influence of proteins extract from helianthus annuus … · influence of proteins extract from...

JPCS Vol(6) ● Jan-March 2013 www.arpapress.com/Volumes/JPCS/Vol6/JPCS_6_02.pdf

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INFLUENCE OF PROTEINS EXTRACT FROM HELIANTHUS ANNUUS

L. SEEDS ON BLOOD VOLUME OF REPRODUCTIVE ORGANS IN

PREGNANT MICE

Muhanad A. A. AlBayaty, Farid J.AL-Tahan & Huda F. Hasan

Dept. of Veterinary Physiological and Pharmacological, Collage of Veterinary Medicine, University of Baghdad.

ABSTRACT

L .arginine-NO pathway has been characterized as domination of blood volume and plays an important role in the

provocation of uterine function. Several sources of L.arginine, Helianthus annuus L. seeds had a great potential

dietary protein rich with L.arginine. The study protocol consist (420) pregnant mice separated into early and late

pregnancy groups each one were divided in to four groups: (1-crude extract of Helianthus annus, 2- partial purified

extracts of Helianthus annus, 3-L.arginine and 4-L.NAME) each 10 pregnant mice given the following dose

(100,150,200,250 and 300mg/kg B.W of crude extracts, purified extracts and L.arginine, L.NAME were given

(50,75,100,125 and 150) mg/kg B.W daily dose intraperitonally, finally normal salin were given to 20 pregnant mice

served as control each 10 for early and late respectively. The results displayed: 1- crude and partial purified protein

extraction of Helianthus annus contained L.arginine 35% and 85,4% respectively. 2- There were gradual

increase in blood volume of ovaries and uterus depend on dose increment and had highly correlated with increase of

hormones (estrogen and progesterone) of both extracts of Helianthus annus and L.arginine treated groups in early

and late pregnancy except in groups treated with dose 300mg/kg B.W. 3-The histology was illustrated the increase

blood vessels and vascular density of uterus and ovaries in each L.arginine and both Helianthus annus extracts

groups during late pregnancy. While in L.NAME groups had decrease in vascular density, blood vessels and micro

capillaries.

1. INTRODUCTION

Helianthus annuus plant is a strategic cultivated plant inIraq and the world (1). Several notions and reportes in

literatures revealed that Helianthus annuus are the seeds richest known source of the vital essential amino acid like

L.arginine (Madhusudhan,et al.,1999). L.arginine is considered as precursor of NO which had potential functions in

the uterus include vasodilatation and suppression of myometrial contractility during pregnancy (3).

The protocol of the present study was designed to shed the spots light on the effect of extracted L.arginine (crude

and purified ) from cheap source Helianthus annuus L , L.arginine and L.NAME on blood volume of pregnant mice

as possibility of maximizing of set points of fertility of breed which can be used as booster of local breeds fertility

characterization with preserving their biological function. This study was aimed to determine of whether differences

in utero-ovarian blood volume and uterine mass under influence of Helianthus annuus L.protein extract offering

various pharmacological profile develop uterine capacity and to examine the relationship between uterine blood

volume and l.arginine containing Helianthus annuus L.in early and late pregnancy.

2. MATERIALS AND METHODS Helianthus annuus L. Extraction and protein precipitation: 1-Preparation of the Helianthus annus seeds:( one

kilogram) were washed and dried then ground by electrical grinder. 2- Defatting of ground flower seeds powder by

petroleum ether in Sochxeltapparatus. 3- Crude protein extract according to method of (4). 4- Partial Purification

of protein to L.arginine according to method of (5 and 6). The concentration of L-arginine in partial purified protein

extract was determined according to (Bremer, et al.,2008 and Mc Donald, et al.,1997).

Experimental animals:

The total number of experiments reached to four hundred and twenty female mice, their weight was about (33-34 )g.

These mice grew up under same suitable condition at 21-24C◦, light (12) hrs. daily and kept in plastic cages were

cleaned daily, and food pellets and drinking water was presented adlibitum.

Protocol of experimental:eachgroups of (L.arginine ,crude extract and L.arginine extract of Helianthus annus )

was divided into five sub groups according to dose adminstration (100, 150, 200, 250and 300) mg/Kg B.W and

group of L.NAME was divided into five doses sub groups (50,75,100,125,150) mg/Kg B.W :

The first group: control group treated orally with normal saline.

The second group: crude extract of Helianthus annus treated orally for (1-6) days in early pregnancy and (7-19)

days in mid and late pregnancy.

