The effect of intra‐peritoneal administration of Papaver bracteatum Lindl. extract on development of NMRI mice oocytes treated with Doxorubicin

Abstract

Purpose

The effect of water–alcohol Papaver bracteatum Lindl. extract on development of mice oocytes treated with Doxorubicin (dox) was examined in this study.

Methods

The mice were classified into four groups. Control group, mice injected intraperitoneally (IP) with saline. Extract group alone, mice treated with 200 mg/kg of body weight (bw), IP, twelve consecutive days. Dox group alone, mice were given dox, IP, 10 mg/kg bw. Experimental group treated with extract and dox together. Effect of the extract on the development of mice oocytes treated with dox were evaluated through assisted reproductive technology techniques (ARTs).

Results

Developmental rate and blastocyst formation was improved by using the extract. A significant increase in in vitro developmental competence in comparison with dox group (P < 0.05) was observed.

Conclusions

The results of this study indicated that P. bracteatum Lindl. extract could prevent dox toxicity of dox affecting both follicle or oocytes, and therefore it can result in improved embryo development which was observed in mice treated with dox plus P. bracteatum Lindl. compared to mice treated with dox alone.

Introduction

Fertility preservation and continuity of reproduction is always one of the most important challenges of mankind throughout history, and humans have tried to remove the barriers to this goal. Previous studies demonstrate that some diseases and even some medical treatments cause infertility. Radiotherapy treatments and chemotherapy may induce ovarian toxicity. Despite considerable advances in cancer therapy, premature ovarian failure (POF) and infertility are well‐known side effects observed in young girls and reproductive‐age women treated for cancer. One of the most important effects of chemotherapy which can induce ovarian toxicity is excessive production of reactive oxygen species (ROS). Doxorubicin is not an exception to this principle. Doxorubicin (dox), [(8 S‐cis)‐1o‐[(3‐amino‐2, 3, 6‐tri‐deoxy‐alpha‐l‐lyxohexopyranosyl) oxy]‐7, 8,9,10‐tetra hydro‐6, 8,11‐tri hydroxy‐8‐(hydroxyacetyl)‐I‐methoxy‐5,12‐naphthacenedione] [1], commercially and clinically known as adriamycin, is used in a wide range of therapeutic activities against various cancers. A single injection of dox can lead to reduction in both ovarian size and weight [2], as well as cause apoptotic changes in the pregranulosa cells resulting in follicular damage [3, 4]. Dox can reduce the storage of primordial, antral and primary follicles even up to a month after treatment. Recent findings have shown that the population size of each type of ovarian follicles after dox administration was significantly reduced when compared to the control group. Histological examinations also revealed positive staining of TUNEL and active caspase‐3 in the dox treated group [2]. Yeh and co‐workers [5] have shown that dox retards testicular growth and impairs spermatogenic function and can cause testicular atrophy in mice. Previous investigations demonstrated other side effects of dox, including decreased ovulation rate, which can cause infertility in women and laboratory animals [2]. Therefore, the administration of antioxidants throughout the course of chemotherapy to support oocyte and ovarian function will be more important than ever. In this regard, and bearing in mind the direct effects that chemotherapy may have on follicles and oocytes, there is still a need for clinical advancements in the field of assisted reproductive technologies (ART). In vitro fertilization (IVF), which is one of the most common routes of ART, can be used in order to try and select the highest quality oocytes for fertilizations. Oogenic cells are susceptible to dox‐induced oxidative stress and apoptosis. Papaver bracteatum Lindl. is named after J. Lindley, who first recognized it as an independent species in 1821 [6]. Papaver bracteatum Lindl. (Papaveraceae) is a sturdy perennial herb indigenous in Iran and many other regions in the world [7]. Chemical studies have demonstrated the presence of rhoeadic acid [8, 9], thebaine [10, 11], papaveric acid [12], and anthocyanins [13] as the major compounds of the extract of Papaveraceae family. Phenolic compounds and alkaloids of the P. bracteatum Lindl. can be used as an antioxidant. Replacing the natural antioxidants present in medicinal plants instead of synthetic antioxidants may prevent oxidative stress. In recent years investigators have demonstrated that anthocyanins have antioxidant, anti‐inflammatory, antimicrobial and anti‐carcinogenic properties. P. bracteatum extract with an anti‐oxidant property increases oocyte quality. It is likely that the extract effects are caused by the antioxidative properties of some phytochemicals of the extract. The purpose of the present study was to assess whether or not the effects of doxorubicin toxicity on these follicles and oocytes could be prevented by extract. The present study was designed to examine the possibility of an ameliorating action of the water–alcohol extract on doxorubicin‐induced detrimental effects in mouse models, and implies that the extract may be a promising adjuvant agent that may attenuate the toxicity of doxorubicin.

