Ginger causes subfertility and abortifacient in mice by targeting both estrous cycle and blastocyst implantation without teratogenesis

Available online 31 January 2018 In Press, Accepted ManuscriptWhat are Accepted Manuscript articles? Phytomedicine Author links open overlay panelReda H.ElMazoudyabAzza A.Attiab https://doi.org/10.1016/j.phymed.2018.01.021 Get rights and content Abstract Background Due to renowned medicinal properties, Ginger rhizomes (Zingiber officinale Roscoe) used traditionally in the treatment of arthritis, rheumatism, muscular aches, constipation, indigestion, hypertension, dementia, fever, and infectious diseases. As an antiemetic, Ginger is consumed by approximately 80% of pregnant women to treat nausea and vomiting of early pregnancy. Purpose The aim of this study is to evaluate the impact of ginger extract on the oestrous cycle and implantation in female mice. Study design and methods Four experimental episodes were identified. One considered the main study of outcomes and lasted 90 days; one lasted 35 days and considered the oestrous cycle; while the third and fourth intended antifertility and abortifacient and continued 20 days for each. Mice dosed Ginger orally at 0, 250, 500, 1000 or 2000 mg/kgbw/day (GNC, GN1, GN2, GN3, GN4, respectively). Results GN3 and GN4 dams showed maternal toxicity. High dose significantly reduced the number of live fetuses and increased fetal death and resorption. Mice treated with 2000 mg/kgbw/day displayed significant decreases in implantation sites. At a dose of 2000 mg/kgbw/day, Ginger prolonged the length of oestrous cycle with a significant decrease in the duration of diestrous-metestrus (luteal) phase, prolonged proestrus-estrus (ovulatory) phase and reduced the number of cycles as well. Therefore, Ginger impairs the normal growth of corpus luteum because of progesterone insufficiency during early pregnancy. The observed-adverse-effect dose set at 2000 mg/kgbw, but no-observed-adverse-effect dose set at 250 and 500 mg/kgbw. Conclusions These findings suggest that Ginger can disrupt the oestrous cycle and blastocyst implantation without teratogenesis. Graphical abstract Image, graphical abstract Download high-res image (258KB)Download full-size image Keywords Ginger Estrous cycle Implantation Fertility Fetus Resorption Abbreviations.: GN: ginger GN1: ginger group 1 GN2: ginger group 2 GN3: ginger group 3 GN4: ginger group 4 H&E: hematoxylin and eosin dpc: day post coitum GD: gestation day SD: standard deviation Mg: gram kg: kilogram P: probability Introduction Since antiquity, Ginger is mainly used worldwide as a popular spice and medicinal plant in Chinese, Ayurvedic and Unani-Tibb medicines (Rong et al., 2009). Globally, ginger (root, extracts, and tinctures) is commercially available as a capsule or syrup form or in candy, cookies, and jam and as a flavouring agent in beverages and many other food preparations (Bryer, 2005). For medical use, Ginger was originally marketed solely as a dietary supplement and in a mixture of other herbs, vitamins, and minerals (Chang et al., 2012). The therapeutic efficacy of Ginger has attributed to its numerous bioactive volatile and non-volatile oils (Jolad et al., 2004), which are known to possess essential activities and may give special prominence to its analeptic and organoleptic effects (Juliani et al., 2004). The active ingredients are structurally and pharmacologically related to its volatile oils, which made up of approximately 1-3% of its weight (Newall et al., 1996). As an antiemetic (Boone and Shields, 2005), Ginger is consumed by approximately 80% of pregnant women for nausea and vomiting remedy of early pregnancy (Ernst and Pittler, 2000), motion sickness (Stewart et al., 1991), postoperative nausea (Visalyaputra et al., 2001), vertigo (Grontved and Hentzer, 1986) and nausea of other etiologies (White, 2007) as well as in pregnant women with severe form of hyperemesis gravidarum (O'Brien and Zhou, 1995). Although, the safety and effectiveness as an alternative to traditional phytotherapy, well-documented data have shown that Ginger per se or its individual bioactive components were cytotoxic and include mutagenicity and antimutagenicity as well as genotoxicity (Celik, 2012). Subsequent cytotoxic studies, however, showed that aqueous and ethanolic ginger extract had antiproliferative and apoptotic properties in a variety of numerous human and rodent cancer cell lines and types (Abdullah et al., 2010). There is a dispute in the teratogenicity of Ginger. A number of studies show that daily doses of ginger extract produced neither embryotoxicity nor teratogenicity up to 1000 mg/kg of rats (Weidner and Sigwart, 2001). Other studies showed that ginger tea caused embryotoxicity associated with an embryonic loss in the prenatal exposure of rats (Wilkinson, 2000 a). Out of 300 non-medical surveys reported that 16 sources of Ginger as unsafe during pregnancy (Wilkinson, 2000 b). A review article reported that, on the treatments for severe nausea and vomiting during pregnancy, ginger showed no evidence of teratogenic abnormalities in infants (Jewell and Young, 2003). In a prospective cohort study, it concluded that ginger does not increase the major deformations over the baseline rate of 1–3% (Portnoi et al., 2003). While the United States FDA (FDA, 2015) considers the Ginger extract “Generally Regarded as Safe” (GRAS), other countries issued warnings on all ginger medicinal products as being unsafe for pregnant women for fetal development (EFFSA, 2009). The phytomedicinal use of Ginger during pregnancy has been reported at high percent not considering abortifacient use, which should not be underestimated (Tiran, 2003). Observations of Shah et al. (2009) on antifertility and abortifacient herbal drugs have demonstrated that Ginger is also used as an abortifacient with Black pepper (Chopra et al; 1969). Although there is no agreement on the correct and the maximum dosage that should be prescribed, there is consent to lessen the possibility of side effects no more than 4000 mg of powdered Ginger or 10,000 mg of fresh Ginger should be taken orally per day (Tiran, 2012). Accordingly, the potential antifertility and abortifacient efficacy of ginger cannot be ignored, especially when Ginger is consumed for a long time and at higher dosages. Furthermore, Ginger has been used not only in one form but also in all types. So, the present study conducted. Materials and methods Materials Processing of Ginger extract Fresh authenticated Ginger (GN) (Zingiber officinale Roscoe, rhizome, family Zingiberaceae) was purchased from a medicinal plant agency in Alexandria, Egypt. One-kilogram cleaned rhizomes were peeled, chopped and dried under the sunlight and then was ground into fine powder. 200 grams of the powder was soaked in one litre of distilled water for 24 h at room temperature. The aqueous extract was filtered by double gauze and concentrated into the final concentrations (250 mg/ml) under reduced pressure (Kamtchoving et al., 2002). HPLC chemo-profiling analyses of Ginger extract The chemo-profiling pattern of Ginger extract was analyzed by an HPLC-UV method. Most of the major active components were terpenes (sesquiterpene) which are zingiberene (15.8%), phenolic compounds such as gingerol (31.5%) and shogaol (27.7%), alkaloids (9.6%), saponins (5.4%). The purity estimated by HPLC-UV was 96.7, 97.2 and 98.3% for [6]-, [8]- and [10]-gingerol, while 97.2, 98.4 and 99.0% for [6]-, [8]- and [10]- shogaol, respectively. Purities of both alkaloids and saponins were 97.5%, and 97.3%, respectively. Animals Screening and accommodation of animals In this study, it used sexually mature and proven fertile female and male mice of the random-bred Crj: CD-1 strain (ICR/CD-1), about 8-10 weeks of age and at least 30 grams in weight. Animals were coming directly from a breeding colony established in the Tudor Bilharz Research Institute (TBRI), Warraq, Giza, Egypt. Initial examination of all female and male mice included external surfaces and orifices, as well as the gross lesions. Thereafter, mice were individually identified, housed (2-3 mice/sex in stainless-steel wire-mesh cages) and left two weeks for acclimation in a controlled and air-refreshed Animal House Unite, Department of Zoology, Faculty of Science, Alexandria University. The temperature was maintained at 23–25 °C, the relative humidity ranged from 45 to 55% and the light were under on/off a 12-h:12-h (L: D cycle). Females were allowed free access soy-free pellets suitable for mice obtained from Agricultural-Industrial Integration Company, Giza, Egypt. Water was also provided ad libitum via an automatic watering system. The soy-free pellets were allowed to decrease the amount of phytoestrogen in the diet and so to reduce the level of estrogen materials (Boettger-Tong et al., 1998). These studies were carried out according to the Good