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Journal of Ethnopharmacology 137 (2011) 1498– 1503

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

jo ur nal homep age : www.elsev ier .com/ locate / je thpharm

eproductive toxicity of Rhizoma Sparganii (Sparganium stoloniferumuch.-Ham.) in mice: Mechanisms of anti-angiogenesis and anti-estrogenharmacologic activities

ie Sun, Shao Wang, Ya-Hui Wei ∗

ey Laboratory of Resource Biology and Biotechnology in Western China, School of Life Science, Northwest University, Xi’an 710069, China

r t i c l e i n f o

rticle history:eceived 9 February 2011eceived in revised form 11 August 2011ccepted 14 August 2011vailable online 22 August 2011

eywords:hizoma Sparganiiibroblast growth factor-1strogen receptor-�: Vascular endothelialrowth factor

a b s t r a c t

Ethnopharmacological relevance: Indications and preliminary studies of Rhizoma Sparganii (RS) suggestits pharmacological mechanism is involved with endocrine/angiogenesis functions. We therefore studiedits potential toxicity on reproduction in mice.Materials and methods: Reproductive toxicity of 100, 200 and 400 mg/kg RS extract were studied in preg-nant ICR mice and its offspring. The embryos’ fibroblast growth factor-1 (FGF-1), vascular endothelialgrowth factor (VEGF) and estrogen receptor-� (ER-�) were evaluated as targets of endocrine/angiogenesisby immunohistochemical test.Results: The offspring of treated mice (100, 200 and 400 mg/kg RS extract) during their pregnancyhad various pathological conditions, suggesting an abnormal FGF signaling phenomenon during preg-nancy. Embryos from the 400 mg/kg group had significantly depressed levels of FGF-1 (P < 0.01) and

umor necrosis factor-� VEGF (P < 0.05) expression levels as compared to controls by immunohistochemical test. Dysplasia in theheart (12.9%), craniofacial region (18.3%) and vertebrae (32.5%) presented in embryos of the 400 mg/kggroup. Furthermore, the ER-� expression was inversely proportional to FGF-1 levels in the same embryo(P < 0.01).Conclusions: These results implicate a FGF signaling abnormality in vivo and indicate that RS has anti-

rogen

angiogenesis and anti-est

. Introduction

Dried rhizome of Sparganium stoloniferum Buch.-Ham. (Rhizomaparganii, RS) is a frequently used traditional Chinese folk medicine.n aqueous extract of RS (RS-W) is widely used in the treat-ent of blood stasis, amenorrhea, functional dyspepsia, and early

tages of tumors especially for hysteromyoma in China (Chineseharmacopoeia Commission, 2010). These indications suggest thathe pharmacological mechanism of RS-W could be related to anti-ngiogenesis and anti-estrogen. It is known that angiogenesis isssociated with wound healing, pregnancy and tumor developmentFerrara and Kerbel, 2005; Fan et al., 2006) and estrogen, involvedith menstrual cycle and accelerating cell division of tumor cells

Imagawa et al., 2002; Maki et al., 2002).

Total flavonoids of Sparganium stoloniferum Buch.-Ham. (RS-F)

ave anti-platelet and anti-thrombotic actions in SD rat. 42 mg/kgS-F daily group showed a lower platelet aggregation rate than0 mg/kg aspirin positive control. And in contrast to 80 mg/kgspirin group, 84 mg/kg RS-F group showed alike protection

∗ Corresponding author. Tel.: +86 29 88302199; fax: +86 29 88303226.E-mail address: weiyahui@nwu.edu.cn (Y.-H. Wei).

378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2011.08.026

toxicity effects in pregnant rodents.© 2011 Elsevier Ireland Ltd. All rights reserved.

activity against thrombosis that is induced by collagen-adrenalineinjection (Lu et al., 1999). Serum from RS-W treated mice reducesestrogen-2, steroidogenic factor-1, 17-beta-hydroxysteroiddehydrogenase-1 and vascular endothelial growth factor (VEGF)secretion from endometrial cells of endometriosis and hysteromy-oma in vitro (Li et al., 2008, 2009). Serum from RS-treated animalscan reduce micro vessels density, inhibit the level of protein andmRNA of VEGF and TNF-� in ectopic endometriotic, and inhibitvascular endothelial cell proliferation induced by VEGF (Ye et al.,2007; Chen et al., 2008). A recent study shows that RS has a betteranalgesic and anti-inflammatory effect in writhing experimentsbut has only a weak toxicity to normal mice because no death orhistopathology was found with the treatment of 7200 mg/kg RS-Wdaily for 14 d (Ma et al., 2009). However, a potential toxicity of RSon the reproductive tract of pregnant female animal has not beenstudied.

