Safety Assessment of Ginkgo biloba-Derived Ingredients as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) assessed the safety of 10 Ginkgo biloba-derived ingredients, which are most frequently reported to function in cosmetics as skin conditioning agents or antioxidants. The Panel reviewed the available data to determine the safety of these ingredients. Because final product formulations may contain multiple botanicals, each containing the same constituents of concern, formulators are advised to be aware of these constituents and to avoid reaching levels that may be hazardous to consumers. The Panel was concerned about the presence of ginkgolic acid in cosmetics. Industry should use good manufacturing practices to limit impurities. The Panel concluded that 5 Ginkgo biloba leaf-derived ingredients are safe in the present practices of use and concentration described in this safety assessment when formulated to be non-sensitizing; data are insufficient to determine the safety of the remaining 5 ingredients under the intended conditions of use in cosmetic formulations.

Cytotoxicity, genotoxicity and gene expression changes elicited by exposure of human hepatic cells to Ginkgo biloba leaf extract.

The use of Ginkgo biloba leaf extract as nutraceutical is becoming increasingly common. As a consequence, the definition of a reliable toxicological profile is a priority for its safe utilization. Recently, contrasting data have been reported on the carcinogenic potential of Ginkgo biloba extract in rodent liver. We measured viability, Reactive Oxygen Species (ROS), apoptosis, colony-forming efficiency, genotoxicity by comet assay, and gene expression changes associated with hepato-carcinogenicity in human cells of hepatic origin (HepG2 and THLE-2) treated with different concentrations (0.0005-1.2 mg/mL) of Ginkgoselect®Plus. Our analyses highlighted a decrease of cell viability, not due to apoptosis, after treatment with high doses of the extract, which was likely due to ROS generation by a chemical reaction between extract polyphenols and some components of the culture medium. Comet assay did not detect genotoxic effect at any extract concentration. Finally, the array analysis detected a slight decrease in the expression of only one gene (IGFBP3) in Ginkgo-treated THLE-2 cells as opposed to changes in 28 genes in Aflatoxin B1 treated-cells. In conclusion, our results did not detect any significant genotoxic or biologically relevant cytotoxic effects and gross changes in gene expression using the Ginkgo extract in the hepatic cells tested.

Mechanism for ginkgolic acid (15 : 1)-induced MDCK cell necrosis: Mitochondria and lysosomes damages and cell cycle arrest.

Ginkgolic acids (GAs), primarily found in the leaves, nuts, and testa of ginkgo biloba, have been identified with suspected allergenic, genotoxic and cytotoxic properties. However, little information is available about GAs toxicity in kidneys and the underlying mechanism has not been thoroughly elucidated so far. Instead of GAs extract, the renal cytotoxicity of GA (15 : 1), which was isolated from the testa of Ginkgo biloba, was assessed in vitro by using MDCK cells. The action of GA (15 : 1) on cell viability was evaluated by the MTT and neutral red uptake assays. Compared with the control, the cytotoxicity of GA (15 : 1) on MDCK cells displayed a time- and dose-dependent manner, suggesting the cells mitochondria and lysosomes were damaged. It was confirmed that GA (15 : 1) resulted in the loss of cells mitochondrial trans-membrane potential (ΔΨm). In propidium iodide (PI) staining analysis, GA (15 : 1) induced cell cycle arrest at the G0/G1 and G2/M phases, influencing on the DNA synthesis and cell mitosis. Characteristics of necrotic cell death were observed in MDCK cells at the experimental conditions, as a result of DNA agarose gel electrophoresis and morphological observation of MDCK cells. In conclusion, these findings might provide useful information for a better understanding of the GA (15 : 1) induced renal toxicity.

Review of Ginkgo biloba-induced toxicity, from experimental studies to human case reports.

Ginkgo biloba seeds and leaves have been used as a traditional herbal remedy for thousands of years, and its leaf extract has been consumed as a botanical dietary supplement for decades. Ginkgo biloba extract is a complex mixture with numerous components, including flavonol glycosides and terpene lactones, and is one of the most widely sold botanical dietary supplements worldwide. Concerns about potential health risks for the general population have been raised because of the widespread human exposure to Ginkgo biloba and its potential toxic and carcinogenic activities in rodents. The National Toxicology Program conducted 2-year gavage studies on one Ginkgo biloba leaf extract and concluded that there was clear evidence of carcinogenic activity of this extract in mice based on an increased incidence of hepatocellular carcinoma and hepatoblastoma. Recently, Ginkgo biloba leaf extract has been classified as a possible human carcinogen (Group 2B) by the International Agency for Research on Cancer. This review presents updated information on the toxicological effects from experimental studies both in vitro and in vivo to human case reports (caused by ginkgo seeds or leaves), and also summarizes the negative results from relatively large clinical trials.

