Evaluate the Influence of Eupatorium adenophorum Extract with Mice Organ

In order to study the influence of extract from Eupatorium adenophorum in mice organs, this experiment will be the basis of further study that make Eupatorium adenophorum become Phyto contraceptive, this experiment take the feeding respectively way after the completion of the 1D, 5D, 10d, 15d of Eupatorium adenophorum mice by intragastrical administration of levonorgestrel group and blank control group. After the same operation in different periods of small rat heart and kidney the uterus, testis, and other organs were observed. The results showed that after extraction of E. adenophorum changes in female mice uterus shape was perfused significantly, showed swelling larger. Data analysis of each viscera coefficient was found E. adenophorum had No obvious effect on the heart, kidneys and testicles of mice. but there are obvious differences date between the treatment group and the blank group. (5d: F=10. 800 P=0. 043 cases) from tissue sections we can see female mice uterus cell morphology changes significantly, there was a similar appearance change in the uterus of the female mice with the estradiol For a male mouse testis of E.adenophorum gavage had No obvious effect. And it is found that the heart, the treated mice kidney, testis, ovary and other organs were observed in each period of time the organization had No obvious change; only female mice uterus tissue sections of individual cells became larger, and the organization of the gap larger. This research shows that E.adenophorum extract has the potential to develop botanical contraceptives, we will conduct in-depth study.


Introduction
The development of agriculture, forestry and animal husbandry are intimidated by plague mice around the word. In recent years, there are 144 million farmers who and 35 million hm2 areas were suffered rat harm each year in China. In addition, the 47 hm2 forest of Northeast China, North china and Northwest China was destroyed. Meanwhile, grassland rodent was increasing. Especially, the mouse can spread many diseases so that it threatened human health all the time. According to statistics, a total of nearly three hundred million people died of plague, it harms far beyond the war. Therefore, how to control the population of rats has become a common problem in the world. In general, there are three methods to control the mice population: physical, chemical and biological. The physical methods mostly used rattraps or mousetraps, but those equipment are suitable for indoor is Not suitable for large area in the outdoor. It's not only will cause some more damage to the environment, but also do harm to other animals by chemical methods [1][2][3]. Besides, the chemical can stimulate the rapid propagation of murine, and even recovery the original population number in a short time. At last, the biological methods mainly lead into the natural enemies which has a certain function in a low population density, but it doesn't has much effects when the rats outbreak. Using the above methods have Not achieved remarkable results, so we need change the method and a research direction is murine chemo sterilant. At present, there are some plant rodenticide, such as cabergoline, Tripterygium wilfordii and so on. This study is conducted to observe the effect of E. adenophorum extract on each organ in mice and then evaluating the probability of becoming rodenticides or mice chemo sterilant.

Experimental animal
Fifty-day old mice (female 32, male 16) each are healthy. They were purchased from the Chongqing mouse breeding base; the average weight was 50. 5± 4. 7g.

Experimental reagents
Eupatorium adenophorum extract (ethanol extraction); Levo Norgestrel quinestrol whose manufacturers as Huarun Zizhu Pharmaceutical Co. Ltd and each active ingredient is 9mg; xylene, formaldehyde is used to soak mouse organs. Physiological saline, distilled water; arowana edible oil is used for dissolving Levonorgestrel quinestrol (each with 3 ml edible oil) [9]. At first, make it dry in the shade and chop it. Then, using 500ml alcohol to extract 100g grass clippings, meanwhile, adjust the rotating speed of rotary evaporator to 55r/min, and 60℃. One hour later, filtering, extracting, separating alcohol out. Next, adjusting the rotating speed to 90r/min and 65 ℃ so that the alcohol evaporates completely. Finally, using the propanetriol to dissolve the remaining, and then set the volume at 100ml.