JPCS Vol(6) ● Jan-March 2013 Al-Bayati et al. ● Reproductive Organs in Pregnant Mice

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The third group: partial purified containing L.arginine extract of Helianthus annus treated orally for (1-6) days in

early pregnancy and (7-19) days in mid late pregnancy.

The forth group: L.arginine treated orally for (1-6) days in early pregnancy and (7-19) days in mid and late

pregnancy

The fifth group: L.NAME treated intrapretonially for (1-6) days in early pregnancy and (7-19) days in mid and late

pregnancy

EXPERIMENTAL PARAMETERS

Blood volume (in both early and late pregnancy experiments): The blood volume was determined by several

steps according to (7) blood sample was collected by a cardiac puncture at the time after scarified. Blood samples

(1ml) was diluted with 1ml of tris buffer (pH 7.4) and was homogenized and centrifuged. Supernatant was separated

and stored at -18C◦.The sample was thawed and diluted with 4ml Tris buffer (pH7.4). The absorbance was measured

by spectrophotometer, manually scanning of absorbance was done by increase wave length gradually and plotted the

absorbance wave length until sutible curve of blood absorbance was obtained.

Calculation of corrected blood Hb (∆A Two maximal peak (449) and (535) wave extracted from absorbance

curve, corrected absorbance (∆A) was calculated for tissue by the equsion as in figure (5) :

∆A= A449 – A535+A518

2

Figure (1): Absorbance curve of the heart blood, two maximal Peak at (449 and 535) nm, the elimination of the

irrelevant background of absorbance was also measured at 518 nm. This and subsequent figures of wavelength scans

were exact tracings of spectrophotometer printouts.

Step 2-Hb stain standard curve :

100mg of Hb stain was dissolved in 10 ml of distilled water with shaking then diluted to 0.5% before measuring the

absorbance. Calibration curve was done by using serial concentration of Hb. The optical density, the data was

resolved in regression between Hb and optical density, by using simple line regression

0

0.5

1

1.5

2

2.5

3

518

535

449

.

.

.

.

.

.

.

.

.

.

∆A

Hae

mo

glo

bin

Ab

sorb

tio

n (

AO

)

Wave length (nm)500

I550I

600I

650I

700I


10

Figure (2): standard curve of Hb stain.

Step 3-Calibration curve of bloodvolume-Hb stain : 1ml of blood sample from heart was taken, this sample

was diluted with 1ml distilled water before measured the absorbance to give the first dilution, this procedure

repeated frequently until reaching to ten dilutions to give standard curve of heart blood, the diluted blood

samples were measured absorbance, these absorbance were calibrated in certain point in Hb-stain standard

curve (step 2) and extracted Hb concentration , then calibration curve Hb concentration (mg/ml) versus blood

volume was depicted.

Step 4-Ovarian and uterine Blood volume determination:

The ovary and uterus in early and late pregnant mice were excised from the body after two ligation from cervix and

ovarian terminal and blotted on a paper towel to remove blood from the surface, each of the uterus and ovary was

placed in (1ml) 0.05 M (hydroxymethyl) amino methane (tris) buffer (pH 7.4) and homogenized for 30 sec. in glass

homogenizer .The homogenate was centrifuged for 20 min. The supernatant fluids, which comprised each ovary or

uterus were transferred to polyethylene tube and stored at -18C◦ until a complete series of experimental samples

could be studied simultaneously. These samples were thawed and diluted 4-folds with Tris buffer (pH 7.4) before

their absorbance were measured by spectrophotometer. The absorbance value was setting in step 2 to check the

concentration of Hb and calibrated in step 3 to extracted blood volume.

Histological sectioning of ovarian and uterus tissue: Specimens of both ovaryand uterus (five specimens for dose 200mg/kg in each group) of animal were taken after

isolated from the other organs and kept in 10% formalin for fixation, processed routinely in histokinette, and

embedded in paraffin wax which cut at 5micrometer thickness by microtome and staind with haematoxylin and

eosin then examined under light microscope. (8).

Vascular parameters: They were measured according to (9):

1- Vascular density (VD): number of microvessels present in selected area, calculated as:

Micro-vessel number = Microvessel area+ residual stromal area X1000

2- Vascular area ratio (VAR): the area occupied by microvessels within the selected area.