The embryo has many developmental mechanisms such as hor-mone secretion, growth factor expression and angiogenesis in

common with the tumor. As a result, anti-tumor drugs targetingon those common developmental mechanisms always show tox-icity on embryos. A recent study demonstrates that the primarycause of thalidomide-induced limb malformations is the loss of the

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orming limb vasculature (Therapontos et al., 2009) and now it haseen used clinically as anti-cancer drug targeting the inhibition ofumor vessel development (McMeekin et al., 2007). Although nooxicity has been reported in the normal animal study with RS, ittill has a potential reproductive toxicity because of its gynecologi-al indications. Therefore, study of anti-angiogenesis/anti-estrogenoxicity of RS-W in pregnant animal is important issue for its safety.o investigate the toxicity mechanism of endocrine/angiogenesisction for RS-W in developing embryos, we first studied the repro-uctive toxicology in pregnant ICR mice and offspring. The levels ofEGF and FGF-1 in embryos were evaluated as targets of angio-enesis. Based on the inhibition of FGF-1 expression, we alsonvestigated RS-W toxicity on the ER-� expression in embryo bymmunohistochemistry (IHC).

. Materials and methods

.1. Herbal preparation

Rhizoma Sparganii was purchased from Yi-Kang Chain Medicineo. (Xi’an, China), and was ground into powder. This drug qualityas consistent with the Good Supplying Practice (GSP) regulated

y the Food and Drug Administration of China. A voucher specimenas deposited in Key Laboratory of Resource Biology and Biotech-ology in Western China. The RS powder (0.1 kg/L) was immersed

n boiling distilled water 20 min and then kept at 80 ◦C for 4 h asescribed in Chinese traditional methods. The supernatant wasollected and dried at 80 ◦C under 0.7 Pa pressure as RS-W, andhe yield was 9.81% (dry weight/dry weight). The extract contain-ng 61.26% starch polyose and 38.23% active compounds that were

ainly composed of Grailsine-Al-glucoside, flavonoids and a fewhenylpropanoid glycosides (Shirota et al., 1996; Zhang et al., 1996;hirota et al., 1997; Sun and Wei, 2011). The RS-W was freshly pre-ared every day, and dilutions were made into distilled water. Inhis study, mice were treated by oral gavage with daily doses of00, 200 and 400 mg/kg RS water extract.

.2. Animals

ICR mice were obtained from the Jiaotong University Laboratorynimal Center (Xi’an, China; license number, SCXK2007-001). Eighteeks old with weights of 23.00 ± 1.50 g. All mice were reared at

temperature of 25 ± 1 ◦C with a 12 h light/dark cycle and wereree to standard mouse chow (Jiaotong University Laboratory Ani-

al Center) and water according to standard procedures during thentire experiment. The study was approved by the Animal Experi-ental Committee of Northwest University, Xi’an, China.

.3. Animal experiment

Adult male mice (n = 25) were kept singly in a cage for 3–5 daysrior to mating. Adult female mice (n = 175) were kept in largeage (each, n = 25) and were checked every afternoon. Those instrus were allowed to mate overnight (one male and one femaleer cage). The following morning, female mice with testicularlugs were kept as pregnant models (each group consisted about0 mice). Noon of this day was considered to be 0.5 days post-oitum (dpc) and the birthday of offspring as day 1.0 postpartumdpp) (Nagy et al., 2006). Mice were randomly allocated to con-rol or treatment groups, and were given 0.2 ml water, 100, 200 or00 mg/kg RS-W once daily by gavage method.

In this investigation, pregnant mice were given distilled waternegative control) and RS-W (100, 200 and 400 mg/kg) daily from.5 dpc until birth (18.5–19.5 dpc) (Jin, 2005; Nagy et al., 2006).he weaning weight, number of surviving offspring, sex ratio and

cology 137 (2011) 1498– 1503 1499

abnormal development data of offspring were collected on 21 dpcand data were analyzed statistically (t-test between two groups).

