An in-vitro study of ginsenoside Rb1-induced teratogenicity using a whole rat embryo culture model

L.Y. Chan1, P.Y. Chiu and T.K. Lau

Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong

1 To whom correspondence should be addressed. e-mail: lyschan{at}cuhk.edu.hk


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Ginseng is a commonly used herbal medicine worldwide. However, there is limited information regarding its effects on the developing embryo. METHODS: The effect of ginsenoside on the developing embryo during the critical period of organogenesis was investigated using a whole rat embryo culture model. Embryos were exposed to various concentrations of ginsenoside Rb1 and scored for growth and differentiation at the end of the culture period. RESULTS: Median total morphological scores in embryos exposed to 30 µg/ml of ginsenoside Rb1 was significantly lower (P < 0.05) than that in control embryos (35 versus 45). Morphological scores for flexion, forelimb and hindlimb were also significantly reduced. The median total morphological scores further decreased to 28 when the concentration of ginsenoside Rb1 was increased to 50 µg/ml. At this concentration, the embryonic crown–rump length and somite number were also significantly reduced compared with control embryos (2.8 versus 3.0 mm and 16.0 versus 21.0, respectively). CONCLUSIONS: Our study has demonstrated that ginsenoside exerts direct teratogenic effects on rat embryos. Until more is known about the effects of ginsenoside in women of reproductive age, we suggest its use should be treated with caution.

Key words: ginseng/ginsenoside Rb1/teratogenicity/whole rat embryo culture


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ginseng is a commonly used herbal medicine around the world. Ginsenosides are believed to be the major active agents of ginseng (Gillis, 1997Go).

Women frequently consume herbal medicine during pregnancy. In a recent survey, 9.1% of pregnant women reported use of herbal supplements (Gibson et al., 2001Go), including ginseng. In Asian countries, up to 10% of women had taken ginseng during their pregnancy (Chin, 1991Go). Although it is a general belief that ‘natural’ herbal medicines are better and safer than conventional medicines, many herbal medicines are in fact associated with serious toxic effects (De Smet, 1995Go). For example, Pennyroyal, a widely available herbal medicine, is hepatotoxic, neurotoxic and teratogenic (De Smet, 1995Go). Despite widespread usage of ginseng during pregnancy, information concerning the potential effect of ginseng on developing fetus is lacking. The aim of the present study was to investigate the direct effect of ginsenoside Rb1, one of the most important active components of ginseng, on the rat embryo during the critical period of organogenesis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Timed-gestation pregnant Sprague–Dawley rats were supplied by the Animal House of the Institute as the embryo donors in this study. The day on which spermatozoa were found in the vaginal smear was defined as day 0 of pregnancy. The institutional Animal Research Ethics Committee approved this study.

Whole embryo culture
The whole embryo culture system was based on a previously described model (New, 1978Go). Animals were killed by diethyl ether overdose (Merck, Lindenplatz, Germany) at gestational day 9.5 between 9 and 10 a.m. in the morning, and embryos were explanted. To minimize variation, only embryos with crown–rump length of 1.5 ± 0.3 mm were used for experiment. Embryos were then cultured for 48 h using a rotating-bottle culture unit (BTC Engineering, Bolton, UK), rotating at a constant rate of 60 revolutions/min. Three to five embryos were placed in one culture bottle that contained 1 ml culture medium/embryo.

Each milliliter of culture medium contained: (i) equal volume of Sprague–Dawley rat serum and Dulbecco’s modified Eagle’s medium (Gibco-BRL, Gaithsburg, MD USA); (ii) penicillin G (Sigma, Poole, UK) and streptomycin sulfate (Sigma) at final concentrations of 60 µg/ml ad 100 µg/ml, respectively; and (iii) ginsenoside Rb1 (Sigma) at different final concentration depending on the study group.

During the period of culture, the system was continuously aerated with initially a gas mixture of 5% CO2, 5% O2 and 90% N2 for 24 h, followed by 5% CO2, 20% O2 and 75% N2 for the next 8 h, and 5% CO2, 40% O2 and 55% N2 for the remaining 16 h. The switching of aerating gas was performed automatically by a timer-controlled system. Different types of gas mixtures were premixed and prepared commercially.