Determined estrogen and progesterone levels: Determination the baseline of circulating serum levels of progesterone and estrogen (ten sample for each dose). The

hormonal measurements were done in each of treated and control pregnant mice groups. The quantitative analysis

was done in clinical Laboratory of Radio Active Isotope (Specific Al-Huda Laboratory).

Statistical analysis:

The ready program SAS (2001) was used in statistical analysis for study the control and treated groups was subject

to analysis two way of variance. A probability of P<0.05 was assumed to denote significant differences. LSD test

was used for comparison between groups and stages

Result Extraction: The sunflower seeds of Helianthus annus were extracted to yield approximately 10% of total solid and

the results crude extract contained 35% L.arginine and partial purified extract contained 85% of L.arginine,

Blood volume in uterus and ovaries: The blood volume showed in tables(1 and 2) significant (P<0.05) increase in

y = 0.280x + 0.054

R² = 0.986

0

0.5

1

1.5

2

0 1 2 3 4 5 6

abso

rban

ce (

AO)

Heamoglobin stian concentrations (mg/ml)

y=a+bx


11

early and late pregnancy periods as compared with the control group, on the other hand, L.NAME groups displayed

a significant (P<0.05) decrease as compared with control groups, In other wise results displayed falling blood

volume rate in dose 300 mg\Kg during late period of pregnancy.

Table (1): The effect of L.arginine, crude extract , partial purified protein extract of Helianthus annuus seeds and

L.NAME in uterine Blood volume.(ml/gm) in pregnant mice with different doses during early and late periods of

gestation.

(1:A): In early pregnancy

(1:B): In late pregnancy

*Total number animals 10 / dose .

*Different capital letters mean significant difference (P<0.05)between column numbers.

*Different small letters mean significant difference (P<0.05)between row numbers.

*The control group in early pregnancy was same in all groups and also in late pregnancy

Groups

Doses

mg/kg

L.arginine treated

group

Helianthus annuus

crude extract

treated group

Helianthus annuus

partial purified

extract treated

group

Doses of

L.NAME

L.NAME treated

group

0 0.00238±0.0005 Aa 0.00238±0.0005Aa 0.00238±0.0005Aa 0 0.00238±0.0004Aa

100 0.00365±0.0003 Ba 0.00244±0.0004Ab 0.00311±0.0002Ba 50 0.000430±0.5495Bc

150 0.00539±0.0002 Ca 0.00341±0.0002Bb 0.00420±0.0001Cc 75 0.000147±0.0248Cd

200 0.01221±0.0002 Da 0.00726±0.0004Cb 0.01164±0.0001Da 100 -0.00198±0.0002 Dc

250 0.01208±0.0019 Da 0.01192±0.0003Db 0.01199±0.0001Dba 125 -0.00182±0.0001 Dc

300 0.01195±0.0001 Da 0.01047±0.0002Da 0.01138±0.0027 Da 150 -0.00176±0.0002 Db

Groups

Doses

mg/kg

L.arginine treated

group

Helianthus annus

crude extract

treated group

Helianthus annus

partial purified

extract treated

group

Doses of

L.NAME

L.NAME treated

group

0 0.02105±0.0004 Aa 0.02105±0.0004Aa 0.02105±0.0004Aa 0 0.02105±0.0004 Aa

100 0.02459±0.0003 Ba 0.02168±0.0002Bb 0.02292±0.0026Bc 50 -0.00530±0.0002Bd

150 0.03827±0.0002 Ca 0.02515±0.0001Cb 0.02720±0.0002Cc 75 -0.00196±0.0001 Cd

200 0.09091±0.0002 Da 0.00726±0.0004Cb 0.08903±0.0001Da 100 -0.00294±0.0002 Dc

250 0.09077±0.0001 Da 0.03403±0.0002Db 0.09070±0.0003Da 125 -0.00272±0.0002 Dc

300 0.08836±0.0003 Ea 0.09064±0.0005Eb 0.08796±0.0002Aa 150 -0.00286±0.0004 Dc


12

Table (2) The effect of L.arginine , crude extract , partial purified protein extract of Helianthus annus seeds and

L.NAME in ovaries Blood volume(ml/g) in pregnant mice with different doses during early and late periods of

gestation.




*The control group in early pregnancy was same in all groups and also in late pregnancy.

