Phytomedicine 21 (2013) 90–97

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Reproductive and developmental toxicity of the Ginkgo biloba special extract EGb 761® in mice Egon Koch a,∗ , Michael Nöldner a , Jost Leuschner b a b

Dr. Willmar Schwabe GmbH & Co. KG, Preclinical Research, 76227 Karlsruhe, Germany LPT – Laboratory of Pharmacology and Toxicology GmbH & Co. KG, 21147 Hamburg, Germany

a r t i c l e

i n f o

Article history: Received 1 July 2013 Received in revised form 29 July 2013 Accepted 19 September 2013 Keywords: Ginkgo biloba EGb 761® Teratogenicity Reproductive toxicity Developmental toxicity Mouse Extract characterization

a b s t r a c t Extracts from leaves of Ginkgo biloba are among the most widely used and best investigated phytopharmaceuticals worldwide. Almost all clinical trials and the majority of preclinical studies have been performed with a specifically defined extract (EGb 761® ) standardized to contain confined concentrations of active ingredients and limited quantities of potentially harmful substances. Besides pharmaceutical grade extracts poorly characterized Ginkgo preparations are now increasingly appearing on the market as nutraceuticals. While the safety of EGb 761® has been evaluated in an extensive set of toxicology studies, adverse effects of Ginkgo extracts of non-pharmaceutical quality on reproductive functions in mice have been reported in several publications in recent years. As this species has not previously been used in reproductive toxicity studies with EGb 761® , the present investigation was conducted to examine the influence of EGb 761® (100, 350 and 1225 mg/kg/day) on embryo-fetal development in mice during the critical period of organogenesis. During external and internal inspection of the fetuses as well as examination of skeletal and soft tissues no embryotoxic properties were noted. In particular, the incidence of malformations, variations or retardations was not increased and the general condition of dams was not influenced. Thus, the no-observed-effect level (NOEL) was above 1225 mg/kg/day for the dams and the fetuses. © 2013 Elsevier GmbH. All rights reserved.

Introduction Beginning in the mid-1960, extracts from leaves of Ginkgo biloba have been developed as medication for the treatment of Claudicatio intermittens, tinnitus, vertigo and dementia of the vascular or Alzheimer type by Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe (Germany). The development program led to a patented manufacturing process providing the specifically defined extract EGb 761® . The sophisticated production process has been designed to enrich substances that contribute to pharmacological and clinical effects (e.g. flavone glycosides and terpene lactones) and remove pharmaceutically problematic constituents (e.g. proanthocyanidins) and especially constituents with potential side effects (e.g. ginkgolic acids). Accordingly, EGb 761® is specified to contain 22.0–27.0% Ginkgo flavone glycosides and 5.0–7.0% terpene lactones, consisting of 2.8–3.4% ginkgolides A–C and 2.6–3.2% bilobalide, and less than 5 ppm ginkgolic acids (Lang et al., 2013). In addition, the raw material used for extract manufacturing is strictly controlled for contamination by environmental pollutants, pesticides, heavy metals, fungi etc. In every aspect, EGb

∗ Corresponding author. Tel.: +49 0721 4005 356; fax: +49 0721 4005 150. E-mail address: [email protected] (E. Koch). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.09.004

761® complies with monographs, such as those of the German Commission E (Blumenthal et al., 1998), the WHO (1999), the European Scientific Cooperative on Phytotherapy (ESCOP, 2003) or the European Pharmacopoeia (Europäisches Arzneibuch, 2013). The annual sales of Ginkgo products have been reported to amount to several hundred million euros (Kakigi et al., 2012). Due to these promising economic prospects, poorly defined Ginkgo preparations are now increasingly appearing on the market as teas, energy drinks, food supplements or cosmetics. While EGb 761® has been developed as a pharmaceutical active ingredient, many of these products have been found to be of questionable quality and variable composition (Kressmann et al., 2002; Gawron-Gzella et al., 2010; Tawab et al., 2010). Since Ginkgo leaves of high quality are short in supply and sold on high prices adulterations are common. In a recent investigation, eight out of 18 analyzed Ginkgo biloba supplements were found to be adulterated. The most frequent form of adulteration is the addition of inexpensive flavone glycosides or aglycones (Harnly et al., 2012). But such extracts have also been observed to contain high concentrations of potentially allergic and genotoxic ginkgolic acids (Kressmann et al., 2002). Herbal medicines exhibit pharmacological effects by the interaction of many compounds. Thus, the pharmacological action of botanical drugs is due to the combined effect of multitudinous constituents, which may interact in a positive or negative manner