2.4. Immunohistochemistry

Rabbit anti-FGF-1 (bs-0229R), rabbit anti-TNF-� (bs-0078R)and rabbit anti-estrogen receptor-� (bs-0122R) were pur-chased from Biosynthesis Biotechnology (Beijing, China, alldiluted to 1:300). Rabbit anti-VEGF (BA0407, diluted to 1:100)and Streptavidin–Biotin-enzyme Complex-AP (SA1052-rabbitIgG)were purchased from Boster Biological Technology (Wuhan, China).

Pregnant mice (n = 31) were sacrificed by cervical dislocation on13.5 dpc. Embryos in 400 mg/kg group (n = 240) and control group(n = 272) were dissected and fixed in 4% paraformaldehyde, afteralcohol dehydration, were embedded in paraffin. The IHC experi-ments were performed by routine methods. Embryo sections, 7 �min thickness, were stained with primary antibody at 37 ◦C for 1.5 h,secondary antibody for 30 min, and finally with BCIP/NBT substratefor 15 min.

2.5. Cytokines statistical analysis

The average gray scale value of different embryo sections wereobtained using Image-Pro Plus 6.0 and two embedded sections ofdata per embryo with the same mean identification area (P > 0.05)were collected for cytokine expression analysis; t-tests were usedto compare groups. The correlation analysis of cytokine expressionin each individual embryo was analyzed using SPSS 13.0 (two-tailed). Data analyses of development toxicity were performedusing a statistical t-test.

3. Results

3.1. Reproductive toxicology of RS-W in mice

Except the mean litter size, mice from the 100 mg/kg and200 mg/kg groups (n = 20, each) showed no toxicity in offspring ascompared to the control group (n = 20, Table 1). Mice treated with400 mg/kg RS-W showed more abortion than in the normal preg-nant mice. But their mean birth weight of offspring and the meanweaning weight of offspring were similar to that of the controlgroup (P > 0.05). All three groups of mice receiving 400 mg/kg RS-Wexhibited a shorter pregnancy time (18.0–19.0 dpc) and had feweroffspring than those in the control group (Table 1). The mean num-ber of surviving offspring in the 400 mg/kg group was significantlylower than that of control group (P < 0.001). This result suggestedthat, in the 400 mg/kg group, approximately 23.9% of embryos hadbeen absorbed during 7.5–19.0 dpc. Furthermore, the surviving off-spring of the 400 mg/kg group had a higher male to female ratiothan the controls (Table 1). Some of the offspring presented withumbilical tumors, dysplasia of the phalanges, and reduced mobility(Fig. 1). Those mice that gave birth to offspring with dysplasia alsohad the shortest gestation times (18.0–18.5 dpc).

3.2. Immunohistochemistry of cytokines

Although pregnant mice treated with 400 mg/kg RS-W (n = 15)presented 6.7% fetal atrophy on 13.5 dpc, the mean embryo num-ber (16 ± 0.85) was not statistically different from the control group(n = 16, 17 ± 0.85, P > 0.05). This suggests most of the destroyedembryos in the 400 mg/kg group had not been absorbed on 13.5 dpcyet.

Embryo sections clearly showed antigen–antibody reactionsthat were especially well defined around the spinal vertebral body.The average VEGF and FGF-1 levels were obviously reduced in400 mg/kg groups compared to controls. In contrast, TNF levels

1500 J. Sun et al. / Journal of Ethnopharmacology 137 (2011) 1498– 1503

Table 1Effect of RS-W treatment on reproduction.

Group (number) Pregnancy times (dpc)(number)

Mean litter size (x̄ ± SD) Sex ratio(male/female)

Accumulatedabnormalities (number)

Mean birth weight(g) (x̄ ± SD)

Mean weaningweight (g) (x̄ ± SD)

0 mg/kg ≤18.5, 18.5–19, ≥19G1 (20) 20 16.35 ± 1.39a 1.03 (166/161) None 1.85 ± 0.17a 11.46 ± 0.92a

100 mg/kg ≤18.5, 18.5–19, ≥19G2 (20) 20 15.20 ± 1.48b 1.00 (152/152) None 1.83 ± 0.18a 11.47 ± 0.99a

200 mg/kg ≤18.5, 18.5–19, ≥19G3 (20) 20 14.75 ± 1.62 1.02 (149/146) None 1.85 ± 0.18a 11.42 ± 0.89a