Groups

Doses

mg/kg

L.arginine treated

group

Helianthus annus

crude extract

treated group

Helianthus annus

partial purified

extract treated group

Doses of

L.NAME

L.NAME treated group

0 0.00032±0.0002 Aa 0.00032±0.0002Aa 0.00032±0.0002 Aa 0 0.00032±0.0002 Aa

100 0.00124±0.0012 Ba 0.00057±0.0002Bb 0.00097±0.0001 Bc 50 -0.00530±0.0002 Bd

150 0.00499±0.0016 Ca 0.00169±0.0001Cb 0.00432±0.0011 Cc 75 -0.00196±0.0013 Cd

200 0.00914±0.0011 Da 0.00580±0.0016Db 0.00898±0.0014 Da 100 -0.00294±0.0017 Dc

250 0.00893±0.0017 Da 0.00818±0.0021Ea 0.00861±0.0014 Da 125 -0.00242±0.0011 Db

300 0.00903±0.0001 Da 0.00803±0.0001Ea 0.00836±0.0018 Da 150 -0.00216±0.0017 Db

Groups

Doses

mg/kg

L.arginine treated

group

Helianthus annus

crude extract

treated group

Helianthus annus

partial purified

extract treated

group

Doses of

L.NAME

L.NAME treated

group

0 0.00218±0.0005 Aa 0.00218±0.0005Aa 0.00218±0.0005Aa 0 0.00218±0.0005 Aa

100 0.00263±0.0011 Ba 0.00250±0.0002Bb 0.0025±0.0002 Bc 50 -0.00345±0.0001Bd

150 0.00478±0.0014 Ca 0.00301±0.0016Cb 0.00405±0.0002Cc 75 -0.00388±0.0001 Cd

200 0.05689±0.0022 Da 0.01100±0.0017Db 0.05598±0.0026Da 100 -0.00462±0.0011 Dc

250 0.05650±0.0011 Da 0.05570±0.0013Eb 0.05584±0.0045Da 125 -0.00430±0.0001 Dc

300 0.05300±0.0071 Ea 0.09064±0.0005Eb 0.08796±0.0002Aa 150 -0.00286±0.0004 Dc


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progesterone and estrogen hormones during early and late period of pregnancy mice :The result in (tables 3

and 4) showed that levels of progesterone and estrogen increased significantly (P<0.05) in treated groups (L.arginine

and both extracts) during early and late period of pregnancy as compared with the control groups in addition the

estrogen controversially was gradual decrease in as flow up of increase dose, while L.NAME groups showed a

significant (P<0.05) decrease of estrogen and progesterone hormones as compared with the control, L.arginine and

both extracted groups.These results showed a significant (P< 0.05) increase in estrogen hormone in dose 300mg/kg

of Larginine and purified extract during late period of pregnanc.y

Table (3): The effect of L.arginine , partial purified extract and L.NAME on progesterone hormone (Pg/ml) with

different doses in early and late period of pregnancy:



* Different capital letters mean significant difference (P<0.05)between column numbers.


*The control group in early pregnancy was same in all groups and also in late pregnancy.

Groups

Doses

mg/kg

L.arginine treated

group

Helianthus annus partial purified

extract treated group

Doses of

L.NAME

L.NAME treated group

0 1.93±0.08Aa 1.93±0.08Aa 0 1.93±0.08Aa

100 2.00±0.34Ba 1.95±0.29Ab 50 0.71±0.15Bc

150 2.37±0.44Ca 2.10±0.51Ba 75 0.26±0.61Bb

200 2.68±0.63Da 2.30±0.31Ba 100 0.09±0.36Cb

250 2.70±0.60Da 2.40±0.44Ba 125 0.03±0.62Cb

300 2.85±0.37Da 2.5±0.52Ba 150 0.01±0.04Cb

Groups

Doses

mg/kg

L.arginine treated

group

Helianthus annus

partial purified

extract treated

group

Doses of

L.NAME

L.NAME treated

group

0 2.93±0.17Aa 2.93±0.17Aa 0 2.93±0.17Aa

100 2.98±0.08Aa 2.95±0.07Aa 50 0.45±0.31Bb

150 3.65±0.15Ba 3.50±0.27Ba 75 0.10±0.45Bb

200 5.94±0.37Ca 4.8±0.11Ca 100 0.06±0.50Cb

250 5.97±1.81Ca 4.9±0.98Ca 125 0.03±0.91Cb

300 1.8±0.33Da 1.7±0.61Da 150 0.02±0.09Cb


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Table (4):The effect of L.arginine , partial purified extract and L.NAME on estrogen hormone (Pg/ml) with different

doses in early and late period of pregnancy:



Angiogensis and vascular density:The results appeared an increase of angiogenesis, and vascular density in uterus

and ovaries listed in (tables 5 and 6) respectively.