Materials and methods

Drug

Doxorubicin hydrochloride (Adriamycin), commercially known as Ebedoxo, was obtained from Helal Ahmar Pharmacy, Tehran, Iran. This drug must be protected from light and sub‐zero temperatures. One millilitre of Adriamycin contains 2 mg doxorubicin hydrochloride. DOX was administered at a dose of 2.5 mg/kg of body weight, i.p. a cumulative dose of 10 mg/kg of body weight, for 4 times, every 3 days, during the 12 days. (days 2,5,8 and 11).

Plant material and extract preparation method

Plant material

Papaver bracteatum Lindl was collected from Poloor region (in the north of Iran). The plant materials were transferred to the department of Pharmacognosy at Shahid Beheshti University to be authenticated by Mr. Kamalinejad (Faculty of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran) [14, 15].

Preparation of the extract

The extract was prepared as follows: 500 ml of 50 % ethanol (V/V) in water was added to 100 g of the dried petals powder and soaked at room temperature for 5 days. After filtration, ethanol was evaporated at low pressure at 33 °C. The extraction yield was 15 g from 100 g of the dried petals of P. bracteatum. The soft extract was dissolved in normal saline and was immediately administered IP to the mice, expressed as mg of extract per kg body weight [14, 15].

Animals

Outbred male and female Naval Medical Research Institute (NMRI) mice (purchased from Pasteur Institute, Tehran, Iran) at 6–8 weeks old were divided into four groups. The mice were housed in air conditioned compartments with controlled light and temperature within the animal house of the Royan institute. The temperature was maintained at 20 °C, and a light/dark cycle of 12:12 h was set. Humidity was maintained at a minimum of 50 %, and the mice were fed on a standard pelleted diet and tap water and were treated with utmost human care during the study period. Animal care was in accordance with institutional guidelines and was approved by the local authorities. The mice were randomly assigned to four groups (30 mice per group) according to the pharmacological treatment they received. In the control group (Cont), mice were treated with injected IP with normal saline alone. In the extract of P. bracteatum group alone, mice were treated with P. bracteatum extract, IP, at the dose of 200 mg/kg of body weight, daily, for 12 consecutive days [16]. In the Dox group alone, mice were given dox, IP, at the dose of 2.5 mg/kg of body weight, every 3 days, for four total shots and an accumulated dose of 10 mg/kg of body weight, during the 12 days (days 2,5,8 and 11). The experimental group (P. bracteatum extract plus dox) consisted of mice treated with P. bracteatum extract and dox together. P. bracteatum extract (200 mg/kg/day, i.p. for 12 days) was administered 1 day before dox and when the two drugs were given in combination, the extract of P. bracteatum was given 3 h before each dox injection [17]. Finally, mice were sacrificed following superovulation, 15 days after the first administration of P. bracteatum extract by cervical dislocation.

Evaluation of in vitro fertilization and embryo development of in vivo matured oocytes

We investigated ovulation rate in 7–8 weeks old NMRI female mice in all treated groups. Female mice were induced to superovulate by IP injection of 7.5 IU pregnant mare serum gonadotropine (PMSG) at day 12, at the last day of injection, of P. bracteatum extract and 7.5 IU human chorionic gonadotropine (HCG) 48 h later. The female mice were sacrificed by cervical dislocation 16–17 h after hCG administration. Cumulus‐enclosed oocytes were isolated from the oviduct ampullae with insulin syringe. Cumulus removal was safely performed by gentle pipetting in human tubal fluid (HTF) medium [18] supplemented with 4 mg/ml bovine serum albumin (BSA; Sigma, USA) using a series of finely drawn glass Pasteur pipettes and MII oocytes required for each treatment were collected and transferred to 100 μl HTF medium supplemented with 4 mg/ml BSA droplets. Spermatozoa were obtained from male mice at 8 weeks of age: after being sacrificed by cervical dislocation, the vas deferens and cauda epididymides were dissected out, disrupted with the needle of an insulin syringe, the spermatozoa were then released into 1 ml of HTF medium supplemented with 4 mg/ml BSA. After dispersion, the concentration was adjusted to a final value of 2 × 10⁶ sperm/ml. An incubation period of approximately 45 min at 37 °C in 5 % CO₂ was considered sufficient for sperm capacitation. Then MII oocytes and 2 × 10⁶ capacitated sperm/ml were transferred into the HTF medium supplemented with 4 mg/ml BSA droplets for fertilization and covered with mineral oil. Approximately 4–6 h after IVF, the oocytes were washed, and both male and female pronuclear (2PN) were transferred into KSOM medium [19] supplemented with 4 mg/ml BSA. KSOM medium was applied through in vitro development (IVD) until the blastocyst stage. The oocytes were monitored by an inverted microscope, and the percentage of 2PN formation was recorded to evaluate the fertilization rate. After the IVF process, the embryos were examined for 96 h until they reached the blastocyst stages. During IVD, the numbers of 2‐cell, 4‐ to 8‐cell, morula and blastocyst embryos were recorded.

