Ruta graveolens aqueous extract retards mouse preimplantation embryo development

Reproductive Toxicology 17 (2003) 667–672

Ruta graveolens aqueous extract retards mouse preimplantation embryo development Jorge L. Gutiérrez-Pajares∗ , Lidia Zúñiga, José Pino Laboratorio de Reproducción y Biolog´ıa del Desarrollo, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Peru Received 23 September 2002; received in revised form 26 June 2003; accepted 11 July 2003

Abstract This work was undertaken to examine possible embryotoxicity of Ruta graveolens (rue), a plant used by indigenous communities for the purposes of therapeutic and fertility regulation. Superovulated mice were mated and isolated after copulation. They were given aqueous extract of R. graveolens (5, 10, and 20% w/v) or plain water (control) orally for 4 days. Ninety-eight hours post-human chorionic gonadotrophin (hCG), embryos were flushed from oviducts and uterine horns to assess their state of development and extent of embryo transport. Ingestion of rue at 10 and 20% resulted in a high proportion of abnormal embryos (36.7 and 63.6%, respectively, P < 0.05). Cell number was diminished (P < 0.01) and embryo transport was slightly delayed in the highest dose group. These findings demonstrate that oral administration of R. graveolens extract can interfere with preimplantation development and embryo transport. © 2003 Elsevier Inc. All rights reserved. Keywords: Ruta graveolens; Preimplantation; Embryo; Embryotoxicity; Mouse; Rue; Cell number; Embryo transport

1. Introduction A large number of plants have been used by indigenous people as part of folk medicine [1,2], including some that are used to control human fertility [3,4]. Ruta graveolens L. and Ruta chalepensis are alleged to be anti-inflammatory, antipyretic, antiparasitic, antihelmintic, and antinoniceptive [1,5–7]. In rats, the aqueous extract of rue has also shown a hypotensive effect that could be explained by a direct effect on the cardiovascular system [8]. Banerji and Banerji [9] reported that R. graveolens in combination with calcium phosphate could be considered as a potent cysticidal agent with very little or no side effects. Ethnobotanical reports from Europe and America have indicated that R. graveolens “rue” is consumed by indigenous people to promote menstruation or fetal expulsion and as an abortifacient [2–4,10]. Results from laboratory animal studies have been inconclusive. For example, the administration of R. graveolens for the first 10 days of pregnancy caused a significant fertility decrease in female rats [11,12] whereas its administration to female hamsters from days 1 to 6 of pregnancy had no antifertility effect [12]. In these species the time of administration comprised the preimplan∗ Corresponding author. Present address: Laboratorio de Endocrinolog´ıa, Facultad de Ciencias Biol´ogicas, Pontificia Universidad Cat´olica de Chile, Alameda 340, Santiago, Chile. Tel.: +56-99181374; fax: +56-22225515. E-mail address: [email protected] (J.L. Guti´errez-Pajares).

0890-6238/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2003.07.002

tation period plus 5 or 2 days, respectively, of the postimplantation period. Preimplantation embryo development occurs prior to maternal recognition of pregnancy. Chemical substances that may interrupt preimplantation embryo development can be found in nature, such as alkaloids, that occur in different plants [13]. Thus, rue consumption during this early period of gestation could lead to preimplantation losses, implantation failures, or alterations in the physiological processes underlying maternal recognition of pregnancy. The present investigation was undertaken to evaluate whether the aqueous extract of R. graveolens (rue) affects preimplantation embryo development and embryo transport in the mouse. 2. Materials and methods 2.1. Animals Inbred Swiss albino mice were maintained under standard conditions (ambient temperature 22–23 ◦ C; 14 h light:10 h dark cycle, lights on at 7:00 a.m.) with free access to commercial pellet diet (Purina, Perú). Nulliparous female mice were 6–8 weeks of age and male mice were 8–10 weeks old and of proven fertility. Animal care was in accordance with the Guidelines for Animal Experiments in Universidad Nacional Mayor de San Marcos.