Table (5): The effect crude and partial purified protein of Helianthus annus, L.arginine and L.NAME on blood

vessels (angiogenesis) during late pregnancy:

Groups

Doses

mg/kg

L.arginine treated

group

Helianthus annus

partial purified

extract treated

group

Doses of

L.NAME

L.NAME treated

group

0 4.63±0.38Aa 4.63±0.38Aa 0 4.63±0.38Aa

100 8.96±1.30Ba 8.50±1.62Ba 50 1.7131±2.70Bb

150 8.25±1.16Ba 8.00±0.99Ba 75 0.63±1.30Cb

200 6.07±0.84Ca 6.30±0.74Ca 100 0.23±0.98Db

250 5.90±5.90Ca 6.20±6.09Ca 125 0.08±0.53Db

300 5.26±0.20Ca 5.50±0.64Ca 150 0.03±0.71Db

Groups

Doses

mg/kg

L.arginine treated

group

Helianthus annus

partial purified

extract treated

group

Doses of

L.NAME

L.NAME treated

group

0 2.06±0.21Aa 2.06±0.21Aa 0 2.06±0.21Aa

100 5.38±1.00Ba 4.9±2.16Ba 50 0.49±1.73Bb

150 5.01±0.92Ba 4.5±0.88Ba 75 0.11±2.18Cb

200 4.71±0.67Ca 4.1±0.85Ba 100 0.04±0.72Db

250 4.0±1.08Ca 4.0±0.99Ba 125 0.02±0.74Db

300 9.5±0.31Da 9.3±0.61Ca 150 0.01±0.90Db

Control treated group L.NAME

treated

groups

L.arginine

treated group

Partial

purified

extract treated

group

Crude extract

treated group

Blood vessels

7.16 X106

±

5.81Ad

3.87 X 106

±

0.10Ac

26.61 X106

±

5.21Ab

27.04 X106

±

1.80Ab

23.2 X106

±

2.59Aa

Uterine area

6.26 X 106

±

d 1.88B

2.64 X 106

±

0.20Bc

18.80 X 106

±

0.93Bb

16.51 X 106

±

0.65Bb

14.8X106

±

2.07Ba

Ovarian area


15

Table (6): The effect crude and partial purified protein of Helianthus annus, L.arginine and L.NAME on vascular

density during late pregnancy

Figures (3): The uterine Cross section (B) of L.arginine treated groups, (C) of partial purified extract of Helianthus

annus treated group and (D) , crude extract of Helianthus annus treated groups display distribution of blood vessel

and micro blood vessel in endometrium and myometrium and branches with high density of vascularization, and

showed different of micro capillary of arterioles of endothelium cells. Showed flatten cell contain RBCs with

association invasive pattern of endothelial cell to new formation arterioles. In (A) control treated group showed

normal distribution of blood vessel and less vascularization density while in (E) L.NAME treated groups showed

decrease blood vessel in endometrium and very low of vascular density . (Heamatoxiline and Eosin).

A X225

B X225

Control treated

group

L.NAME

treated

groups

L.arginine

treated group

Partial purified

extract treated

group

Crude extract

treated group

Vascular

density

7.59±1.36d

3.67±0.12c 23.60±2.58b 21.81±1.89b

14.58±1.62a

Uterine

area


16

C X225

D X225

E X 225


17

Figures (4): A cross section in ovary A2) represented L.ar ginine treated group , (B2) partial purified extract of Helianthus

annus treated group and (C2) crude extract of Helianthus annus treated group display distribution of blood vessel and micro

blood vessel and branches with high density of vascularization , and showed different of micro capillary of arterioles showed

flatten cell contain RBCs with association invasive pattern of endothelial cell to new formation arterioles and (D2)

represented control treated group indicated normal distribution of blood vessel while (E2) L.NAME treated group showed

decrease in blood vessel and vascularization density to induced sinusoid (Heamatoxiline and Eosin).

A2 X225

B2X 225

C2X 225


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D2X 225

E2X 225

DISCUSSION The extraction: the results of crude extract and partial purified extract agreed with those reported by Pearce, (4).

