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
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Abstract |
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Key words: ginseng/ginsenoside Rb1/teratogenicity/whole rat embryo culture
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Introduction |
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Women frequently consume herbal medicine during pregnancy. In a recent survey, 9.1% of pregnant women reported use of herbal supplements (Gibson et al., 2001), including ginseng. In Asian countries, up to 10% of women had taken ginseng during their pregnancy (Chin, 1991
). 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, 1995
). For example, Pennyroyal, a widely available herbal medicine, is hepatotoxic, neurotoxic and teratogenic (De Smet, 1995
). 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.
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Materials and methods |
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Whole embryo culture
The whole embryo culture system was based on a previously described model (New, 1978). 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 crownrump 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 SpragueDawley rat serum and Dulbeccos modified Eagles 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 crownrump length were measured. Embryonic morphologies were studied according to a standard morphological scoring system (Van Maele-Fabry et al., 1990), which gives a numerical score (of 05) 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 KruskalWallis test; the least significant difference test was used as an a posteriori test, when a difference was found with KruskalWallis test. All analyses were performed using the Statistical Package for Social Sciences for Windows version 10.0 (SPSS Inc., Chicago, IL, USA).
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Results |
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Discussion |
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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., 1995). Rb1 is the representative ginsenoside from the panaxadiol group. It is also the major ginsenoside in North American ginseng (Kitts et al., 2000
). 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., 1996
; 1997
), 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, 1997). Previous studies had shown that different ginsenoside might have different or even antagonistic actions (Corthout et al., 1999
). 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.
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References |
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Submitted on January 14, 2003; resubmitted on April 28, 2003; accepted on June 18, 2003.