Laboratory Practice Guidelines and Applicable Animal Welfare Legislation (OECD, 1981). Experimental procedures and rules Dosing ranges and grouping design After acclimation, ICR mice were divided into five assigned groups and daily gavaged with distilled water or ginger. Four different experimental groups with doses of the aqueous ginger extract prepared according to the volume level (250 mg/ml). Females of group 1(GN1) were administered 250 mg/kgbw. Group 2 (GN2) was given 500 mg/kgbw and group 3 (GN3) mice were given a ginger extract at 1000 mg/kgbw while mice of group 4 (GN4) were received 2000 mg/kgbw extract orally. While females in the control group (positive control) received the distilled water (vehicle) corresponding to those given in ginger-treated groups. Dosing intervals Main study (long-term study, sub-chronic study): females continued with treatments for 90 days and throughout mating and gestation. Estrous cycle: for two weeks before evaluating vaginal cytology and throughout a 20 day of the evaluation period (35 days total). Antifertility (Pre-implantation loss): females continued with treatments for 20 days and throughout mating. Abortifacient (Post-implantation loss): females continued with treatments for 20 days and throughout gestation. Toxicological aspects Clinical toxicity, body and organs weight and food consumption For clinical morbidity and mortality, females screened twice daily in the morning and evening. Cage-side observations made in 1, 2 and 4 hours after the first dose on the first experimental day and once daily then and any abnormal findings, if any, recorded. Individually, hazardous sings included changes in physiological and autonomic activities, as well as a change in behaviour, were monitored daily. Initial and final body weights were recorded during the experimental duration. Organ weight of the ovary recorded at necropsy. Individual food consumption estimated before dosing and weekly thereafter (Melin et al., 2016). Experimental episodes Episode I: Main study (long-term study) (90 days) Fertility, reproductive performance, and developmental outcomes Before onset this experimental episode, females showed at least constant length of three successive regular oestrous cycles of 4-5 days. The research protocol was strictly followed throughout the experiments by the guidelines for the Care and Housing of Laboratory Animals of United States Environmental Protection Agency (USEPA) (USEPA, 1998). Females in control and treated groups (n = 25 per group) were co-housed (2:1/cage) overnight with an untreated fertile male. Fertilization proved by the formation of a copulatory plug, which defined as the first day of gestation (GD1). On GD20, all dams from each group euthanized by ether anaesthesia. Each late-gestational fetus with placenta was then dissected out of the uterus, separately weighed and evaluated. Viable and dead fetuses counted and recorded. Viable fetuses were evaluated by gestational day, number of somites, development of the branchial arches, the territory of heart and limb bud formation, and deepening of the lens pit. Early and late resorbed fetuses demonstrated by extreme tissue autolysis. Episode II: Estrous cycle (35 days) Regular cycling female mice were assigned into control and treated groups (n = 10 per group). Throughout evaluation period, every morning between 8:00 to 8:30 a.m. oestrous cycle phases were screened according to procedures described by Marcondes et al. (2002). Briefly, epithelial cells were collected daily from the vaginal opening, smeared on a glass slide, air-dried and then stained with Wright-Giemsa. Thereafter, the vaginal cytology was assessed, and the phases of the oestrous cycle were identified according to the specifications of Byers et al. (2012). Episode III: Pre-implantation loss (post-coital anti-implantation, 6th dpc) Successful implantation sites investigated in female mice dosed with Ginger and saline control (n= 10 per group). Females on proestrus stage bred to untreated males overnight in a ratio of 2:1 and the day one of gestation (GD1) was designated by the copulatory plug. On GD6, females injected into the tail vein with 0.1 ml of 1% Chicago Sky Blue 6B dye (Sigma) to stain uterine deciduas (Chien et al., 2013). One minute thereafter, females were laparotomised under light ether anaesthesia. The two horns of gravid uteri were then examined for the number of implanted blastocysts on the uterine epithelium