The survey of the asparagus effect on the folliculogenesis with cyclophosphamide medication post-treatment in rats

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Vol-7, Special Issue-Number4-July, 2016, pp539-544 http://www.bipublication.com

Research Article

The survey of the asparagus effect on the folliculogenesis with

cyclophosphamide medication post-treatment in rats

Hojatollah Karimi Jashni1, Hossein Kargar Jahromi2*,

Belghis Soleymani3 and Maryam Sahebi3

1_{Zoonoses Research Center, Jahrom University of Medical Sciences,Jahrom, Iran.} 2_{Research Center for Noncommunicable Diseases,}

Faculty of Medicine,Jahrom University of Medical Sciences, Jahrom, Iran.

3_{Student Research Committee, Jahrom University of Medical Sciences, jahrom, Iran}

Corresponding Author: E-mail: [email protected]

ABSTRACT:

Field and objective: asparagus racemosus (A. racemosus) belongs to the family Liliaceae and commonly known as Satawar, and it is a very strong antioxidant with positive effects on the fertility process in female gender. The objective of the present study is the survey of asparagus extract effect on folliculogenesis process in rats after treatment with cyclophosphamide drugs.

Materials and methods: the present experimental research was conducted on 48 adult female Wistar rats. The rats were grouped into 5 empirical and one control cohorts. The experimental groups 1 and 2 received 200 mg/kg/bw and 400 mg/kg/bw, respectively, of the asparagus extract orally and the experimental groups 3, 4 and 5 all were injected intra-peritoneally with 150 mg/kg/bw cyclophosphamide medication. Then experimental groups 4 and 5 orally received 200 mg/kg/bw and 400 mg/kg/bw, respectively, asparagus extract on a daily basis. Blood samples were obtained from all of the groups after 4 weeks after being subjected to anesthesia. The blood serum was isolated and the samples were sent to the laboratory for measuring the serum concentration of the hormones estrogen and progesterone, MDA, ROS and TCA. The ovaries were also studied by taking advantage of light microscope after being removed, weighted and sectioned.

Findings: ovary average weight, the number of the primary, secondary and tertiary follicles in the experimental group 3 (cyclophosphamide cohort) were reduced in contrast to the control group. But, in experimental groups 1 and 2 (200mg and 400 mg asparagus, respectively) and experimental groups 4 and 5 (cyclophosphamide plus asparagus cohorts) the ovary average weight and the number of the ovarian follicles were increased. Estrogen, progesterone and TAC hormones average serum concentrations declined in the group received cyclophosphamide in comparison to the control group. But, in the groups received asparagus along with cyclophosphamide these concentrations were increased in comparison to the cyclophosphamide group (P<0.05).

Conclusion: The present study indicated that asparagus extract reduces the detrimental effects of cyclophosphamide drugs on ovary and the folliculogenesis process in rats through increasing the antioxidant indices.

Keywords: asparagus, cyclophosphamide, folliculogenesis, rats

INTRODUCTION:

Cyclophosphamide, as a strong anti-carcinogen or carcino-preventive drug, is currently being used in the treatment of different types of cancers such as leukemia and Hodgkin’s lymphoma [1]. This drug also possesses suppressive effects on immunity

system in organs transplantation and toxic effects on all of the body tissues particularly on sex organs [1, 2]. The results of the studies performed

on the female gender indicate that

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stress causes disorders in folliculogenesis process and decreases the number of natural ovary follicles [3]. On the other hand, it has been found out that cyclophosphamide side effects can be

reduced by simultaneous prescription of

antioxidants, [4]. In between, the use of the herbal ingredients with antioxidant characteristics is of a considerable importance.

Asparagus is a perennial herbaceous dioecious plant, from the Asparagaceae family and Asparagus order [5]. Asparagus grows all around the world and eastern Mediterranean to Caucasus mounts are cited as the place of origin for such a

plant [6]. Asparagus contains Saponins,

Flavonoids, Asparagine, Coniferines, Inosite, tannin, Gallic Acid, Succinic Acid and various vitamins [7]. The results of the numerous studies conducted on asparagus therapeutical strength show that this plant possesses antioxidant characteristics and it indicates free-radicals controlling effect [8], it is anti-carcinogenic [9] and it exhibits liver-protective attributes [10]. Moreover, it has become clear that this plant boosts sexual stamina [11] and it has positive effects on folliculogenesis process in female gender [12].