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2.2. Aqueous extract Ruta graveolens was purchased from commercial sources (Lima, Perú). Leaves were dried in a stove at 37 ◦ C for at least 4 days. The aqueous extract was prepared daily by boiling the dried leaves in water (0.5, 1 or 2 g per 10 ml) at 60–70 ◦ C for 15–20 min. The extract was filtered and completed to a final volume of 10 ml for administration to each female each day. 2.3. Experimental procedure Female mice were superovulated by intraperitoneal (i.p.) injection with 5 IU of pregnant mare’s serum gonadotrophin (Sigma Chemical Co., 16:00 h) followed at 48 h by an injection of 5 IU of human chorionic gonadotrophin (hCG) (Pregnyl, Organon). The same day of hCG injection the females were caged with males overnight (1–2 females per male, 19:00 h). The following morning they were examined for a vaginal plug as an indication that copulation had occurred (day 1 of pregnancy). Pregnant females were individually isolated in polypropylene cages and given ad libitum 10 ml of 5% (R5, n = 6), 10% (R10, n = 6), or 20% (R20, n = 6) (w/v) of the extract or plain water (control, n = 5) as the only source of water for 4 days. Treatment began at 18 h post-hCG and a fresh supply of extract was replaced every 24 h for the next 3 days. Ingested volumes were recorded every day. 2.4. Evaluation of embryo morphology and transport In the afternoon of the 4th day of pregnancy, 98 h post-hCG, females were killed by cervical dislocation and embryos were flushed from the excised oviducts and uterine horns, separately, with phosphate buffer saline (pH 7.4) supplemented with 4 g/l bovine serum albumin (Sigma Chemical Co.). Embryos were examined under a phase-contrast microscope and classified by developmental stage as retarded (less than 9 cells per embryo), uncompacted morula (more than 8 cells with no sign of compaction), compacted morula, and blastocyst. Compacted morula and blastocysts were further classified as normal or abnormal: normal embryos had no more than one extruded blastomere and had 70–100% active cells as determined by their refringency [14,15]; all other embryos were considered abnormal. The number of embryos belonging to each given class was divided by the total number of embryos found in a female (embryo class index). Embryo transport was scored by counting the number of embryos flushed from the oviducts versus uterine horns. 2.5. Cell number Tarkowski’s method [16] was used to count the number of cells per embryo. Briefly, each embryo was incubated in sodium citrate 1% for 10–15 min and then placed on a

slide with a minimal amount of liquid. A drop of fixative (methanol:acetic acid, 3:1) was applied directly to the embryo. Slides were gently air dried, stained with 5% Giemsa stain for 15 min, and evaluated under a light microscope. 2.6. Statistical analysis “Embryo class index” data were transformed with the arcsin function before analysis. One-way ANOVA and Dunnet’s Multiple Comparison test were used to compare both embryo class index and cell number. Kruskal–Wallis Nonparametric ANOVA test and Dunn’s Multiple Comparison test were used to analyze embryo transport. Linear regression analysis of the group means and Pearson coefficient of correlation for the number of blastocysts, abnormal embryos, and cell number were evaluated with the program MedCalc v. 5.0 (Mariakerke, Belgium). The remaining analyses were done using INSTAT v. 2.04a (GraphPad Software, San Diego, USA). An alpha level of P < 0.05 was considered to be statistically significant. 3. Results Females that drank extract of rue showed no overt sign of intoxication and the weight gain did not differ from the control group during this early period of pregnancy. All females drank most of the plain water or extract irrespective of the treatment group. Ingested volumes ranged from 8.6 ± 0.1 ml/day to 8.8 ± 0.2 ml/day (P > 0.05). Superovulation yielded a total number of 103, 109, 119, and 95 embryos for the control, R5, R10, and R20 groups, respectively. Fig. 1 shows examples of embryos recovered from control animals and after 80 h of maternal exposure to R. graveolens aqueous extract. Regardless of the quality, there was a dose-dependent tendency to increase the percentage of embryos at the morula stage and to decrease those that had reached the blastocyst stage (Table 1). The decrease in the proportion of blastocysts and increase in the proportion of morula were both significant for the R20 group. The percentage of embryos that developed to normal morula and blastocyst stages is shown in Fig. 2. There was a dose-dependent decrease in the percentage of normal blastocysts (y = 0.8524 − 0.2288 arcsin x; P < 0.0001) with high linear correlation (r = −0.85). The non-linear residual variation among group means was not significant (P = 0.36). Ingestion of rue could explain 73% of the decrease in the number of normal blastocysts. Concomitantly, the proportion of abnormal embryos increased also in a dose-dependent manner (y = −0.7765 + 0.1717 log x; P = 0.003) with linear correlation (r = 0.65) and non-significant remaining non-linear variation among group means (P = 0.79). The R10 and R20 groups had increased percentages of abnormal compacted morula and blastocyts. This explains the decrease in the percentage of blastocysts and compacted morula 16 and 11.8% for the R10 group, and 28 and 26.9% for R20 group, respectively (Fig. 2).