indicated by blue staining. Anti-implantation efficiency was evaluated according to Khanna and Chaudhary (1968). The number of corpora lutea in the ovary was recorded. Episode IV: post-implantation losses and abortifacient (6-10th dpc) As the methodology described above, regular cycling females in control and treated groups (n= 10/group) were cohabited (2:1/cage) overnight with an untreated fertile male. Mating confirmed by observation of a copulatory plug, which recorded gestation day one (GD1). During the abortifacient episode, females observed for vaginal bleeding. On GD10, all dams from each group euthanized by ether anaesthesia. Embryos at mid-gestation (GD10) collected out of the uterus and assessed. Resorbed embryos identified by the disintegration of normal embryonic tissue and abnormality of deciduae as well. All live embryos estimated. Ovarian tissue processing and histopathology On the scheduled day of the experiment, all female mice from each group anaesthetized with sodium pentobarbital (50 mg/ml/kg) (Waynforth, 1988) and sacrificed thereafter. For histopathological preparations, left and right ovarian tissues were randomly assigned to fixed in 10% neutral formalin, dehydrated through graded alcohols, embedded in paraffin wax, serially sectioned at 5 µm, placed on microscopic slides, and finally stained with hematoxylin and eosin (Suvarna et al., 2013). Histopathological examination and then assessment performed. Statistics Statistical analyses were performed using SPSS/Version 17 software, USA (1989–2002) LEAD Tech. Inc. All quantitative data expressed as means ± SD. Differences were considered as significant when P ≤ 0.05. The maternal toxicity parameters (Body weight change, absolute ovary weight, mortality, and abortion) for dose groups were compared utilizing the one-way analysis of variance procedure (ANOVA) followed by Tukey's multiple comparisons. Pairwise Mann–Whitney's test was used for comparisons of the values of the number of implantations, the number of resorptions, the number of live fetuses, and post-and pre-implantation loss to the control. Fetal weights were analyzed using a nested ANOVA followed by Tukey's multiple comparisons. Sex ratio and litter size statistics were compared with the Kruskal-Wallis test. The number of oestrous cycles was compared to the control in each group by the Kruskal-Wallis test, followed by Dunnett-type multiple comparison methods. The values of phases of oestrous cycles were analyzed using GraphPad Prism software 7 followed by Dunnett's Multiple Comparison test. Results Maternal toxicity All assigned pregnant females survived until the scheduled time of necropsy on GD20, except for one mortality from GN3 (1000 mg) and two from GN4 (2000 mg) dose group at days 18 and 16, respectively. No females in preterm birth recorded from the dose groups. No incidences of salivation, tremors, diarrhoea and localized alopecia and abrasions observed during the dosing period among female mice. Also, there were no gross lesions notice in any of the mice from the Ginger-treated groups (P ≤ 0.05). Body weight changes significantly reduced in a dose-dependent pattern over the 90 days in females treated with 1000 and 2000 mg (GN3 and GN4) than vehicle control (GNC) (P ≤ 0.05; Table 1). Despite significant differences between the females dosed with 1000 and 2000 mg and vehicle control group, average food consumption was alike (P ≤ 0.05; Fig. 1). Table 1. Toxicological aspects of female mice orally gavaged Ginger for 90 days. Aspects Ginger groups GNC (positive control) GN1 GN2 GN3 GN4 Mortality 0 0 0 1 2 Abortion incidence % 0 0 0 0 20 Body weight change (g/100g) 101.2 ± 9.8 110.9 ± 11.7 99.0 ± 9.0 46.0 ± 4.9A 33.0 ± 2.8A Ovarian weight (mg) 0.27 ± 0.2 0.26 ± 0.2 0.25 ± 0.1 0.21 ± 0.3 0.21 ± 0.2 The data are presented as mean ± SD, n = 25 female mice/group. A: Significant difference as compared with control group P ≤ 0.05. Eventually females sacrificed at 91th day. A, Significantly different from control at P ≤ 0.05 Fig 1 Download high-res image (110KB)Download full-size image Fig. 1. Average of food consumption (grams) of female mice orally gavaged Ginger for 90 days. (n = 25; mean ± S.D). *Significantly different from control at P ≤ 0.05. Regularity of estrus cycle By the 20 days of evaluation, it was evident that Ginger in the GN4 group disrupted the regularity of oestrous cycle with a significant reduction in the number of normal oestrous cycles (Fig. 