2.4.2.
Gavage time arrangement, 30 female rats and 15male rats divide into three groups which are Eupatorium adenophorum group, Levonorgestrel quinestrol group and blank group, and 10 female mice and 5male mice each group. Continued routine feeding for one week, feeding many times according to arrangement time with 0. 5ml. The time was 1D, 5D, 10D and 15D. What's more, killing two female mice and one male mouse of each group by removal of vertebra in order to anatomical observation and make tissue sections before the 5D, 10D and 15D feeding [4][5][6][7]. Especially, ensure adequate daily feed and water during the test. Specific schedule follow Table 1 Description: The A was stand for taking two female rats and one male rat to anatomical observation before gavage, and others was according to the amount to feed.
The B means to remove two female rats and one male rat to anatomical observation The C was intragastrical administration as usual. (1) Fixed: Fixed mouse organs with formaldehyde solution after observation in live and different degrees (cut size is about 3mm×3mm×5mm).
(3) Slice: take the embedded various organs to tissue sections and sliced thickness is about 8 micron each.
(4) Dewaxing: after slicing, the slice was fixed on the slide glass in order to dewax. Specifically, for (2) in the reverse order, and rinse 2min with distilled water at last.
(6) Eosin stained 15min, and washing with distilled water for a short time.
(7) Dehydration and mounting: Firstly, sequential immersion in 75% ethanol, 80% ethanol, 95% ethanol (two times), 100% ethanol (two times), each time 5min. Next, soaking 15min in the xylene solution and repeat once. At last, drying 24 hours in the oven at 40 degrees Celsius.  (Figures 1 and 2).  In order to be able, understand, I made the following two tables to reflect the changes in the organs of each male and female mice during different periods (table two) and in the same time difference drugs make different results (table three). There are obvious changes with the number of +, No significant change is used to. -

Determination of organ coefficient
The experimental data were statistically analysed by Excel 2010, and the data analysis method which named single factor analysis of variance (One-way ANONA) was used to analyze the conspicuousness of variable difference. When P is less than 0. 01, the difference is very significant. When P is less than 0. 05, the difference is significant. The mean is mean error ± standard error (Mean ± SE).

Histological sections observation
Tissue sections are generally used to study the microstructure of organ and cells, and can be preserved for a long time. It is widely used in medicine, biology, embryology and other fields. According to the contrast, there were no significant changes in the heart and kidney tissue sections of the male and female rats in each group. The renal 10D tissue section of kindey of each group was shown below:  We found that the female rat uterine cells of E. adenophorum group and levonorgestrel easters group in different periods have different degrees swelling, taking 10d as a case, after the same magnification observation can obtain the following comparison chart:

Appearance change observation
After killing mice and observe organs, we found that the heart, kidney and other organs showed no obvious changes in appearance by compared the combination of E. adenophorum levonorgestrel group, but in the uterus comparison, we found that the group of levonorgestrel group and E. adenophorum group were bigger than the blank group, edema phenomenon. From the reference literature, the effective component of levonorgestrel quinestrol is similar to the EP-1 of (a rodent contraception) which will make the endometrial transformation, thinning, loose interstitial edema, thickening of mucus secretion, being Not conducive to sperm penetration thus causing infertility. From the appearance changes and morphologic changes of E. adenophorum group and the group had the similar effect of levonorgestrel quinestrol. The E. adenophorum group and the levonorgestrel quinestrol group had the similar effect from the appearance and morphologic changes [10][11].

Comparative analysis of organ coefficient
From the results of viscera coefficient, there was no significant difference in the heart, kidney and testicular. However, there was a significant difference between each treatment group and the blank group at each time. From the analysis of variance of E. adenophorum group and control group, the mean values of P were about 0. 05-0. 10 so that there is No significant difference. There are some differences of E. adenophorum group and control group by the picture analysis. This shows that the extract of E. adenophorum spreng has some effects on female mice uterus, but the affect is lower than levonorgestrel and quinestrol.

Histological observation
From the result of histological observation, it hasn't obvious changes of the heart, kidney and testis tissue cells from different agent's mice at the same time in the same group, but uterine tissue sections showed uterine cells occurred swell, and cell gap is also larger. The experimental results demonstrate that the mechanism of levonorgestrel and E. adenophorum don't influence the morphology (size, color and texture) of heart, kidney and testis, but there is a certain effect on the uterus [12][13].
The above three indexes showed that E. adenophorum extract had no obvious effect on mouse heart, kidney and testis, but has effect on the uterus which appearance show edema thickening; tissue showed cell edema and big gap. The uterus is the reproductive organs of female mice, so infer the levonorgestrel quinestrol and E. adenophorum extract have a certain influence on fertility of female