Experimental groups
During the first part of the experiment, embryos were randomly assigned to one of the three study groups. Group 1 is the control group, without ginsenoside. Embryos in groups 2 and 3 were exposed to ginsenoside Rb1 at a concentration of 5 and 50 µg/ml, respectively. Based on the result of the first part of the experiment, the second part was performed to investigate the lowest teratogenic concentration of ginsenoside Rb1, and the following concentrations were used: 0 (control), 15, 30 and 40 µg/ml.

Morphological assessment
Embryos were examined after 48 h of culture at the equivalent of 11.5 days of gestation by a researcher who was not aware of the study group assignment. Mean yolk sac diameter and crown–rump length were measured. Embryonic morphologies were studied according to a standard morphological scoring system (Van Maele-Fabry et al., 1990Go), which gives a numerical score (of 0–5) to 17 morphological features depending on their stage of development. Only viable embryos were included in the analysis. Viability was based on the overall appearance of the embryo and the presence or absence of a heartbeat or circulation.

Statistical evaluation
Between group differences were analysed by Kruskal–Wallis test; the least significant difference test was used as an a posteriori test, when a difference was found with Kruskal–Wallis test. All analyses were performed using the Statistical Package for Social Sciences for Windows version 10.0 (SPSS Inc., Chicago, IL, USA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the first part of the experiment, rat embryos were exposed to 0, 5 or 50 µg/ml of ginsenoside Rb1. Three non-viable embryos were excluded in the 50 µg/ml group. Kruskal–Wallis test revealed significant between group differences in total morphological score, number of somites, crown–rump length and yolk sac diameter (Table I). Post-hoc test showed that embryos exposed to high concentration of ginsenoside Rb1 (50 µg/ml) had significantly lower total morphological score, number of somites and crown–rump length (Table I) than those in the control group.


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Table I. Developmental characteristics of rat embryos exposed to high ginsenoside Rb1 concentration
 
When embryos were exposed to a lower concentration of ginsenoside Rb1 (15, 30 and 40 µg/ml), there was no significant difference between study and control groups in yolk sac diameter, crown–rump length and somite number. However, there was a significant difference among the four groups in total morphological score (Table II). Post-hoc analysis showed that embryos exposed to ginsenoside Rb1 at concentrations of 30 and 40 µg/ml had a significantly lower total morphological score compared with the control group. Regarding individual morphological features, Kruskal–Wallis test and post-hoc analysis also revealed that these embryos had significantly lower scores for flexion, heart, limbs and eyes development.


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Table II. Developmental characteristics of rat embryos exposed to low ginsenoside Rb1 concentration
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In many countries, including the UK and USA, herbal medicine such as extracts of ginseng are placed on the market as food supplements. As a result, these ‘food supplements’ are available over the counter and manufacturers are not required to submit proof of safety and efficacy before marketing. Although there are numerous reports in the literature concerning the potential beneficial effects of ginseng, much less is know about the potential toxicity of ginseng. Previously reported potential adverse effects of ginseng include hypoglycaemia, an increased risk of bleeding and a decreased anticoagulant effect of warfarin (Ang-Lee et al., 2001Go). There are no data in the literature concerning the potential effect of ginseng on the developing fetus.

In the present study, we investigated the direct effect of ginsenoside Rb1 on rat embryos during the critical period of organogenesis. Ginsenoside can be divided into two major groups, namely panaxadiol and panaxatriol (Attele et al., 1995Go). Rb1 is the representative ginsenoside from the panaxadiol group. It is also the major ginsenoside in North American ginseng (Kitts et al., 2000Go). Our study showed that ginsenoside Rb1 has a significant effect on the morphogenesis of rat embryos. Exposure to ginsenoside Rb1 at concentration of >=30 µg/ml resulted in a significant reduction of total morphological score and scores for some individual features. The importance of this concentration in human pregnancies is uncertain. We were unable to retrieve any information on plasma concentration of ginsenoside after oral ingestion in human from the medical literature. The only pharmacokinetic studies of ginsenoside in human are by Cui and colleagues (Cui et al., 1996Go; 1997Go), which showed that ginsenoside is present in urine in human after oral ingestion. Further investigation is necessary to evaluate the pharmacokinetics and placental transfer of ginsenoside in human.