Statistical analysis

The ANOVA and Duncan protected least‐significant tests, using Statistical Analysis System (SAS) v1.9 program, were used for all statistical analysis. All percentages of values were subjected to arc sine transformation prior to analysis. All data were expressed as mean ± SEM. A probability of P < 0.05 was considered to be statistically significant.

Results

According to our results, the injection of dox alone reduced the quantity and quality of embryos that were already formed with treatment of P. bracteatum extract. In the group of P. bracteatum extract, 2PN formation improved; however, the improvement was not significant compared to the control group (31.2 ± 5.3 vs. 29 ± 6.51), and 2PN formation ameliorated extract plus dox group in comparison with dox group alone (24.1 ± 1 vs. 20.5 ± 3.2). Most of the 2PN belongs to the P. bracteatum group and the dox group holds the least amount of 2PN (Table 1). The 2‐cell rate compared to the Dox group significantly (P < 0.05) increased when 200 mg/kg P. bracteatum extract was injected to mice treated with dox alone. Therefore, the most embryos in the dox group, were arrested at the 2‐cell stage after 48 h in KSOM medium (Table 1). In addition, there was a significant decrease in the rate of embryo formation at the 4–8cell, morula, blastocyst and hatched blastocyst stages in the dox group (P < 0.05) compared to the other groups. On the other hand, a significant (P < 0.05) increase was observed in the rate of embryo formation of the mentioned stages in mice treated with P. bracteatum extract and dox together, in comparison with dox group alone. However, no significant difference was detected when compared to the cont and P. bracteatum groups (Table 1). The observation in Dox group showed that the rate of progress decreased considerably at the morula stage. Therefore the numbers of embryos that reached the blastocyst and hatched blastocyst stages had significantly (P < 0.05) decreased. On the other hand, the rate of fertilization and the number of oocytes reaching the blastocyst stage were significantly higher in the P. bracteatum extract plus dox group than in the dox group alone; (41 vs. 16, respectively, P < 0.05). Finally, 14.36 ± 2.25, 11.04 ± 1.97, 5.3 ± 1.78, and 10.05 ± 2.36 blastocysts from the cont group, P. bracteatum group, Dox group, and PapaverDox group, respectively, reached the hatching stage after 96–120 h of fertilization, and no significant difference was observed in the experimental group (PapaverDox) when compared to the control and papaver groups.

Table 1.

Effect of the P. bracteatum Lindl. extract (Papaver), Doxorubicin (Dox), and both (P + D) on in vitro fertilization rates and in vitro embryo development

Groups	No. of matured oocytes (in vivo)	After 4–6 h	After 24 h	After 48 h	After 72 h	After 96 h	After 96‐120 h
		2 PN (mean ± SEM)	2 Cell (mean ± SEM)	4–8 Cell (mean ± SEM)	Morula (mean ± SEM)	Blastocyte (mean ± SEM)	Hatched blastocyst (mean ± SEM)
Control	587	29 ± 6.51a	62.75 ± 5.21ab	39.27 ± 6.02a	30.54 ± 5.12ab	22.99 ± 3.09a	14.36 ± 2.25a
Papaver	603	31.2 ± 5.32a	67.02 ± 4.34a	43.78 ± 4.23a	32.71 ± 3.44a	22.35 ± 3.68a	11.04 ± 1.97ab
Dox	421	20.5 ± 3.2a	48.4 ± 4.1b	19.77 ± 2.08b	17.44 ± 2.03b	8.9 ± 1.7b	5.3 ± 1.78b
P + D	473	24.1 ± 4.93a	60.33 ± 4.85ab	36.45 ± 4.56ab	28.97 ± 3.74ab	17.31 ± 2.53ab	10.05 ± 2.36ab

Percentage of embryos expressed as mean ± SEM. All experiments were repeated ten times. Different superscripts indicate significant differences (P < 0.05)