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(Efferth and Koch, 2011). For this reason, the whole extract needs to be considered as the active pharmaceutical ingredients. The composition of a herbal extract depends on numerous variables which are mainly related to (I) the raw material used for extraction, e.g. the correct species, the parts of the plant used, the location where it was grown, the growth conditions or the time when it was harvested and (II) the extraction process, e.g. extraction solvent(s), extraction temperature, methods of purification, steps taken to enrich pharmacodynamically active compounds or to remove potentially harmful constituents. Only if the whole process is strictly controlled a defined extract with acceptable batch-to-batch consistency will be obtained, which is a prerequisite for reproducible pharmacological action as well as clinical efficacy. Even slight deviations in the starting material and/or in the extraction process will almost certainly lead to significant differences in the composition of the extract. Therefore, pre-clinical and clinical studies performed with one specific extract cannot be transferred to other preparations from the same plant (Hoerr and Weber, 2013). The safety and tolerability of EGb 761® has been proven in an extensive set of toxicology studies, in a great number of clinical trials as well as during more than four decades of therapeutic use (Lang et al., 2013). The toxicology program was conducted under good laboratory practice (GLP) conditions and included acute, subacute and chronic toxicity studies as well as investigations on genotoxicity, carcinogenicity, and reproduction toxicity. In particular, no effect on male and female fertility, pregnancy, embryonic, fetal as well as peri- and postnatal development of the offspring was observed following oral administration of EGb 761® at doses of 100, 400 and 1600 mg/kg/day to rats and 100, 300 and 900 mg/kg/day to rabbits (Spiess and Juretzek, 2012). In recent years several studies have been published which report adverse effects of Ginkgo extracts of non-pharmaceutical quality on reproductive functions in mice (Al-Yahya et al., 2006; Zehra et al., 2010; El’Mazoudy and Attia, 2012). This animal species is not commonly used in reproductive toxicity studies for regulatory purposes. It is well known that different species may display variable levels of susceptibility notably with respect to reproductive and developmental toxicity. This may be due to species-specific differences in exposure relative to critical periods of development, pharmacokinetics, developmental patterns, placentation, or modes of action. For the assessment of risk to humans, it is generally considered appropriate to use the most sensitive animal species as humans appear to be particularly vulnerable to the majority of agents known to cause reproductive toxicity (EPA, 1996). Thus, the present study was conducted in compliance with GLP standards to examine the influence of EGb 761® on embryo-fetal development in mice during the critical period of organogenesis (6th to 15th day of pregnancy) by oral administration to the dams. Materials and methods Ginkgo biloba extract EGb 761® For the study a single batch (No. 454) of EGb 761® was used. The extract was standardized to contain a content of 24.7% flavone glycosides, 5.7% terpene lactones (including 2.95% ginkgolides A, B and C and 2.75% bilobalide), and 0.4 ppm ginkgolic acids. The extract corresponded to all specifications with respect to microbial quality as well as the content of residual solvents, heavy metals, and environmental pollutants.