Ausência de toxicidade reprodutiva e sistêmica em ratos wistar tratados com extrato seco de gingko biloba durante o período de desmame à puberdade / Absence of reproductive and sistemic toxicity in rats treated with Gingko biloba extract from weaning to puberty

Introdução: O Gingko biloba (EGb) é um fitoterápico usado há séculos, porém com poucos estudos referentes a seus efeitos sobre o período pós-natal. Estudos dessa natureza vêm sendo preconizados pela Agência Europeia de Medicina, visto que muitos órgãos completam seu desenvolvimento nesse período, inclusive o sistema reprodutor. Objetivo: Avaliar o efeito do extrato seco de EGb sobre o desenvolvimento do sistema reprodutor de ratos, tratados desde o desmame até o fim da puberdade. Métodos. Ratos Wistar foram tratados com 25mg/kg/massa corporal (EGb 25); 50 mg/kg (EGb 50) e 100 mg/kg (EGb 100). Controle (C ­ 0,1ml água destilada), por gavage dos 25 aos 45 dias de vida pós-natal. Variáveis observadas: indícios clínicos de toxicidade sistêmica, peso corporal, descida dos testículos, evolução da morfologia da glande, peso de rins, baço e fígado e dos órgãos do sistema reprodutor. Hematimetria, Concentração de hemoglobina. Concentração de espermatozoides na secreção epididimária. Resultados: Não foram encontradas diferenças significativas em quaisquer das variáveis. Conclusão: A exposição ao extrato seco de EGb durante o período pré-puberal e puberal em ratos Wistar não altera o desenvolvimento do sistema reprodutor masculino.

Introduction: Gingko biloba extract (EGb) is a phytotherapic that has been used for centuries but there is no studies concerning their effects during the postnatal period. This kind of research had been suggested by the European Medicine Agency since there are organs that complete their development in this period, including reproductive organs. Purpose: To evaluate the effect of EGb dry extract upon the rat reproductive system from weaning to 45 postnatal days. Methods: Wistar rats were treated with 25mg/kg/body weight (EGb 25); 50 mg/kg (EGb 50) and 100 mg/kg (EGb 100). Control (C 0,1ml distilled water). Variables: clinical signs of systemic toxicity, body weight, testicles descent, evolution of glans morphology, kidneys, liver, spleen and reproductive organs weights. Hematimetry. Haemoglobin concentration. Sperm concentration in the epidydimal secretion. Results: No significant differences were observed in none of the observed variables. Conclusion: The EGb dry extract exposition to prepuberal and puberal rats do not alter the reproductive system development.

Efeito do extrato de ginkgobiloba (EGb) sobre a toxicidade sistêmica e órgãos do sistema reprodutor masculino de ratos Wistar adultos / Effect of ginkgobiloba extract (EGb) on systemic toxicity and organs of the male reproductive system of adult Wistar rats

O Extrato de Ginkgobiloba (EGb) é um dos fitoterápicos mais consumidos no mundo. Entretanto ainda há escassez de ensaios toxicológicos em animais e o risco à exposição humana principalmente pelos compostos alquilfenóis, representados pelos ácidos ginkgólicos, que podem causar quadros alérgicos e serem compostos mutagênicos e carcinogênicos. O presente trabalho teve o objetivo de avaliar a toxicidade sistêmica do EGb. Oitenta ratos Wistar de três meses de idade foram tratados com água destilada (Grupo Controle) e extrato aquoso de Ginkgobilobanas seguintes doses: 3,5 (EGb 3,5); 7,0 (EGb 7,0) e 14,0mg/kg (EGb 14,0) uma vez ao dia, por 56 dias consecutivos. Foram avaliados semanalmente, o peso dos animais (g) e a estimativa de consumo diário de ração (g). Indícios de sinais de toxicidade sistêmica como perda de peso, piloereção, diarreia, cromodacriorreia, estereotipias, alterações da atividade locomotora e comportamentais e mortes também foram monitorados. Após anestesia, o sangue dos animais foi coletado para avaliação de hemograma completo e dosagem bioquímica de ureia, creatinina e alanina aminotransferase (ALT). Após a eutanásia, os animais foram submetidos à necropsia e os testículos esquerdo e direito, epidídimo esquerdo, vesícula seminal repleta, próstata ventral, rins esquerdo e direito, fígado e baço foram removidos e pesados em balança de precisão. Durante todo o procedimento experimental não foram observados nos animais sinais clínicos de toxicidade sistêmica e mortes. Houve diferenças estatísticas da estimativa de consumo de ração na sexta semana e oitava semanas de avaliação, embora sem diferença no peso corporal. Não houve diferença no peso dos órgãos e na análise bioquímica sérica. Na avaliação hematológica dos animais, houve diferença estatística significativa na hemoglobinometria em que o grupo EGb 14,0 apresentou-se estatisticamente superior ao grupo EGb 3,5.A concentração de hemoglobina globular média também apresentou diferença estatística significativa, em que o EGb 3,5 apresentou médias inferiores aos grupos EGb 7,0 e EGb 14,0 e o grupo controle apresentou média inferior ao grupo EGb 14,0. Sugere-se que o EGb no presente trabalho, e com as doses utilizadas, não causou toxicidade sistêmica e nem provocou alterações em órgãos de ratos Wistar.