3). The average length of the oestrous cycle in the GN4 Ginger group was significantly prolonged (10.05 ± 0.8) days (P ≤ 0.05) compared with (4.99 ± 0.5) days recurrent and successive oestrous cycles in GNC control female mice (P ≤ 0.05) (Fig. 2). While in other treated groups, oestrous cycle recurred at approximately five-day intervals (P ≤ 0.05) (Fig. 2). Proestrus and estrus stage significantly prolonged in the group treated with 51.4 mg/kgbw Ginger compared to the treated groups and control, indicating that females tend to spend a more time in pre-ovulatory phase than control one (Figs. 4A, B). Moreover, the duration of the metestrus and diestrus stage significantly decreased by dose 2000 mg (Figs. 4C, D). Diestrus index was also decreased after the administration of 2000 mg Ginger treatment when compared with those of the corresponding parameters of the control mice (Fig. 5). Fig 2 Download high-res image (109KB)Download full-size image Fig. 2. Mean length of estrous cycle of female mice after 20-day evaluation of oral Ginger treatment. (n = 10; mean ± S.D.). *Significantly different from control at P ≤ 0.05. Fig 3 Download high-res image (120KB)Download full-size image Fig. 3. Mean number of recurrent and successive estrous cycle within 20 days in female mice orally gavaged Ginger. (n = 10; mean ± S.D.). *Significantly different from control at P ≤ 0.05. Fig 4 Download high-res image (507KB)Download full-size image Fig. 4. Effect of Ginger on subsequent estrous phases for 20-day evaluation. A) Proestrus; B) Estrus; C) Metestrus; D) Diestrus. The duration of estrous stages disrupted by Ginger, proestrus and estrus cycles show prolongation, diestrus and metestrus exhibit decrease in their duration and the overall cycle duration prolonged. (n= 10; mean ± S.D.) Fig 5 Download high-res image (109KB)Download full-size image Fig. 5. 20-day evaluation of diestrus index of female mice orally gavaged Ginger. (n = 10; mean ± S.D.) *Significantly different from control at P ≤ 0.05. Pre-implantation loss (Post coitum anti-implantation) The total number of ovarian corpora lutea of groups GN1, GN2, and GN3 for two weeks and throughout mating, up to GD6 of the evaluation were not significantly changed than control (P ≤ 0.05;Table 2) so, Ginger at these doses did not impair fertility and the inseminated females were pregnant. After treatment with 2000 mg/kgbw (GN4), however, the number of corpora lutea was significantly diminished compared to control (P ≤ 0.05;Table 2). Statistically, percent of successful blastocyst implantation site insignificant between GNC control, and GN1, GN2, and GN3 treated mice in the 2 weeks of dosing period (P ≤ 0.05; Table 2). With an increase in the oral dose of the Ginger (2000 mg), the percentage of implantation failure was significantly increased by 36% (P ≤ 0.05; Table 2), which may reflect dose-dependent antifertility (anti-implantation) effect. Moreover, the total number of uterine implants in GN4 group exhibited an increment in the percentage of the rate of the preimplantation loss (16.59 %) compared to control (P ≤ 0.05; Table 2). The preimplantation loss was, however, not different from each other in the GN1, GN2, and GN3 groups (P ≤ 0.05; Table 2). Table 2. Effect of Ginger rhizome extracts on post-coital anti-implantation in female mice for 2 weeks and throughout mating, up to GD6 of evaluation. Estimated parameters Ginger groups GNC (positive control) GN1 GN2 GN3 GN4 No. of corpora lutea/ovary 14.68 ± 1.1 15.02 ± 1.2 15.44 ± 1.1 14.39 ± 1.3 10.97 ± 0.9 A Blastocyst implantation sites/litter 14.47 ± 1.2 14.78 ± 1.1 15.16 ± 1.4 14.14 ± 1.3 9.15 ± 0.9 A Pre-implantation loss% 1.43 ± 0.1 1.60 ± 1.2 1.81 ± 1.4 1.73 ± 0.2 16.59 ± 3.4 A The data are presented as mean ± SD, n = 10 female mice/group (n = 10; mean ± S.D.) A, Significantly different from control at P ≤ 0.05 Post-implantation loss (mid-gestational abortifacient) At mid-gestational day (GD10), Ginger showed abortifacient activity in a dose-dependent manner that evident by the number of implants and litters. In GN4, the number of viable embryos was significantly reduced by 37.5% (P ≤ 0.05; Table 3) accompanied by an increase in the average per litter of resorbed embryos (P ≤ 0.05; Table 3). The increasing trend in post-implantation loss was found by 13.3 % in the 2000 mg Ginger-treated animals (P ≤ 0.05; Table 3). 