400 mg/kg ≤18.5, 18.5–19, ≥19G4 (20) 5, 15 10.45 ± 1.86d 1.31 (115/88) Umbilical tumor (4) 1.87 ± 0.19a 11.42 ± 0.94a

G5 (20) 3, 17 11.10 ± 1.76d 1.20 (126/105) Dysplasia of phalange (7) 1.86 ± 0.21a 11.50 ± 0.88a

G6 (20) 20 10.80 ± 1.92d 1.20 (116/97) 1.86 ± 0.21a 11.45 ± 0.95a

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1–G6: Eight weeks old female ICR mice with testicular plugs were randomly alloG4–G6). Mice in treatment groups were given freshly prepared RS-W by gavage m

< 0.001.

id not differ between groups (Fig. 2A–F). The FGF-1 expressionas seriously even completely inhibited in 400 mg/kg embryos

Fig. 2D–F).Besides, contrast to the control group, embryo morphological

hanges are also notable in 400 mg/kg treatment groups (Table 2).ypoevolutism of the heart and abnormal morphology of theasal cavity also appeared in embryos from the 400 mg/kg groupFig. 2G–I). Abnormal vertebral body morphology appeared in00 mg/kg embryos that had reduced FGF-1 expression, which indi-ates dysostosis of the spinal bone (Fig. 2D–F, K and L).

A study shows that 17�-estradiol regulates FGF-1 mRNAs in aell-specific manner in vitro, and up-regulates normal cell (NMEC)GF-1 mRNA (except 1.B spliced) as well as protein expressionYazidi et al., 1998). Thus, we analyzed embryos for ER-� expressiono explore whether RS-W might act via an anti-estrogenic mecha-ism. As a result, the ER-� expression was significantly enhanced

n the 400 mg/kg group compared to controls (Fig. 2A). And higheruantity of ER-� was correlated with lower level of FGF-1 in samembryos (P > 0.01). The mean ER-� gray value around spinal verte-

ral, craniofacial, lingual areas in the sections of 400 mg/kg groupas statistically different to that of the negative controls (Fig. 2J).bnormalities of the vertebral body, heart and nasal cavity werebvious in the ER-� stained sections (Fig. 2G–I).

ig. 1. Pathological defects in 400 mg/kg RS-W-treated mice offspring. (A and C) DysplasF) Normal phalanges in the offspring.

to control group (G1), 100 mg/kg group (G2), 200 mg/kg group (G3) or 400 mg/kg. Same letters (b, c) mean P > 0.05; (a, b) P < 0.05; (a, c) P < 0.01; (a, d), (b, d), (c, d)

Although skeletal, myocardial and craniofacial developmentdefects were observed in some of the embryos from the 400 mg/kggroup, similar defects were not observed in weaned offspring. Itsuggests the severity of such defects lead to an embryo abortionlater.

3.3. Pathology in the female parent

In the one and a half months following offspring lactation, thefemale parents (n = 60) in 400 mg/kg RS-W-treated group showedhigher mortality than that of the groups that received only the vehi-cle (n = 20, Table 3). All the ailing mice present as reduced bodyweight, poor appetite, reduced activities and tachypnea beforedeath. After dissection, diseased mice showed a gland hyperplasiaaround the uterus. In some individuals, the hyperplasia presentedintestinal invasion and even neoplasms.

4. Discussion

A previous study has shown no toxicity of RS in normal mice (Maet al., 2009), but in the present study, there is a dose-dependentincrease on mean offspring litter size of 100, 200 and 400 mg/kgRS extract treated mice. Offspring of 400 mg/kg groups showed

ia of phalange in the right foot. (E and H) Left foot hyperplasia. (B and G) Syndacty.

J. Sun et al. / Journal of Ethnopharmacology 137 (2011) 1498– 1503 1501

Fig. 2. Effect of Rhizoma Sparganii on the expression of cytokines in embryos. (A) Cytokines analysis between 400 mg/kg and negative control groups. (J) Estrogen receptoreav

TE

xpression level in different section of 400 mg/kg group embryos. *P < 0.05 and **P < 0.0ntibodies (G–I, K and L). B, D, G and K are control (Abbreviation: a, atrial; c, craniofacentricular).

able 2ffect of RS-W on 13.5 dpc embryonal morphology.