The Ginkgobiloba Extract (EGb) is one of the most commonly consumed herbal in the world. However there are still few toxicity tests on animals and the risk of human exposure mainly by alkyl compounds, represented by acids, which can cause allergies and are mutagenic and carcinogenic compounds. This study had the objective of evaluate the systemic toxicity of EGb. Eighty Wistar rats, three months of age were treated with distilled water (Control Group) and aqueous extract Ginkgobilobanas following doses: 3.5 (EGb 3.5); 7.0 (EGb 7.0) and 14,0mg / kg (14.0 EGb) once a day for consecutive 56 days. Were evaluated weekly animal weight (g) and the estimated daily intake (g). Evidence of systemic signs of toxicity such as weight loss, piloerection, diarrhea, stereotypies and behavioral changes in motor activity and deaths were also monitored. After anesthesia, the animals were collected for evaluation of complete blood count and biochemical analysis of urea, creatinine and alanine aminotransferase (ALT). After euthanasia, the animals were autopsied and the left and right testis, left epididymis, seminal vesicle filled, ventral prostate, left and right kidneys, liver and spleen were removed and weighed on a precision scale. Throughout the experimental procedure were not observed in animals clinical signs of systemic toxicity and deaths. Were no statistical differences in the estimate of feed intake in the sixth week and eighth week evaluation, although no difference in body weight. There were no differences in organ weight and serum biochemical analysis. Hematological evaluation of the animals, there was a statistically significant difference in Hemoglobinometry where 14.0 EGb group was statistically higher than the EGb group 3,5. A mean corpuscular hemoglobin concentration also showed a statistically significant difference in the EGb 3 5 showed an average lower than 7.0 and EGb groups EGb 14.0 and the control group showed less than 14.0 EGb group. It is suggested that EGb in this work, and the doses used, did not cause systemic toxicity nor caused changes in organs of Wistar rats.

Toxicity and carcinogenicity studies of Ginkgo biloba extract in rat and mouse: liver, thyroid, and nose are targets.

Ginkgo biloba extract (GBE) is a popular herbal supplement that is used to improve circulation and brain function. In spite of widespread human exposure to relatively high doses over potentially long periods of time, there is a paucity of data from animal studies regarding the toxicity and carcinogenicity associated with GBE. In order to fill this knowledge gap, 3-month and 2-year toxicity and carcinogenicity studies with GBE administered by oral gavage to B6C3F1/N mice and F344/N rats were performed as part of the National Toxicology Program's Dietary Supplements and Herbal Medicines Initiative. The targets of GBE treatment were the liver, thyroid, and nose. These targets were consistent across exposure period, sex, and species, albeit with varying degrees of effect observed among studies. Key findings included a notably high incidence of hepatoblastomas in male and female mice and evidence of carcinogenic potential in the thyroid gland of both mice and rats. Various nonneoplastic lesions were observed beyond control levels in the liver, thyroid gland, and nose of rats and mice administered GBE. Although these results cannot be directly extrapolated to humans, the findings fill an important data gap in assessing risk associated with GBE use.

[Herbal drug-drug interaction and adverse drug reactions]. / Phytopharmaka als Ursache von unerwünschten Arzneimittelwirkungen.

Herbal medicines are used by many patients. Their known or potential adverse events should be taken into account during treatment with herbal medicines. In this article adverse effects of commonly used herbs are presented. St. John's wort is known to be a potent inducer of cytochrome P450 (CYP) 3A4 leading to reduced blood concentrations of a number of CYP3A4 substrates. For many other combinations evidence is sparse but due to a number of case reports of adverse interactions they should only cautiously be combined with certain critical dose drugs until their risk is fully assessed. Pertinent examples are the immunostimulant Echinacea which could decrease the effect of immunosuppressants. Ginseng and ginkgo should not be combined with anticoagulants. Excessive sedation may occur with concomitant use of valerian and barbiturates.