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for the study. All animals were allowed at least 7 days for adaptation. Except during the mating period, the mice were kept singly in makrolon cages under standardized environmental conditions with a room temperature of 22 ± 3 ◦ C, a relative humidity of 55 ± 15% and a 12 h dark/12 h light cycle (about 150 lx at approximately 1.50 m room height). Granulated textured wood was used as bedding material. The cages were cleaned and changed once a week. Drinking water and commercial feed (sniff® M-Z V 1121, SSniff GmbH, Germany) was offered daily ad libitum. Mating procedure Sexually mature male mice of the same breed served as mating partners. The female breeding partners were randomly chosen. Matings were monogamous with 1 male and 1 female animal placed together in one cage during the dark period. The date of conception was ascertained by establishing the presence of a copulatory plug and/or sperm. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). Treatment protocol The extract was given to groups of each 25 mice at dosages of 100, 350 and 1225 mg/kg/day to get at least 20 dams with litters per group for evaluation of reproduction toxicity. Doses were selected based on a previous dose-range-finding study. An additional control group was treated with vehicle only (20 ml/kg tap water). The extract was administered daily from day 6 to day 15 of pregnancy by gavage in a volume of 20 ml/kg in tap water as vehicle. In-life observations and necropsy During the study animals were individually observed at least once daily for any clinical signs and food and water consumption were monitored throughout the study. Body weight was determined daily. On the 17th day of gestation, the mice were sacrificed and ovaries and uteri were removed. Post-mortem examination included macroscopic inspection of internal organs and placentae of the dams. In case of findings, the affected maternal tissue was preserved in 7% buffered formalin for possible future histopathological examinations. The fetuses were removed and the following examinations were performed: macroscopic inspection of the placentae, the number of fetuses (alive and dead), the number of placentae, sex and viability of fetuses, number and size of resorptions, number of corpora lutea, number and location of implantations and fetuses in the uterus, gravide uterus weight, and weights of the placentae and fetuses. The fetuses were inspected externally and internally for damages, especially for malformations. Half of the fetuses in each litter were examined for skeletal anomalies. The thorax and peritoneal cavity were opened and the location, size and condition of the internal organs were determined. Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications. The skeletal system was examined for the determination of the number and type of retardations, variations as well as malformations. The remaining 50% of the number of fetuses in each litter were examined for soft tissue anomalies. Statistics

Animals One hundred virgin female CD-1 mice (Charles River, Sulzfeld, Germany) with a body weight between 22.4 and 28.8 g were used

For all numerical values, homogeneity of variances was tested using the Bartlett chi-square test. When the variances were homogeneous, the Dunnett test (p ≤ 0.01) was used to compare the

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experimental groups with the control group. In case of heterogeneity of variances, the Student’s t-test was carried out; limit of significance was p ≤ 0.01. For the comparison of classification measurements (for example rates of malformation, resorption, retardation, and variation) the Fisher’s exact test (n < 100) or chisquare test with Yates’ correction for continuity (n ≥ 100) (p ≤ 0.05 and p ≤ 0.01) was employed. Results General examination of dams No changes of behavior or external appearance were noted in the dams after oral treatment with 100, 350 or 1225 mg EGb 761® /kg/day. None of the dams died prematurely. The feces of all dams were of normal consistency throughout the experimental period, and no test item-related influence was noted on food and water consumption of the dams in all dose groups. Minimal changes noted in comparison to the control group are judged as spontaneous findings. Thus, the food intake of the high dose dams (1225 mg EGb 761® /kg/day) was statistically significantly reduced (at p ≤ 0.05) by up to 14% compared to the control group on gestation days 7, 11 and 12, but this is regarded to be within the normal range of variation. No influence of treatment was noted on the body weight of the dams and the body weight gain was similar to that of controls at all dose levels. Necropsy revealed no pathological findings in the dams treated with 100, 350 or 1225 mg EGb 761® /kg/day. The gravid uterus weight, the carcass weight and the net weight change were not influenced in all treatment groups (Table 1). Reproduction data No test item-related influence on the prenatal fetal development was detected at 100, 350 or 1225 mg EGb 761® /kg/day with respect to the number of corpora lutea, implantation sites, resorptions and live fetuses or the values calculated for the pre- and post-implantation losses. A total post-implantation loss consisting of eight early resorptions was noted in one dam treated with 350 mg EGb 761® /kg/day. A total loss of implants is known to occur spontaneously in mice of this strain and hence, this finding is considered as spontaneous. Slightly but statistically significantly increased incidences (p ≤ 0.05) were noted for the ratio of early and total resorptions (including the aforementioned total postimplantation loss) vs. implantations at 100, 350 or 1225 mg EGb 761® /kg/day. Hence, the post-implantation loss was increased (7.5%, 8.7% or 8.6%) when compared to the incidentally very low value of the control (2.9%). However, the data observed for the post-implantation loss in the test groups are well within the historical control data range (3.2–15.5%) (Table 1). Examination of fetuses The mean fetal weights were not influenced by the administration of 100 or 350 mg EGb 761® /kg/day as compared to the control group. Slight but statistically significantly decreased body weights (p ≤ 0.05) compared to the control were noted for the male and all viable fetuses at 1225 mg EGb 761® /kg/day (Table 2). However, all values were still within the range of spontaneous variability. No dead fetuses were noted at 100 or 350 mg EGb 761® /kg/day. In the high dose group one dead fetus was observed, this incidence is within the range of variability and hence is considered as spontaneous. Laparotomy revealed no twins at all dose groups (Table 3). The sex distribution of the fetuses in all test groups was within the normal range of variance (Table 1). No test item-related external malformations were noted in all treatment groups. External malformations in the form of cleft