2000 mg dose of Ginger also resulted in 20 % abortion versus control, as indicated by the number of females with vaginal bleeding and/or preterm birth (Table 1). Table 3. Effect of Ginger rhizome extracts on the mid-gestational abortifacient activity in female mice for 2 weeks and throughout mating and gestation, up to GD10 of evaluation. Estimated parameters Ginger groups GNC (positive control) GN1 GN2 GN3 GN4 No. of mid-gestational viable fetuses/litter 14.59 ± 1.02 14.26 ± 1.08 14.18 ± 1.08 13.75 ± 1.03 9.11 ± 0.58A No. of resorptions/litter 0.72 ± 0.1 0.63 ± 0.2 0.61 ± 0.3 0.79 ± 0.1 2.28 ± 0.2A Post-implantation loss% (abortifacient activity) 6.17 ± 0.60 6.25 ± 0.60 6.03 ± 0.58 7.90 ± 0.76 13.32 ± 1.02A The data are presented as mean ± SD, n = 10 female mice/group (n = 10; mean ± S.D.) A, Significantly different from control at P ≤ 0.05; Fertility, reproductive performance, and developmental outcomes Susceptibility of oral administration of different doses of Ginger for 90 days on reproductive performance, fertility, and intrauterine fetal development outlined in Table 4. Female copulation index was significantly reduced at 2000 and 1000 mg/kgbw groups, whereas the female pregnancy index was significantly decreased only in the 2000 mg group, comparable to the untreated group. So, there was evidence of infertility in the GN4 dose group, which may indicate that Ginger may affect other vital processes in female reproduction, including blastocyst implantation, placentation and/or organogenesis. Also, the number of implantation sites and live fetuses for experimental females of 2000 mg group were lower than those for the control and other treated females. A Ginger-associated increase in fetal resorption (Fig. 6D).and post-implantation loss noticed only in the 2000 mg group as compared to control rats given saline (Fig. 6A). Despite there were signs of fetotoxicity were recorded in GN4 group, no evidence of fetal malformations observed. The gross developmental toxicity was significantly evident with growth retardation (Fig. 6B, C), reduced pup weight, and delayed in the crown-rump length (Table 4; Fig. 6B, C). Table 4. Effects of Ginger on fertility, reproductive performance and developmental toxicity of female mice after 90 days of Ginger oral administration. Estimated parameters Ginger groups GNC (positive control) GN1 GN2 GN3 GN4 Pregnancy behavior + ve + ve + ve - ve - ve Copulation rate (%) 25/25 (100) 25/25 (100) 25/25 (100) 20/25 (80) 17/25 (68) Pregnancy rate (%) 25/25(100) 25/25(100) 25/25 (100) 20/20 (100) 12/17 (71) Preterm delivery 0 0 0 0 0 Litters (fetal number) 25 (375) 25 (378) 25 (368) 18 (261) 12 (27) No. of implants/uterus 15.33 ± 1.1 15.45 ± 1.2 15.00 ± 1.01 14.95 ± 0.98 6.05 ± 0.76 Total No of implants 383 386 375 333 73 Viable fetuses/uterus 15.00 ± 1.3 15.11 ± 1.1 14.70 ± 2.01 14.51 ± 1.3 2.21 ± 0.3A Dead fetuses/uterus 0.11 ± 0.1 0.12 ± 0.1 0.10 ± 0.1 0.11 ± 0.1 1.54 ± 0.2A Early resorptions/ uterus 0.10 ± 0.1 0.11 ± 0.1 0.10 ± 0.1 0.16 ± 0.1 1.70 ± 0.3A Late resorptions/ uterus 0.12 ± 0.1 0.11 ± 0.1 0.10 ± 0.1 0.17 ± 0.1 0.60 ± 0.1A Resorption/uterus 0.22 ± 0.1 0.22 ± 0.1 0.20 ± 0.1 0.33 ± 0.1 2.30 ± 0.4A Post-implantation loss % 2.15 ± 0.3 2.20 ± 0.3 2.00 ± 0.3 2.76 ± 0.3 63.47 ± 4.5A Viability ratio % 97 ± 7.5 97 ± 7.3 98 ± 7.6 97 ± 6.9 37 ± 6.6 Fetal body weight (g) i- Male 1.10 ± 0.2 1.10 ± 0.3 1.14 ± 0.1 1.15 ± 0.1 0.94 ± 0.2 ii- Female 0.96 ± 0.1 0.95 ± 0.2 0.98 ± 0.2 0.99 ± 0.2 0.85 ± 0.3 Crown Rump Length (CRL) (mm) i- Male 1.94 ± 0.3 1.92 ± 0.4 1.89 ± 0.1 1.63 ± 0.2 0.97 ± 0.2A ii- Female 1.42 ± 0.1 1.42 ± 0.2 1.53 ± 0.2 1.32 ± 0.3 1.00 ± 0.1A Sex ratio i- Male/litter 5.3 ± 0.8 5.5 ± 0.9 5.7 ± 0.6 4.5 ± 0.5 1.2 ± 0.3 ii- Female/litter 3.7 ± 0.2 3.6 ± 0.1 4.0 ± 0.1 5.3 ± 0.5 1.0 ± 0.1 Total Males 133 138 143 112 7 Total Females 92 90 100 133 6 Male: Female 133: 92 138: 90 143: 100 112: 133 7: 6 i-% male 57 58 57 44 19 ii- % female 43 42 43 56 81 No. of malformed fetuses /litter 0.32 ± 0.01 0.30 ± 0.01 0.21 ± 0.02 0.25 ± 0.01 0.48 ± 0.03 The data are presented as mean ± SD, n = 25 female mice/group. A; Significant difference as compared with control group P ≤ 0.01, P ≤ 0.05. Eventually females sacrificed at 91th day. A, Significantly different from control at P ≤ 0.05 Fig 6 Download high-res image (2MB)Download full-size image Fig. 6. Susceptibility of oral administration of different doses of Ginger for 90 days on intra uterine fetal development in mice. A, control fetus; B, mid dose group; D and C, high dose. Fetotoxicity of Ginger was clearly