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Fig. 1. Effect of Ruta graveolens on preimplantation embryos. Superovulated pregnant mice were exposed to R. graveolens extract during the first 4 days of pregnancy. Embryos were recovered 98 h post-hCG. (A) Normal expanded blastocyst, labeled as “a”, from female mice that ingested plain water. (B) Abnormal compacted morula “b” and uncompacted morula “c” from mice treated with 10% of R. graveolens. Arrow shows extruded blastomere.

ANOVA showed a significant reduction in cell number (P < 0.05) and this effect was moderately dose-dependent (y = 47.7083−6.3398x; P = 0.001), with linear correlation (r = 0.46) and non-significant non-linear variation (P = 0.79) (Table 2). Embryo transport was slightly delayed in all groups that received the extract of rue (Table 3). Control females had no embryo in their oviducts at 98 h post-hCG, whereas 7.4% of

the embryos were present in this segment of the reproductive tract in the R20 group (P < 0.05). 4. Discussion This study is based on the in vivo administration of rue extract so the effects observed involve the potential direct

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Table 1 Developmental stage of embryos Treatment group

Number of dams

Control R5 R10 R20

5 6 6 6

Number of embryos

103 109 119 95

Embryonic developmenta Retarded

Uncompacted morula

Compacted morula

Blastocyst

9.7 6.4 5.9 3.2

0.0 0.0 3.4 7.4

15.5 27.5 31.9 44.2

74.8 66.1 58.8 45.3

(10) (7) (7) (3)

(0) (0) (4) (7)∗

(16) (30) (38) (42)∗∗

(77) (72) (70) (43)∗

Superovulated pregnant mice were exposed to Ruta graveolens extract at 5% (R5), 10% (R10), or 20% (R20) (w/v), or plain water (control) during the first 4 days of pregnancy. Embryos were recovered 98 h post-hCG. a Percentage (number of embryos). ∗ P < 0.05 ∗∗ P < 0.01.

Fig. 2. Distribution of embryos based on their morphology. Superovulated pregnant mice were exposed to Ruta graveolens extract at 5% (R5), 10% (R10), or 20% (R20) (w/v), or plain water (control) during the first 4 days of pregnancy. Embryos were recovered 98 h post-hCG. Normal embryos were determined as mentioned in Section 2. ∗ P < 0.05. ∗∗ P < 0.01.

action of active components of ingested rue on the reproductive system as well as potential systemic effects through participation of the digestive system and liver metabolism, as occurs naturally. The aqueous extract was chosen because it is the way indigenous women use it [1]. In humans, 0.5 g of the plant twice daily is suggested for therapeutic

usage [10]; however, higher doses ingested to induce abortion may cause multiple organ system failure and death [17]. Oral administration of rue leaves to goats caused ataxia, frequent urination, dyspnoea, and death [18]. Meanwhile, administration of ether extract of rue to growing rats for 3 weeks caused no harmful effect in liver and kidney function

Table 2 Number of cells per embryo

Table 3 Embryo transport in female mice

Treatment group

Number of dams

Number of embryos assessed

Number of cells (mean ± S.E.M.)