The estimated result of the two body enzymatic and non-enzymatic defensive systems is an indicator of the ovary tissue antioxidant capacity [13]. Total antioxidant capacity (TAC) is an estimation of the various antioxidants potential combination acting interactively. It seems that measuring this index is more better than the individual and antioxidant status indices (such as antioxidant enzymes SOD, CAT and GPX), since complicated reactions occur between antioxidant and oxidative forces in vivo and what is measured by such an index is the pure result coming from such actions and reactions [14]. Reactivated

oxygen species (ROS) and the serum

Malondialdehyde level (MAD) are considered as important lipid peroxidation bio-indicators [15]. Therefore, according to asparagus antioxidant attributes, in the present article besides the tissue

evaluations and hormone assays three

aforementioned factors will also be evaluated in the following section in order for the exact performance of this herb to be scrutinized regarding the cyclophosphamide medication toxic effects.

MATERIALS AND METHODS:

There was made use of 48 healthy adult rats in the age range of between 2 to 3 months and with the weight ranging from 150g to 170g in the present empirical study. During the experiment period, the mice were kept in 12 hours light/dark cycles. To adjust to the environment the rats were kept this way for one week then they were divided into 6 random groups.

To prepare the asparagus extract, firstly the asparagus roots after being cleaned in laboratory environment were completely dried and grinded to powder. The resulting powder was admixed with ethyl alcohol, 96%, with a ratio of 5 times the plant powder volume and it was stirred in rotodoxy device for 24 hours in room temperature and in the end a uniform solution was obtained. In the next stage, the solution was filtered and it was dried for 48 hours in dry environment condition to be transformed into alcohol-free solid extract. 200mg and 400mg of the solid extract were dissolved to 1cc twice distilled water and they were kept in the refrigerator till the consumption time [16]. The first cohort (control) had free access to enough compressed food and water. The

experimental groups 1 and 2 received,

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10%, to be sent to the laboratory for preparing tissue incisions. After molding, serial tissue incisions, with a 5 µm thickness, were prepared and three fields of each slide were studied under light microscope by taking advantage of scalar graticule, and number of the primary, secondary and tertiary follicles was counted and the average number of follicles was determined.

Data were analyzed by the use of SPSS software and one way variance analysis (ANOVA) and the

significance level was found to be P≤ 0.05.

Findings:

Based on the results illustrated in table 1, the average ovary weight is indicative of a reduction in experimental group 3 (cyclophosphamide) in comparison to the control group (P<0.05). But, in experimental groups 4 and 5 a statistically significant increase was shown in comparison to

experimental group 3 (cyclophosphamide)

(P<0.05). The average number of primary, secondary and tertiary follicles was also

suggestive of a significant reduction in

experimental group 3 (cyclophosphamide) in

comparison to the control group (P<0.05). But, the number of the primary and secondary follicles was indicative of a significant increase in experimental group 5 respective to the experimental group 3 (cyclophosphamide). Moreover, the average number of the tertiary follicles in experimental groups 4 and 5 was shown to have a significant increase in contrast to experimental group 3 (P<0.05). Based on the results given in table 2, the level of TAC serum concentration in experimental group 3 (cyclophosphamide) was indicative of a significant decrease in comparison to the control group (P<0.05). But, in experimental groups 4 and 5 it was shown to have a significant increase in

contrast to the experimental group 3

(cyclophosphamide) (P<0.05). The levels of ROS and MDA serum concentration were shown to have a significant increase in experimental group 3 (cyclophosphamide) in comparison to the control group (P<0.05). But, in experimental groups 4 and 5 it was shown to have a significant decrease compared to the experimental group 3 (P<0.05).

Table 1: the average weight and the number of the ovary follicles in various experimental groups Variable

Groups

Ovary weight (mg)

The average number of the primary

follicles

The average number of the secondary

follicles

The average number of the tertiary

follicles

Control group 33.16 ± 3.7 6.86±1.1 3.6 ± 1 3.6 ± 1

Experimental group 1

(asparagus extract 200 mg/ kg) 36.7 ± 3.5 9.2 ± 1.4* 4.2 ± 1 3.5± 0.5

(asparagus extract 400 mg/ kg) 34.5 ± 4.8 11.1 ± 1.4* 4.3 ± 0.8 4.5 ± 0.5

(cyclophosphamide) 28 ± 6 3.5 ± 1.1* 1.3 ± 0.48* 1.3 ± 0.48*

Experimental group 4 (cyclophosphamide + asparagus extract 200 mg/kg)

35.1 ± 4 # 5 ± 0.9 2.1 ± 1.1 2.1 ± 0.8#

25.3 ± 5.9# 6.6 ±1.3# 3.1 ± 0.7# 2.6 ± 0.5#

* sign indicates the difference with the control group.