identified by growth retardation, delayed in the crown-rump length (arrow), and resorption (arrowhead). Original magnification, 40X. Ovarian histopathology Mice ovary have suffered histopathological damage in GN4 group after 90 days of experimental treatment. The histopathological detailed inspection showed dispersed ovarian follicle atresia (Fig. 7A, C). Atretic follicles had cell debris (Fig. 7B, D) and hemorrhage in the antral cavity (Fig. 7D). In degenerative antral follicle granulosa cells degenerated and desquamated into the antrum (Fig. 7B). On the other hand, ovaries of the GN1, GN2 and GN3 groups appeared no clear histopathological alterations compared to the GNC ovary. Ovarian polycystic characterized by degeneration of primary follicles with pyknotic nuclei were clearly noticed at 2000 mg dosed females. Deteriorated follicles were observed as a detaching of layers of granulosa cells from the basal membrane by dilation of zona pellucida and with evidence of apoptosis (Fig. 7B). the non-visibility of the follicular nuclei was also noticed in damaged ova (Fig. 7B). Because of histopathological damage of follicles and granulosa cells prevalent in ovarian tissue, it is evident that Ginger possesses anti-ovulation properties. These are possibly due to the involvement of saponin in Ginger. Fig 7 Download high-res image (2MB)Download full-size image Fig. 7. Photomicrographs of mice ovary showing histopathological damage in GN4 group after 90 days of Ginger treatment (A, B, C, D panel). The histopathological detailed inspection showed dispersed ovarian follicle atresia (A). Atretic follicles had cell debris (arrow) and hemorrhage in the antral cavity (H). In degenerative antral follicles granulosa cells degenerated and desquamated into antrum (arrow head). Degenerated primordial follicles with pyknotic nuclei forming ovarian polycystic (P). H&E. Discussion In present findings, doses of 1000 and 2000 mg/kgbw/day resulted in maternal toxicity, with increased mortality and significant decreases in maternal weight gain and feed consumption. There was also disruption in the regulation of the estrous cycle, implantation and fetal resorption without teratogenicity in the 2000 mg/kgbw/day dose group. These results contrast with the published studies that reported that Ginger has no maternal toxicity (Weidner and Sigwart, 2000; Wilkinson, 2000a), or no evidence of overt mortality (Rong et al., 2009), while being compatible with the data indicated that no potential teratogenicity noted at the ginger dosages (Wilkinson, 2000a; Weidner and Sigwart, 2001). These disparities might be related to the type of ginger preparations used and the doses administered. Conversely, other results obtained higher weight gain after feeding rainbow trout with ginger diet (El-Desouky et al., 2012; Talpur et al., 2013). These maternal effects are consistent with an alkaloid - mediated toxicity in the sense of taste and appetite (Yakubu and Musa, 2012), or consistent with toxic effects of Ginger on the gastric mucosa (Dissubandara and Chandrasekara, 2007). No significant adverse effects noticed in the 250 and 500 mg/kg/day dose group. Despite no clinical evidence of harm observed, safety concerns still exist in the published literature relevant to this herb regarding its use by pregnant women (Keating and Chez, 2003). The incidence of vaginal bleeding monitored in the 2000 mg/kgbw/day treated females could be attributable to anticoagulant activities of (8 - (Gingerol, (8) - Shogaol, (8) – Paradol of Ginger constituents (Mishra et al., 2012) and bleeding effects (Spolarich and Andrews, 2007) through the decrease of platelet thromboxane production (Nurtjahja-Tjendraputra et al., 2003). This episode before laparotomy, not only suggests abortion but also fetotoxicity (Yakubu et al., 2010). On the other hand, as a contraceptive (Dabhadkar and. Zade, 2013), alkaloids, flavonoids, and saponins have oxytocic effects and known as promoters of abortion (Saraiya et al., 1998). Furthermore, the abortive activity might be mediated by high levels of estrogen, which could be explained due to the phytoestrogenicity of flavonoids (ElMazoudy and Attia, 2012), because estrogens are known to increase uterine contractility and cause abortion (Shibeshi et al., 2006). These effects may in part contribute to its anti-implantation and abortifacient activities. A possible interpretation of the anti-implantation and abortifacient effects exerted by