palate, encephalocele or hydrocephalus were noted in individual fetuses of the dose groups treated with EGb 761® and in the control group (Table 3). None of the malformations noted are considered to be related to the treatment with EGb 761® as these findings are known to occur spontaneously in this mouse strain. Macroscopic inspection and internal examination after dissection of the fetuses revealed no test item-related findings at all dose levels or in the control group. Skeletal examination revealed no test item-related malformations, variations and retardations at any tested dose level. A domed head noted in one fetus of the high-dose confirmed the aforementioned spontaneous external malformation in the form of hydrocephalus. A slightly but statistically significantly increased incidence (p ≤ 0.05) was noted for retarded ossification of metacarpalia (absence of ossification) at 100 mg EGb 761® /kg/day. This increase compared to the control is not considered to be test item-related but is considered to be spontaneous as above all no dose-response was noted and altogether decreased incidences (p ≤ 0.01) for total skeletal retardations were noted at 100 mg EGb 761® /kg/day. Similarly, decreased incidences were observed for lumbar vertebral bodies (less than 6 lumbar vertebral bodies ossified) and sternebra(e) (reduced in size) at 1225 mg EGb 761® /kg/day. These decreases compared to the control are not considered to be test item-related but are considered as spontaneous (Table 4). No test item-related malformations or variations were noted during soft tissue examinations of the fetal organs at 100, 350 or 1225 mg EGb 761® /kg/day. Soft tissue variations in the form of dilatation of the renal pelvis or the 1st, 2nd or 4th cerebral ventricle and hemorrhagic foci in the liver were noted in individual fetuses at the tested dose levels and/or in the control. These findings are common in this mouse strain and hence are regarded to be spontaneous (Table 5).

Discussion The present study was conducted to examine the influence of EGb 761® (100, 350 and 1225 mg/kg/day) on embryo-fetal development in mice during the critical period of organogenesis. The investigation was initiated in response to two recently published papers that reported on deleterious effects of Ginkgo extracts of non-pharmaceutical quality on reproductive functions in mice (Zehra et al., 2010; El’Mazoudy and Attia, 2012). During external and internal inspection of the fetuses as well as after examination of skeletal and soft tissues no embryotoxic properties were noted following administration of EGb 761® to the dams. In particular, the incidence of malformations, variations or retardations was not increased and the general condition of pregnant females was not influenced. Hence, a no-observed-effect level (NOEL) above 1225 mg/kg/day for the dams and the fetuses was established. The actual experiment supplements earlier studies on reproduction toxicity of EGb 761® which were performed with oral doses of 100, 400 and 1600 mg/kg/day in rats and 100, 300 and 900 mg/kg/day in rabbits. From the present and these previous studies it can be concluded that EGb 761® does not possess any embryotoxic and teratogenic properties and in general has no adverse effect on fertility, embryo-fetal as wells as peri- and postnatal development. In contrast to the findings with EGb 761® , Zehra et al. (2010) observed teratogenic effects in mice after administration of a Ginkgo biloba extract which is stated to be standardized to contain 24% flavone glycosides and 6% terpene lactones. Unfortunately, further information to more precisely characterize this extract (e.g. kind and concentrations of extraction solvent, drug to extract ratio) are not reported. The authors administered the extract orally to

