte and ovarian GSH levels and serum
catalase activity that can be inferred by the reduction of their levels
in PCOS group. Also, there was an increase in lipid peroxidation
that can be reflected by increasing serum and ovarian MDA levels
as consequence of PCOS.
Administration of licorice roots, marjoram, DPP, and mix of
herbal extracts maintained the antioxidant status and lipid
peroxidation that were manifested by the marked increase in
erythrocyte and ovarian GSH levels and serum catalase activity and
a marked decrease in serum and ovarian MDA levels. Mixture of
herbs has the best effect in increasing erythrocyte GSH
(18.04mg/dl) and serum CAT (711.95 U/L) activity and reducing
serum level of MDA (69.34mmol/L). Marjoram extracts has the
best effect in increasing ovarian levels of GSH (36.17mg/g tissue)
and reducing ovarian MDA (141.31nmol/g tissue). These herbs
contain compounds that have antioxidant capacity to scavenge ROS
and free radical. Herbal flavonoids have been attributed to their
antioxidant properties through several possible mechanisms, such as
their ability to scavenge free radicals, break radical chain reactions,
directly reducing peroxides, and stimulating the anti-oxidative
defense enzyme activities.
(7) Ovarian caspase3-protein:
PCOS control rats showed prevalence of apoptosis in ovarian
cells which detected by the elevation of ovarian caspase3- protein
level. The anti-apoptotic effect of licorice roots, marjoram, DPP,
mix of herbs, as well as metformin appeared as a reduction of
ovarian caspase3- protein level as compared to PCOS control
group. The present results revealed that herbal administration were
more effective than metformin due to their high antioxidant
contents. The best reduction in ovarian caspase-3 protein
(4.17pmol/L) level was in mixture of herbs group.
(8)Lipid profile measurements:
Disturbance in serum lipid profile was illustrated in PCOS
control group which detected by the elevation of serum TC, TAGs,
LDL-C, and VLDL-C levels with reduction of HDL-C level. The
present study proved the hypolipidemic effect of licorice roots,
marjoram, DPP, mix of herbs, and metformin, as their
administration caused significant decrease in serum TC, TAGs,
LDL-C, and VLDL-C levels, and noticeable elevation of HDL-C
level. Mixture of herbs has the best effect in increasing serum level
of HDL-C (55.99mg/dl) and reducing serum levels of TC (157.2
mg/dl) and LDL-C (80.78 mg/dl), while marjoram extract has the
best effect in reducing serum levels of TAGs (100.57 mg/dl) and
VLDL-C (20.11 mg/dl).
Phytosterols present in herbs are well known for their ability
to inhibit absorption of cholesterol and lowering of serum
cholesterol by preferential uptake in the gut for plant sterols versus
cholesterol, and improving elimination of cholesterol.
(9) Microscopic examination of ovaries samples:
Ovaries section from PCOS-control rats showed marked
ovarian cyst, congestion and necrosis of interstitial blood vessel and
interstitial inflammatory cells infiltration also vacuolation of lutein cells of multiple corpora lutea. On the other hand, administration of
licorice roots, marjoram, DPP, mix of herbs, and metformin showed
strands of fine fibroblasts proliferation in between the follicles and
show numerous follicles of different types and multiple corpus
luteum. The therapeutic effects of herbal extracts were
characterized by the restoration of estrus cyclicity, the reduction of
blood glucose level and oxidative stress as well as the improvement
of lipid profile and sex hormones.