high ginger dose (2000 mg/kgbw) could implicate alkaloids, flavonoids, and saponins. Virtually, flavonoids can block and inhibit the cell division (Skibola and Smith, 2000) modulating the cell cycle (Singh and Agarwal, 2006) and can also inhibit enzyme activities, such as protein kinases involved in the regulation of cellular proliferation and apoptosis (Galati and O'Brien, 2004). This could interpret the anti-implantation through failure to form deciduoma in the endometrium, which is essential for blastocyst implantation (Latha et al., 2013). In addition, flavonoids may act as endocrine disruptors as they have preferential competitive binding affinities for estrogen receptors (ERs) (Kang et al., 2006). As such, it seems likely that Ginger mediates its toxic effects on fetogenesis at least in part via its ER selective activities. It demonstrated that alkaloids induced toxicity in fetal and maternal parameters through inhibition of concentration of LH and FSH (Yakubu et al., 2010) and caused fetal death, most likely because of blocking of mitosis with metaphase arrest (Okouneva et al., 2003), and then pregnancy termination (Yakubu and Musa, 2012). Accordingly, alterations in the regular oestrous cycle, reduction in the number of blastocyst implantation may attribute to the antifertility effect of flavonoids and saponins (Wikhe et al., 2012). Furthermore, phytochemical investigations proved that the existence of alkaloids, terpenoids, and saponins cause infertility (Hiremath and Hanumantha, 1990), an anti zygotic and anti-implantation activity (Francis et al., 2002), suggesting that the antifertility effect of Ginger might be due to the presence of one of these bioactive. Also, the antigonadotrophic effects of alkaloids, flavonoids, and saponins have been implicated in irregularity of the oestrous cycle (Oluyemi et al., 2007). The irregularity of the oestrous cycle by shortening the metestrus - diestrus luteal stage and by lengthening of the proestrus - estrus follicular stage, clearly show that Ginger targeted against the hypothalamus-pituitary-ovary axis. This could be attributable to the presence of high level of phytoestrogens like steroidal saponins (Tamura et al., 1997). It was clear that the antifertility effect of saponins and flavonoids based on disturbances in normal cyclicity (Wikhe et al., 2012). In fact, the alterations of estrous cycle and ovarian toxicity profiling in present results may reflect the direct cytotoxic effect of the active Gingerol (Peng et al., 2012) that hypothalamus - pituitary could be also affected by Gingerol stress, a conclusion that should be supported by further studies, involved in cell apoptosis (Yang et al., 2012). So, the derangement of normal cyclicity and increased implantation loss observed in high dose display its antifertility effects. Another side-effect of saponins and alkaloids has been found to possess anti-ovulatory properties (Mukherjee et al, 2006). Moreover, in vitro and in vivo data suggests that alkaloids act an antiangiogenic agent (Eun and Koh, 2004) by inhibiting the VEGF secretion of granulosa cells disrupting follicular angiogenesis (Basini et al., 2007). Conclusion The present study points out that the probable mechanisms of ginger toxicity or some of its constituents: (1) may be mediated essentially via antagonism of the cholinergic M receptor and serotonin receptor-blocking activity, due to the presence of [6]-gingerol, [6]-shogaol, as these constituents could easily cross the blood-brain barrier. This could perturb the release of neurotransmitter levels in the hypothalamus-pituitary-gonadal axis and thereby deteriorate the excretion of gonadotropic hormones and female cycle; (2) could block the gene expressions regulating NO-mediated signaling pathways and controlling angiogenesis that leads to inhibition of cell proliferation and influences cell growth, arrest of cell cycle, apoptosis, and migration implicated in embryogenesis; (3) and the mutagenic, antimutagenic, hypoglycemia, cytotoxic and anti-inflammatory properties interfering with a variety of cellular pathways might be involved in ginger toxicity such as growth retardation induced by suppression of key enzymes controlling carbohydrate metabolism, impaired placental function by inhibition of the arachidonic acid cascade, embryo loss by mutagenicity, and apoptosis through the activation of p53 and increased caspase-3. 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