JPCS Vol(6) ● Jan-March 2013

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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. 8

JPCS Vol(6) ● Jan-March 2013

Al-Bayati et al. ● Reproductive Organs in Pregnant Mice

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

3

449 518

Haemoglobin Absorbtion (AO )

2.5

535 . . . . . . . . . .

2

1.5

∆A

1

0.5

0

I 500

I 550

Wave length (nm)

I 600

I 650

I 700

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

9

JPCS Vol(6) ● Jan-March 2013

Al-Bayati et al. ● Reproductive Organs in Pregnant Mice

y=a+bx

absorbance (AO)

2 1.5

0.054x + 0.280y = 0.986= ² R

1 0.5 0 0

1

2

3

4

5

6

Heamoglobin stian concentrations (mg/ml) 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