Control R5 R10 R20

4 4 4 4

14 25 29 32

49.8 40.2 34.1 29.6

± ± ± ±

3.3 3.8 3.7∗∗ 3.1∗∗

Superovulated pregnant mice were exposed to Ruta graveolens extract at 5% (R5), 10% (R10), or 20% (R20) (w/v), or plain water (control) during the first 4 days of pregnancy. Embryos were recovered 98 h post-hCG. ∗∗ P < 0.01.

Treatment group

Number of embryos

Proportion of embryos present in Oviducts (%)

Uterine horns (%)

Control R5 R10 R20

103 109 119 95

0.0 1.8 2.5 7.4∗

100.0 98.2 97.5 92.6

Superovulated pregnant mice were exposed to Ruta graveolens extract at 5% (R5), 10% (R10), or 20% (R20) (w/v), or plain water (control) during the first 4 days of pregnancy. Embryos were recovered 98 h post-hCG. ∗ P < 0.05.

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[19]. The doses and exposure time in the present study did not show acute toxicity as indirectly measured by female weight loss. It is possible that drying the leaves before extract preparation reduces the amount of volatile oils such as methyl-nonyl-ketone that may induce uterine hemorrhage [20]. The results shown in this animal study should be interpreted cautiously before extrapolating to humans because there is no direct relationship between chemical equivalent dose in different animal species and concentration in blood or tissues, and endogenous metabolic processes are not necessarily the same in animals and human [21]. Although it is common to evaluate xenobiotic effects on mammalian embryos during organogenesis, the effects of these agents on preimplantation development is less commonly studied. Toxic agents or an unfavorable environment during preimplantation stages could alter normal events to arrest embryos or invoke abnormalities that affect subsequent stages of development [22–24]. In the present study, rue aqueous extract ingestion during the preimplantation phase diminished mouse embryo quality and development. Among xenobiotics, some common alkaloids occurring in plant extracts have been shown to lower the rates of implantation success and postimplantation development [23,25]. Flavonoids, acridones, and furanocoumarins are alkaloids that offer a wide spectrum of activities and that occur in R. graveolens. Cellular growth is inhibited by plant flavonoids due to the inhibition of tyrosine protein kinase [26], alteration in protein kinase C and the interaction with calmodulin [27]. Acridones, only offered by the Rutaceae plant family, show mutagenic activity [28]. Bergapten, a furanocoumarin highly present in leaves of R. graveolens [29], is a selective blocker of nerve fiber potassium channels [30]. Additional studies will be needed to determine which, if any, of these alkaloids present in R. graveolens might interfere with normal early embryogenesis. During preimplantation development, the embryo undergoes mitotic cell divisions and cell differentiation. Perhaps the reduction of cells per embryo in rue-treated groups might be explained by the inhibitory action of alkaloids on cell proliferation. It is known that reduced cell number in preimplantation embryos may lead to a decrease in blastocyst formation [31], increased rates of preimplantation, and postimplantation losses [25], or reduced weight of the fetus at term [24,31]. Maternal undernutrition during preimplantation development in the rat can have a lasting effect on fetal and postnatal growth, due to retarded preimplantation cell proliferation [24]. Consistent with a lasting effect of abnormal preimplantation embryo development, a preliminary analysis of two female mice induced to superovulate and treated with the highest dose of rue during the preimplantation phase showed no implanted embryos when analyzed on day 9 of pregnancy (unpublished results). Exogenous estradiol administered to female mice on day 1 of pregnancy caused tubo-uterine closure so that embryos persisted in the oviducts on day 4 of gestation [32]. It is possible that phytoestrogens may be present in rue aqueous

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extract accounting for the slight delay in embryo transport found with the highest dose studied here. It is known that isoflavones and isoflavonoid metabolites can bind to estrogen receptors and invoke minor estrogenic responses [33]. Although no phytoestrogens have been reported in Rutaceae, some steroidal alkaloids are under current study in other plant families [34]. In conclusion, our results demonstrate that ingestion of the aqueous extract of R. graveolens L. during the preimplantation phase alters normal blastocyst formation in mice, explained in part by diminished embryo cell number and retarded embryo development. We speculate that these altered developmental parameters are linked with the action of alkaloids present in rue extract and that phytoestrogens might explain the slight delay in embryo entering to the uterus 98 h post-hCG injection.

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