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Table 1 Summary of data on carcass weight, uterus weight, net weight change, and reproduction of dams. Test group 1 Control

Test group 2 100 mg/kg EGb 761®

Test group 3 350 mg/kg EGb 761®

Test group 4 1225 mg/kg EGb 761®

Carcass weight (g)

Mean S.D.

34.5 2.7

34.0 3.0

34.3 2.8

34.7 2.5

Uterus weight (g)

Mean S.D.

16.6 5.7

13.8 5.9

16.7 6.2

17.1 3.8

Net weight change (g) from day 6

Mean S.D.

4.9 2.1

5.2 2.2

4.9 1.8

5.3 1.6

Females pregnant Aborted Premature birth Dams with viable fetuses Dams with all resorptions

N N N N N

20 0 0 20 0

20 0 0 20 0

21 0 0 20 1

20 0 0 20 0

Pregnant at cesarean section

N %

20 100

20 100

21 100

20 100

Corpora lutea

Mean S.D. Total

12.0 4.0 240

10.6 3.4 211

12.6 3.3 265

13.4 2.4 267

Implantation sites

Mean S.D. Total

11.5 4.4 229

9.9 4.3 197

12.2 3.7 256

13.1 2.6 261

Pre-implantation losses

Mean% S.D.

7.7 13.0

12.2 21.6

6.1 17.3

2.6 6.3

Post-implantation losses

Mean% S.D.

2.9 3.7

7.5 14.2

8.7 21.5

8.6 12.3

Resorptions: Total

Mean S.D. Total Mean% S.D.

0.4 0.5 8 2.9 3.7

0.8 1.5 16* 7.5 14.2

0.9 1.8 19* 8.7 21.5

1.1 1.5 21* 8.2 11.1

Resorptions: Early

Mean S.D. Total Mean% S.D.

0.3 0.5 6 2.2 3.5

0.7 1.1 13* 5.9 10.5

0.8 1.8 17* 8.0 21.6

0.7 1.0 14 5.8 8.2

Resorptions: Late

Mean S.D. Total Mean% S.D.

0.1 0.3 2 0.7 2.2

0.2 0.5 3 1.6 5.1

0.1 0.3 2 0.7 2.3

0.4 0.8 7 2.5 5.9

Dams with viable fetuses Live fetuses

N Mean S.D. Total Mean% S.D. Mean S.D. Total Mean% S.D. Mean S.D. Total Mean% S.D.

20 11.1 4.3 222 97.1 3.7 6.1 3.5 121 48.0 21.4 5.1 2.6 101 49.1 22.9

20 9.1 4.4 181* 92.5 14.2 3.8 2.6 75** 36.4 18.3 5.3 2.5 106** 56.1 17.4

20 11.9 3.6 237* 95.9 5.0 5.9 2.6 117 49.8 19.6 6.0 2.9 120 46.1 20.1

20 12.0 2.9 239** 91.4 12.3 6.1 2.3 121 45.4 14.1 5.9 1.5 118 46.3 12.4

55 45

41 59

49 51

51 49

Females

Males

Percent live females Percent live males * **

Significantly different from control: p ≤ 0.05. Significantly different from control: p ≤ 0.01.

the dams throughout the gestational period at doses of 78 and 100 mg/kg, respectively. On the 18th day of pregnancy the mice were sacrificed, the uteri were removed and the fetuses were dissected. The fetuses were examined for gross abnormalities and their body weight as well as the crown-rump length was measured. Administration of the Ginkgo extract at a dose of 100 mg/kg caused

a significant decrease of fetal weight and crown-rump length when compared to controls and dams treated with a dose of 78 mg/kg. In addition, in this dose group a high frequency of malformations such as round shaped eyes and orbits, syndactyly, malformed pinnae, nostrils, lips and jaws was observed, while no such gross abnormalities were seen in the two other experimental groups.