It should also be noted that the reduction in morphological score is dose dependent. It is therefore possible that lower concentration of ginsenoside Rb1 might have caused less severe abnormalities that would escape detection by our methods of embryo assessment, including morphological and biometrical, which were designed to study gross derangements only.

Ginsenoside Rb1 is only one of the ginsenosides present in commercially available ginseng extracts. More than 20 ginsenosides have been identified (Gillis, 1997Go). Previous studies had shown that different ginsenoside might have different or even antagonistic actions (Corthout et al., 1999Go). Further studies are required to evaluate the potential teratogenic effects of other ginsenosides and their addictive effects on embryogenesis.

Although results from animal teratogenicity studies may not reflect the circumstances in humans, our findings suggest that further investigations and monitoring of embryonic effects of ginsenoside on human pregnancy are warranted. Before more information in humans becomes available, use of ginseng during first trimester of pregnancy should be with caution.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ang-Lee, M.K., Moss, J. and Yuan, C.S. (2001) Herbal medicines and perioperative care. JAMA, 286, 208–216.[Abstract/Free Full Text]

Attele, A.S., Wu, J.A. and Yuan, C.S. (1995) Ginseng pharmacology: multiple constituents and multiple actions. Biochem. Pharmacol., 58, 1685–1693.

Chin, R.K. (1991) Ginseng and common pregnancy disorders. Asia Oceania J. Obstet. Gynecol., 17, 379–380.[Medline]

Corthout, J., Naessens, T., Apers, S. and Vlietinck, A.J. (1999) Quantitative determination of ginsenosides from Panax ginseng roots and ginseng preparations by thin layer chromatography-densitometry. J. Pharm. Biomed. Anal., 21, 187–192.[CrossRef][ISI][Medline]

Cui, J.F., Garle, M., Bjorkhem, I. and Eneroth, P. (1996) Determination of aglycones of ginsenosides in ginseng preparations sold in Sweden and in urine samples from Swedish athletes consuming ginseng. Scand. J. Clin. Lab. Invest., 56, 151–160.[ISI][Medline]

Cui, J.F., Bjorkhem, I. and Eneroth, P. (1997) Gas chromatographic-mass spectrometric determination of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol for study on human urinary excretion of ginsenosides after ingestion of ginseng preparations. J. Chromatogr. B Biomed. Sci. Appl., 689, 349–355.[CrossRef][Medline]

DeSmet, P.A.G.M. (1995) Health risks of herbal remedies. Drug Saf., 13, 81–93.[ISI][Medline]

Gibson, P.S., Powrie, R. and Star, J. (2001) Herbal and alternative medicine use during pregnancy: a cross-sectional survey. Obstet. Gynecol., 97, S44–S45.

Gillis, C.N. (1997) Panax ginseng pharmacology: a nitric oxide link? Biochem. Pharmacol., 54, 1–8.[CrossRef][ISI][Medline]

Kitts, D.D., Wijewickreme, A.N. and Hu, C. (2000) Antioxidant properties of a North American ginseng extract. Mol. Cell. Biochem., 203, 1–10.[CrossRef][ISI][Medline]

New, D.A.T. (1978) Whole embryo culture and study of the mammalian embryo during organogenesis. Biol. Rev., 53, 81–112.[ISI][Medline]

O’Hara, M., Kiefer, D., Farrell, K. and Kemper, K. (1998) A review of 12 commonly used medicinal herbs. Arch. Fam. Med., 7, 523–526.[Abstract/Free Full Text]

VanMaele-Fabry, G., Delhaise, F. and Picard, J.J. (1990) Morphogenesis and quantification of the development of postimplantation mouse embryos. Toxicol. In Vitro, 4, 149–156.[CrossRef][ISI]

Submitted on January 14, 2003; resubmitted on April 28, 2003; accepted on June 18, 2003.