The blood volume:The results of increase blood volume of uterus and ovaries in both proteins extract and

L.arginine treated groups this might be attributed to three important mechanism of L.arginine-NO pathway: 1-

stimulation of β-adrenergic receptors in the uterine vascular beds which reduced basal and agonist-stimulation

inositol phosphate production through activation of K+ channels, associated with increasing cGMP and beside that

the production of cGMP may be due to increase in nitric oxide as a metabolite of L.arginine, all these led to produce

an increased in uterine blood volume this result agreement with (10).

2- regulate cytosolic [Ca++

] lead to the vasodilation increase blood volume was demonstrated by (11).

3-increase progesterone hormone might regulate the cGMP effector system for relaxation in both the myometrium

and blood vessels led to increase blood demined through enlarging the blood vessel capacity then blood volume

(12) .

The gradual increase in blood volume of uterus and ovaries of crude extract indicated that Helianthus annus seeds

might be due to nutritional quality (13) and their potential as dietary protein sources in animal feed is well

recognized (14).Helianthus annus seeds also contained lecithin, tocopherols, carotenoids, waxes and high vitamin E

content (15).

The falling blood volume rate in dose 300 mg\Kg during late period of pregnancy, these results might be explained

by accumulation of free radical (nitrite) in blood by an extremely increase in oxidation of NO to N2O3 due to over

dose causing tissue injury, toxic effect and abortion, these explained agreement with (16). other studies showed a

proportional relationship between abortion and rise of estrogen level and decrease progesterone which agreement

with our result in dose 300mg/kg of Larginine and purified extract during late period of pregnancy(17).

The decrease in blood volume of dose 300mg/kg of crude extract during late periods of pregnancy might be

attributed to the presence of other compounds as like trypsin inhibitor (anti-nutritive factor) had been observed in

Helianthus annus seedsthat presumably might be effect although the ratio of this inhibitor was low, (18).

The decrease in blood volume of uterine and ovaries during early and late pregnancy of NG-nitro-L-arginine methyl

ester L.NAME groups this might be due to direct effect of (L-NAME) on NOS inhibition which prevent conversion

of L-arginine to citrullin which neglected NO relaxing effect lead to which provoke PGF2α that led to stimulate Ca++


19

entry into smooth muscle cells through L-type Voltage-operated Ca channel (VOCCs) and increase in the sensitivity

of contractile apparatus to Ca (19)

The progesterone and estrogen hormone: The high progesterone hormonal level in both protein extracts and

L.arginine treated groups might be attributed to NO decrease prostaglandin F2α (PGF2α)-induced inhibition in

progesterone in ovaries by block the receptors of PGF2α in granulosal cell which appear to mediate the luteolytic

actions of this hormone, attenuating the fall in the serum progesterone concentration (20).

During early pregnancy estrogen might elevate eNOS expression in the myometrial tissues via estrogen receptor

ERα and/or ERβ, and that this elevation of eNOS may play some roles in the maintenance of the pregnancy, this

might explained the increase which happened in estrogen (21).

The gradual decrease in estrogen in treated groups might be attributed to that NO donors inhibited both estradiol

secretion and aromatase activity. Further, aromatase activity in microsomal of granulosa cells was inhibited by

native NO. (22)

The changes of L-NAME groups result caused a decrease in progesterone and estrogen level might be due to

impaired NO production which provoke PGF2α stimulant in order to PGF stimulation led to stimulate Ca++

entry into

smooth muscle cells through L-type Voltage-operated Ca channel (VOCCs) and decrease in progesterone hormone

(19).

Histophysiology: The increase angiogenesis parameter might be attributed to mechanisms associated with

L.arginine-NO pathway was enhanced endothelial cell proliferation, perhaps by increasing the expression of

vascular endothelial growth factors, this pathway also enhances endothelial migration by stimulating endothelial

cells podokinesis, enhancing the expression of α and β receptors and increasing dissolution of the extracellular

matrix via the basic fibroblast growth factor–induced upregulation of urokinase-type plasminogen activator (23).

The increased vascularity of dominant follicles which stimulated by L.arginine-NO pathway attributed to increase

serum gonadotropins (24) in addition to positive relation of uterine and ovarian blood volume (25).

The lowest angiogenesis and vascular density in Helianthus annus crude extract this might attributed to presence of

anti-nutritive factor and ratio of unsaturated fat like trypsin that decrease the blood volume and decrease corpus

luteum angiogenesis.

The decrease of uterine and ovaries angiogenesis in L.NAME groups might be attributed to inhibition of the NOS

which had a stimulatory role of NO in angiogenesis and blocking of vasoactive molecules such as substance P and

prostaglandin E1 (Ziche, et al., 1994).

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