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Table 2 Summary of placental and fetal body weights. Test group 1 Control

Test group 2 100 mg/kg EGb 761®

Test group 3 350 mg/kg EGb 761®

Test group 4 1225 mg/kg EGb 761®

Litter mean S.D. N

0.1085 0.0213 20

0.1168 0.0233 20

0.1124 0.0157 20

0.1011 0.0170 20

Of male fetuses

Litter mean S.D. N

0.1101 0.0217 20

0.1210 0.0268 20

0.1159 0.0143 19

0.1035 0.0194 20

Of female fetuses

Litter mean S.D. N

0.1052 0.0238 19

0.1087 0.0192 19

0.1093 0.0174 20

0.0996 0.0176 20

Litter mean S.D. N

1.030 0.114 20

1.045 0.164 20

1.005 0.071 20

0.953* 0.113 20

Of male fetuses

Litter mean S.D. N

1.045 0.114 20

1.069 0.173 20

1.032 0.077 19

0.967* 0.106 20

Of female fetuses

Litter mean S.D. N

1.015 0.115 19

1.008 0.153 19

0.982 0.065 20

0.942 0.126 20

Placental weights (g) Of all viable fetuses

Fetal weights (g) Of all viable fetuses

*

Significantly different from control: p ≤ 0.05.

However, this study does not fulfill accepted scientific standards in a number of respects and so interpretation of data is rather limited. Besides an insufficient characterization of the extract, the methods employed and the findings obtained are inadequately described (e.g. mouse strain used, nature of findings, effects on general condition of dams which could explain the observed teratogenic effects). As only two instead of three doses were tested a dose-dependency of the effects cannot reliably be concluded. With reference to a previous publication (Al-Yahya et al., 2006), dose selection is claimed to be based on the human equivalent therapeutic dose (HED). However, calculation of the HED in this previous paper is already erroneous. Under the assumption of a human therapeutic dose of 100 mg and body weights of 20 g in mice and 60 kg in humans, the HED in mice would be about 24 mg/kg (Reagan-Shaw et al., 2008) and not 78 mg/kg as calculated by these authors. The basis for the selection of the second dose (100 mg/kg) is entirely obscure, but is said to be derived from the maximal tolerated human dose which is stated to be 1.6 g/kg/day. Anyhow, it is rather unusual that a dose of 78 mg/kg was entirely free of any toxic effect while the slightly higher dose of 100 mg/kg caused severe structural abnormalities. A Ginkgo extract from the same supplier as those employed by Zehra et al. (2010) was also used in another study on reproductive toxicity in male Swiss albino mice (Al-Yahya et al., 2006). Following oral administration at doses of 25, 50 and 100 mg/kg/day for a period of 3 months a significant increase of the weight of caudae epididymis and prostate as well as the frequency of chromosomal aberrations of germ cells was seen in the high and with respect to the epididymis weight also the intermediate dose group. In addition, after mating of female mice with males treated with 100 mg/kg of the extract rate of pregnancy and number of live fetuses was significantly reduced. The fact that this particular extract caused reproductive toxicity in two studies indeed raises suspicion on the quality and safety of this preparation. Recently a further study investigated the effect of treatment with a Ginkgo extract on the sexual cycle and reproductive performance as well as anti-implantation and abortifacient potential in mice (El’Mazoudy and Attia, 2012). The extract was orally