# sign indicates the difference with the cyclophosphamide group.

Table 2: estrogen and progesterone hormones, TAC, ROS and MDA serum concentrations in various experimental groups

Variable Groups

Estrogen hormone (nanogram)

Progesterone hormone (nanogram)

TAC ROC MDA

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(asparagus extract 200 mg/ kg)

Experimental group 2 (asparagus extract 400 mg/ kg)

76 ± 10.7* 50.5 ± 7.2 3.9 ± 0.73* 47.2 ± 3.3 0.52 ± 0.23*

(cyclophosphamide) 25± 5.8* 27.9 ± 5.4* 0.85 ± 0.22* 125.5 ± 14.1* 1.8 ± 0.3* Experimental group 4

(cyclophosphamide + asparagus extract 200 mg/kg)

31 ± 5.7# 34.2 ± 4.6 1.9 ± 0.3# 107 ± 4.8*# 1.2 ± 0.24*#

57 ± 7.8*# 38.4 ± 6.3# 1.8 ± 0.36# 87.7 ± 13*# 0.97 ± 0.24#

* sign indicates the difference with the control group.

# sign indicates the difference with the cyclophosphamide group.

DISCUSSIONS:

Based on the findings of the present study, cyclophosphamide has brought about a significant decrease in the ovary average weight, the average number of the ovary follicles and estrogen and progesterone hormones serum concentration levels in contrast to the control group. These observations conform to the findings obtained by the other researchers regarding the study field. In a study performed by Farrokhi et al in 2007 it became evident that cyclophosphamide causes a detrimental damage to the ovary follicles particularly the primordial follicles [17]. In the study conducted by Lopez et al in 2008 also disorders in ovary performance, the reduction of the number of the follicles and reduction in the ovary weight have been reported as a result of consuming cyclophosphamide medication in rats [3].

In the present study, also in cyclophosphamide group there was observed a significant reduction in TAC serum concentration level and a significant increase was observed in MAD and ROS serum concentration levels which are expressive of the free radicals production, oxidative stress and lipid peroxidation. The results of the studies indicate that oxidative stress created due to cyclophosphamide causes damage to the follicle cells as the source of estradiol production in ovary [18]. Therefore, the decrease in the ovary hormones levels in cyclophosphamide group in

the current study is connected to the damage in the follicle cells secreting such hormones.

On the other hand, in empirical groups receiving cyclophosphamide and various doses of asparagus extract simultaneously (experimental groups 4 and 5) a considerable increase in ovary weight and also in the number of the natural follicles and ovary hormones was observed in comparison to the cyclophosphamide group. In experimental groups 1 and 2 which received 200 mg/kg and 400 mg/kg of only asparagus extract a significant increase in weight and the number of the ovary follicles was observed respective to the control group and this is indicative of the asparagus extract positive effects on the folliculogenesis process. In another study performed on the asparagus extract effects on the ovary tissue it became clear that the asparagus extract brings about an increase in the number of the primary, secondary and graph follicles and also causes a decrease in the number of the atretic follicles in rats [12].

Significant increase in TAC and decrease in MAD and ROS levels in experimental groups receiving

cyclophosphamide and asparagus extract

simultaneously in comparison to the

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are capable of reducing the cyclophosphamie poisonous effects on the female reproduction organs. In the surveys conducted on 23 widely used medicinal plants it was determined that asparagus possesses the highest antioxidant activity [7]. Moreover, the asparagus extract liver-protective attributes have been proved against drugs such as Acetaminophens and this is confirmatory of the asparagus antioxidant activity [22].

Flavonoids, Saponins and vitamins are considered among the antioxidant ingredients of asparagus [7]. The studies performed have shown that

saponins cause a reduction in the

cyclophosphamides detrimental effects on the sex organs [23]. Phenolic ingredients antioxidant activity is also a result of their revitalizing effects which allows them to act as revitalizing,

hydrogen-donating and unbound oxygen

deactivating agents [21].

General Conclusions:

According to the results obtained in the present study it seems that prescribing asparagus

antioxidant medicinal plant along with

cyclophosphamide can be effective on the reduction of the detrimental effects of such a medication on the female reproduction organ in patients with cancer.

ACKNOWLEDGMENTS

The authors appreciate Jahrom University of Medical Sciences for funding the survey.

Conflict of interest

All authors declare that there is no conflict of interest.

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