Discussion

This study evaluated the effect of P. bracteatum Lindl. extract on the development of NMRI mice oocytes treated with doxorubicin. Presently, there is an increasing interest in natural antioxidants present in dietary plants and medicinal plants, which may replace synthetic antioxidants and by doing so contribute to the prevention of oxidative disorders occurring in humans [20]. Anthocyanins are putative antioxidants and have radical‐scavenging effects which can protect cells from oxidative damage [21]. Anthocyanins belong to the widespread class of phenolic compounds collectively known as flavonoids [22]. Phenolic compounds and alkaloids of the P. bracteatum Lindl. can be used as an antioxidant. Phenolic compounds are often related to the antioxidant activity of plants due to their ability to absorb and neutralize free radicals by means of quenching unpaired oxygen radicals or decomposing peroxides [20]. Flavonoids are non‐enzymatic antioxidants and are considered as a class of secondary metabolites widespread in various plants [23]. One of the most actively studied properties of flavonoids, is their protection against oxidative stress [24, 25]. For instance, flavonoids are ideal scavengers of peroxyl radicals due to their favourable reduction potentials relative to alkyl peroxyl radicals [26]. In general, anti‐oxidative effects of the extract are due to its flavonoids. Anthocyanins are in plant pigments that are widely found in many berries, in dark grapes, cabbages and other pigmented food products, plants and vegetables [27, 28]. Antioxidant supplements or food, vegetables, and plants containing antioxidants can be used to reduce oxidative damage induced by reactive oxygen/nitrogen species (ROS/RNS) [65]. Varied medicinal plants are known as a source of natural phytochemicals with antioxidant activities that can protect organisms from oxidative stress as well as from various chronic diseases [66]. Papaver bracteatum, is also known as the Iranian poppy [67]. The results of poppy ethanolic extract indicated the presence of compounds which are able to scavenge free radicals. The free‐radical scavenging activity [68] and antioxidant activity [66] of this extract was determined according to the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assay described previously. The molecules possessing radical‐scavenging properties present in ethanolic poppy extract can quench DPPH free radicals [68]. The antioxidant properties of anthocyanins have been demonstrated by both in vitro and in vivo experiments [29, 30, 31, 32, 33, 34, 35]. Previous studies have demonstrated the antitumor activity and in vivo antioxidant status of Mucuna pruriens (Fabaceae) seeds against Ehrlich ascites carcinoma in Swiss albino mice [36]. Investigations on the effect of botanical components, such as green tea polyphenols [37], demonstrated that supplementation of green tea polyphenols as anti‐oxidants through maturation of bovine oocytes increased blastocyst formation. It was demonstrated that P. rhoeas extract exerted its antioxidative effects on mouse brain tissue [38]. The antioxidative potential of some plants such as P. rhoeas has been evaluated, and it was shown that those plants are scavengers of hydrogen peroxide [39]. Former studies demonstrated that P. bracteatum Lindl. has antioxidative activity [40]. In recent years, investigators have demonstrated that anthocyanins have antioxidant [41, 42, 43], anti‐carcinogenic [44, 45, 46], vasoprotective [47], anti‐inflammatory [48], and antimicrobial [49] properties. Vitamin C is known to have a protective effect within the follicle, as vitamin C deficiency has been reported to result in ovarian atrophy, extensive follicular atresia, and premature resumption of meiosis [50]. Furthermore, anthocyanins are in plant pigments. A recent study by Kostic et al. [49] revealed that red pigment present in the flowers of P. rhoeas L. and the Papaveraceae family originates from anthocyanins, which may act as natural antioxidants. P. bracteatum Lindl. anthocyanins were determined by gas liquid chromatography (GLC) [69] and thin liquid chromatography (TLC) [70]. In this regard, pelargonidin 3‐glucoside, cyanidin 3‐glucoside, and cyanidin 3‐sophoroside are present in P. bracteatum Lindl. as anthocyanins. Pelargonidin 3‐glucoside is present in the P. bracteatum Lindl. petals, and cyanidin 3‐glucoside is present in the blotch at the base of the petal [51]. In former studies, Pei‐Ni Chen and co‐workers shown that treatment with cyanidin 3‐glucoside could decrease the protein levels of CDK‐1, CDK‐2, cyclin B1 and cyclin D1, and cyanidin 3‐glucoside resulted in a strong inhibitory effect on tumor cell growth via G2/M arrest [52]. Cyanidin 3‐glucoside also induced caspase‐3 activation and chromatin condensation [53]. Cyanidin 3‐glucoside has notable antioxidant and anti‐inflammatory properties [54]. In addition, it was demonstrated that antioxidants exerted their antioxidative effects on oocyte quantity and quality in the mouse [71]. It was found that oxidation is affected by antioxidants that can act as radical scavengers [71]. Investigations on the effect of P. rhoeas extract have revealed that extract of P. rhoeas can increase oocyte maturation and subsequent embryo development [15]. Recent studies have indicated the effects of antioxidants on the development of parthenogenetic porcine embryos. Antioxidants increase the average number of total cells at the blastocyst [55]. However, in this study no difference was detected between the P. bracteatum and the control group in the rate and developmental competence of oocytes obtained through IVF. Researches in cancer therapy have shown that majority of women and girls who suffer from cancer also suffer from infertility or premature ovarian failure (POF) difficulty [56]. Therefore, they are demanding fertility preservation after cancer therapy. In this regard, doxorubicin is an anthracycline, antineoplastic antibiotic [57]. Despite previous studies proving the destructive effects of doxorubicin on ovarian tissue [2], it is one of the most effective and common clinical chemotherapy agents that can be used for the treatment of various cancers [57]. Increased generation of ROS and intracellular calcium is one of the most important effects of chemotherapy treatment. It has been previously shown that doxorubicin forms oxidative free radicals which can in turn induce apoptosis. Doxorubicin can transfer an electron to an oxygen molecule (O₂) to form a superoxide negative radical (O−) and other ROS [58, 59] such as hydroxyl radicals (OH·⁻) and hydrogen peroxide (H₂O₂) and superoxide anions (O₂·⁻). Accumulation of ROS in the cytoplasm causes oxidative stress [60]. Ovarian failure is a known side effect observed in women treated for cancer [2], and recently the serious disturbing impact on oogenesis and spermatogenesis have been demonstrated. Antioxidants act as a scavenger to neutralize free radicals and have generated considerable interest in overcoming the adverse and pathological results of oxidative stress. Oxidative stress can cause direct damage to oocytes in developing follicles, spermatozoa in the peritoneal cavity, or the embryo in the fallopian tube [61, 62]. Therefore, it seems that depletion in the in vitro developmental competence of embryos with the effect of doxorubicin may be related to effects demonstrated in previous studies, which are mentioned above. Recently the use of cytoprotective agents such as cyclophosphamide, vincristine, prednisone, and procarbazine has been recommended against chemotherapy. In this research, we evaluated possible protective effects of P. bracteatum Lindl. against toxic effects of chemotherapy by doxorubicin. Studies on the effects of the methanol extract of M. pruriens seeds proved that methanol extract exhibits potential antitumor and antioxidant activities [36]. The rate of 2PN formation and development of embryos treated with P. bracteatum extract and doxorubicin were better than the group receiving injection of doxorubicin alone (Fig. 1). Furthermore, embryo development in the group of mice treated with P. bracteatum Lindl. and doxo approximately reached the same level as the control group. Therefore, we conjecture that the increase in the rate of embryo development in the group treated with P. bracteatum extract and doxorubicin, compared with the group treated with doxorubicin alone, may be due to the anthocyanins present in this plant and it seems that administration of P. bracteatum Lindl. during chemotherapy with doxo may protect the quality of ovarian oocytes. It can be inferred that the possible antioxidant activity of the P. bracteatum extract may be due to their free‐radical scavenging activity, which may be due to the presence of phenolic compounds in the extract. In addition, several studies have reported that the administration of antioxidant activity during chemotherapy does not prevent the antitumor chemotherapy. For instance, green tea enhances the antitumor activity of doxorubicin [63, 64]. With regard to previous studies, and through this research, we can construe that in women and young girls suffering from cancer, and treated with chemotherapy medication and subject to oxidative stress, antioxidant activity of the extract is most desirable, beacuse it can not only partly prevent the destructive effects and toxicity of dox, but it allows the dox to still continue its antitumor activities.

Figure 1.

The rate of embryo development in all of the treatment groups. The horizontal axis indicates the embryo developmental rate in the different groups, and the vertical axis expresses the percentage of embryos as mean ± SEM. All experiments were repeated ten times. Different superscripts indicate significant differences (P < 0.05)

In conclusion, this study provides firm evidence that doxorubicin can adversely damage the ovary tissue and significantly reduce oocyte production through imposing oxidative stress, while P. bracteatum Lindl. pretreatment could effectively prevent these adverse effects. Since doxorubicin‐based chemotherapy is still a necessary and indispensable for treatment of various cancers, and the resultant drug induced infertility is devastating and often unavoidable, our results suggest the co‐administration of P. bracteatum Lindl. extract with doxorubicin may be a promising solution to these otherwise very serious side effects of doxorubicin.

Acknowledgments

This research was supported by a grant‐in‐aid for scientific research from the Royan Institute.