administered at doses of 3.7, 7.4, and 14.8 mg/kg/day for 28 days for evaluation of the reproductive cycle. Some of the animals were than mated with normal fertile males and treatment continued until laparotomy for determination of pregnancy and fertility analysis on day 20. For investigation of potential anti-implantation and abortifacient action pregnant mice were treated from day 1 to day 7 or from day 10 to day 18 of pregnancy, respectively. Ovarian follicle counts, number of corpora lutea, serum progesterone level, copulation and pregnancy rate, weight of the gravid uterus, number of implantations, and number of live fetuses were significantly reduced after administration of 14.8 mg/kg/day. Treatment at this dose also induced disruption of estrous cycle and increased preand post-implantation losses. The authors of this study claim to have used the standardized Ginkgo extract EGb 761® for their investigations. The extract was obtained from an Egyptian pharmaceutical company in form of capsules containing 260 mg. This company, however, is not a licensee of the manufacturer of EGb 761® (Dr. Wolfgang Weber, Senior R + S Manager, International Division, Dr. Willmar Schwabe GmbH & Co. KG, personal communication). Furthermore, EGb 761® is not available as capsules but only as film-coated tablets containing a maximum of 240 mg extract. Analyses of different batches of the preparation from this Egyptian company revealed that the extract is clearly adulterated by the addition of flavones and contains high concentrations of ginkgolic acids (Dr. Friedrich Lang, Head of Analytical Development, Dr. Willmar Schwabe GmbH & Co. KG; personal communication). Hence, the authors definitively did not use EGb 761® for their investigations and by these means not only violate standards of scientific conduct but also infringe trademark right. Since publication lends special credibility to scientific findings it is most regrettable that in this case the peer review system of the publishing journal utterly failed. In this context, it is worth mentioning that even within the US National Toxicology Program (NTP) very comprehensive toxicological studies were conducted with a Ginkgo extract which did not comply with internationally approved monographs (National Toxicology Program, 2013). Despite the limited information in the study report, it is obvious that the extract used was very different

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Table 3 Summary of all fetal external observations.

Litters evaluated Fetuses evaluated Live Dead Total malformations Fetal incidence Litter incidence Affected fetuses/litter Cleft palate Fetal incidence Litter incidence Encephalocele Fetal incidence Litter incidence Hydrocephalus Fetal incidence Litter incidence Total variations Fetal incidence Litter incidence Affected fetuses/litter

N N N N N % N % Mean% S.D.

Litter incidence Twins Fetal incidence Litter incidence

Test group 2 100 mg/kg EGb 761®

Test group 3 350 mg/kg EGb 761®

Test group 4 1225 mg/kg EGb 761®

20 222 222 0

20 181 181 0

20 237 237 0

20 240 239 1

3 1.4 3 15.0 1.1 2.7

1 0.6 1 5.0 0.7 3.2

1 0.4 1 5.0 0.4 1.6

3 1.3 3 15.0 1.1 2.7

N % N %

1 0.5 1 5.0

1 0.6 1 5.0

1 0.4 1 5.0

1 0.4 1 5.0

N % N %

1 0.5 1 5.0

0 0.0 0 0.0

0 0.0 0 0.0

1 0.4 1 5.0

N % N %

1 0.5 1 5.0

0 0.0 0 0.0

0 0.0 0 0.0

1 0.4 1 5.0

N % N % Mean% S.D.

0 0.0 0 0.0 0.0 0.0

0 0.0 0 0.0 0.0 0.0

0 0.0 0 0.0 0.0 0.0

0 0.0 0 0.0 0.0 0.0

2 0.9 1 5.0

0 0.0 0 0.0

0 0.0 0 0.0

1 0.4 1 5.0

N % N %

0 0.0 0 0.0

0 0.0 0 0.0

0 0.0 0 0.0

1 0.4 1 5.0

N % N %

2 0.9 1 5.0

0 0.0 0 0.0

0 0.0 0 0.0

0 0.0 0 0.0

Total fetal external unclassified observations N Fetal incidence % Litter incidence N % Found dead at laparotomy Fetal incidence