Groups (number) Hypoevolutism of heart(number/percentage)

Abnocavity

0 mg/kg (272) 0/0% 0/0%

400 mg/kg (240) 31/12.9% 44/18

1. Sections were stained with anti-VEGF (B and C); anti-FGF-1 (D–F); anti-ER-�ial; d, diencephalons; e, endplate; l, lingual; n, nasal; p, pharyngeal; s, spinal; v,

rmal morphology of nasal (number/percentage)

Abnormal morphology of vertebralbody (number/percentage)

0/0%.3% 78/32.5%

1502 J. Sun et al. / Journal of Ethnopharma

Table 3Toxicity effect of RS-W on mouse mother.

Groups (number) Death count (number) Mortality

0 mg/kgG1 (20) 0 0%

100 mg/kgG2 (20) 0 0%

200 mg/kgG3 (20) 0 0%

400 mg/kgG4 (20) 2 10%G5 (20) 2 10%

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CNagy, A., Gertsenstein, M., Vintersten, K., Behringer, R., 2006. Manipulating the

Mouse Embryo, 3rd ed. Cold Spring Harbor Laboratory Press, USA.

G6 (20) 3 15%

arious pathological conditions, including umbilical tumor, dyspla-ia of phalange and unbalanced sex ratio. Moreover, female parentsf 400 mg/kg group presented shorter pregnant times, and 13.3%ortality (n = 60) which was higher than the 100 mg/kg, 200 mg/kg

nd vehicle groups (respectively, n = 20). In our study, RS-W had strong toxicity in pregnant mice and its offspring. This leads uso the conclusion, that pregnant women should not receive thisreatment.

Although RS-W reproductive toxicity had no relationship withNF by immunohistochemical results, the mean VEGF expressionevel in the RS-W treated embryo was significantly lower thanhe control group whereas the ER expression level was higherhan controls. The results of RS-W study indicated that anti-ngiogenesis/anti-estrogen toxicity was in accordance with thether studies (Ye et al., 2007; Chen et al., 2008; Li et al., 2008,009). Furthermore, we found that the RS treated group had aeduced expression of FGF. FGF is another important growth fac-or of angiogenesis and the downstream growth factor mediatedy estrogen (Fujimoto et al., 1997; Ferrara and Kerbel, 2005).GF signaling plays an important role in osteoblast differentia-ion (Marie, 2003). Other reports have confirmed that FGF-2/-9 cannhance osteogenic potential in vitro and FGFs affect the develop-ent of early hypertrophic chondrocyte (FGF-9/-18), calvaria and

imb (FGF-9/-18/-20), as well as inner ear (FGF-3/8/-10) in mousembryo (Hajihosseini and Heath, 2002; Fakhry et al., 2005; Hungt al., 2007; Zelarayan et al., 2007).

A reversible increased ER-� expression in MCF-7 breast cancerells by estrogen antagonists has been reported (Laios et al., 2003).oreover, estrogen antagonists can reduce estrogen-enhanced FGF

xpression in vitro (Fujimoto et al., 1997). FGF signaling pathwayas a complex relationship with Twist expression and the deficiencyf Twist can hinder the growth of limb buds and cranial suture clo-ure in transgenic mouse embryos (O’Rourke et al., 2002; Zunigat al., 2002; Connerney et al., 2008). Several reports have also shownhat FGFs are required for heart development of mouse embryosnd abnormal FGF signaling can induce defects in avian facial mor-hogenesis in vivo (Lavine et al., 2005; Lu et al., 2008; Szabo-Rogerst al., 2008). In this study, female embryos exhibited dysplasiaf the phalanges (Fig. 1), suggesting a FGF signaling disturbancey RS-W during pregnancy. We also observed heart and facialefects (Fig. 2G–I) that suggest abnormal regulation of FGF expres-ion. Besides, a higher male offspring sex rate, induced cancer-likeesions in treated female parents implicate anti-estrogen activity.hese results confirmed RS has anti-angiogenesis and anti-estrogenoxicity effects in pregnant rodents and implicate a FGF signalingbnormality in vivo.

onflict of interest statement

The authors do not have conflict of interest in this work.

cology 137 (2011) 1498– 1503

Acknowledgments

We thank Jia-Kun Dai and Ding Ma for animal breeding and YangQi, Bing Yan, and Na Tian for technology support.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jep.2011.08.026.

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