Test group 1 Control

from EGb 761® at least with respect to its composition as it contained 31% flavone glycosides, 15% terpene lactones and 10 ppm ginkgolic acids. Thus, the statement that this extract “is similar to the Schwabe extract” is evidently wrong and misleading. It is rather astonishing that the US Senate’s Committee on Appropriations, whose main duty is the allocation of federal funds to government agencies, departments, and organizations, recently considered it necessary to urge the NTP to be highly precise when describing the results of its studies on particular extracts of an herbal species to avoid any possible confusion about the relevance of such studies to other extracts of the species (http://www.ahpa.org/Portals/0/ 13 1607Senate Labor-HHS NTP.pdf, accessed on July 25, 2013). Since herbal extracts exhibit pharmacological and toxicological effects by the interaction of many constituents, variations in their composition will unavoidably lead to differential biological

responses. It is, therefore, inappropriate and scientifically unsound to generally view extracts prepared form a certain plant or plant part as bioequivalent. Reproducible and convincing therapeutic effects can only be achieved if extracts with consistent composition and pharmacological properties are used. This can only be achieved if the raw material and the entire manufacturing process are strictly controlled and standardized. Preclinical and clinical data generated with such a preparation are unique to this product and may not be generalized. Thus, in order to avoid any confusion and misinterpretation it is of paramount importance that in any publication a precise description of the preparation is provided which at least should include the binomial scientific Latin name of the plant species, the plant part used, the nature and concentration of the extraction solvent, the extraction process, the raw material to extract ratio, and the physical state of the product.

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Table 4 Summary of all classified fetal skeletal observations.

Litters evaluated Fetuses evaluated Live Dead

N N N N

Total fetal skeletal malformations Fetal incidence Litter incidence Affected fetuses/litter Total fetal skeletal variations Fetal incidence Litter incidence Affected fetuses/litter Total fetal skeletal retardations Fetal incidence Litter incidence Affected fetuses/litter

Test group 1 Control

Test group 2 100 mg/kg EGb 761®

Test group 3 350 mg/kg EGb 761®

Test group 4 1225 mg/kg EGb 761®

20 111 111 0

20 90 90 0

20 119 119 0

20 120 119 1

N % N % Mean% S.D.

0 0 0 0 0.0 0.0

0 0 0 0 0.0 0.0

0 0 0 0 0.0 0.0

1 0.8 1 5.0 0.8 3.7

N % N % Mean% S.D.

44 39.6 16 80.0 41.4 31.6

32 35.6 14 70.0 30.0 28.4

52 43.7 19 95.0 43.1 26.1

58 48.3 20 100.0 51.6 24.7

N % N % Mean% S.D.

109 98.2 20 100.0 98.5 4.8

81** 90.0 18 90.0 90.0 30.8

118 99.2 20 100.0 99.2 3.7

120 100.0 20 100.0 100.0 0.0

Significantly different from control: ** = p ≤ 0.01, Fetuses affected by several changes will be counted as one fetal incidence Table 5 Summary of all classified fetal soft tissue observations. Test group 1 Control Litters evaluated Fetuses evaluated Live fetuses Dead fetuses

N N N N

Total fetal soft tissue malformations Fetal incidence N % Litter incidence N % Total fetal soft tissue variations Fetal incidence Litter incidence

N % N %

20 111 111 0

Test group 2 100 mg/kg EGb 761® 20 91 91 0

Test group 3 350 mg/kg EGb 761®

Test group 4 1225 mg/kg EGb 761®

20 118 118 0

20 120 120 0

0 0.0 0 0.0

1 1.1 1 5.0

1 0.8 1 5.0

0 0.0 0 0.0

7 6.3 6 30.0

8 8.8 6 30.0

3 2.5 2 10.0

8 6.7 6 30.0

Fetuses affected by several changes will be counted as one fetal incidence.

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Reproductive and developmental toxicity of the Ginkgo biloba special extract EGb 761® in mice.

Extracts from leaves of Ginkgo biloba are among the most widely used and best investigated phytopharmaceuticals worldwide. Almost all clinical trials ...
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