Ginkgo biloba effects on mice fetal liver / Efectos de la ginkgo biloba en el hígado fetal de ratón

Ginkgo biloba is considered to be an alternative drug for various indications; unfortunately very few studies are available on its side effects. This present study describes the harmful effects of Ginkgo biloba on developing fetal liver. Two experimental groups of six pregnant female mice each were given Ginkgo biloba at human therapeutic dose (A) and a higher dose (B) throughout the gestation period. A third group (C) was taken as a control and given distilled water only. Fetal livers were examined and the effects of the drug observed. There were signs of congestion and fatty change along with dilatation of sinusoids in a dose dependent manner concluding that Ginkgo biloba affects fetal liver.

La Ginkgo biloba es considerada, en varias indicaciones, como un medicamento alternativo; sin embargo, existen pocos reportes disponibles sobre sus efectos secundarios. Este estudio describe los efectos nocivos de Ginkgo biloba en el desarrollo del hígado fetal. Dos grupos experimentales de 6 ratones hembras preñadas recibieron Ginkgo biloba en la dosis terapéutica humana (A) y una dosis más alta (B) por el período de gestación. Un tercer grupo control (C) recibió agua destilada. Los hígados fetales fueron examinados y observados los efectos de la droga. Hubo signos de congestión y degeneración grasa, junto con la dilatación de sinusoides en función de la dosis. Como conclusión la Ginkgo biloba afecta el hígado fetal.

Ginkgo biloba induced malformations in mice.

OBJECTIVE: To determine the gross structural malformations to the mice fetuses of the mothers given Ginkgo biloba during pregnancy. STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: The Experimental Research Laboratory, University of Health Sciences, Lahore, from May 2006 to December 2006. METHODOLOGY: The teratogenic effects of Ginkgo biloba extract (78 mg/kg/day and 100 mg/kg/day) dissolved in water were studied on the gross features of mice fetuses. Three groups (A, B and C) of 6 females each were mated with 2 males in two cages with 3:1 ratio of females to males. The first two groups (A and B) served as experimental and the third (C) was used as a control. Pregnancy was confirmed by a vaginal plug and gravid female mice (6) were separated from the males. Group A was treated with human therapeutic dose (78 ppm) while group B was given a high dose (100 ppm). Group C was given water only. Both experimental groups were given the drug orally throughout the gestational period. Result were compared using ANOVA with significance at p < 0.05. RESULTS: Forty-nine fetuses from B and C groups and 50 fetuses from A group were recovered. There was a significant (p < 0.05) decrease in weight and crown-rump length of fetuses in group B as compared to those from group A and C. Further, fetuses from groups A and C did not show any gross abnormalities, whereas those from group B exhibited a high frequency of malformations including round shaped eye and orbits, syndactyly, malformed pinnae, nostrils, lips and jaws. CONCLUSION: The results obtained substantiate the early finding that Ginkgo biloba can be teratogenic when given to pregnant mothers.

Ginkgo biloba leave extract: biological, medicinal, and toxicological effects.

Ginkgo biloba leave extract is among the most widely sold herbal dietary supplements in the United States. Its purported biological effects include: scavenging free radical; lowering oxidative stress; reducing neural damages, reducing platelets aggregation; anti-inflammation; anti-tumor activities; and anti-aging. Clinically, it has been prescribed to treat CNS disorders such as Alzheimer's disease and cognitive deficits. It exerts allergy and changes in bleeding time. While its mutagenicity or carcinogenic activity has not been reported, its components, quercetin, kaempferol and rutin have been shown to be genotoxic. There are no standards or guidelines regulating the constituent components of Ginkgo biloba leave extract nor are exposure limits imposed. Safety evaluation of Ginkgo biloba leave extract is being conducted by the U.S. National Toxicology Program.

Larvicidal activity of extracts of Ginkgo biloba exocarp for three different strains of Culex pipiens pallens.

Ethanolic extracts from the Ginkgo biloba L. exocarp from the Chinese ginkgo were assayed against larvae of three strains of Culex pipiens pallens Coquillett. The chemical compositions were detected using a Hewlett-Packard 6890/5973 mass spectrometric detector. The larvicidal bioassay was carried out according to the recommendations of the World Health Organization. The analysis of the essential oil of ginkgo exocarp showed that its major components are ginkgo acid (85.3%) and ginkgo phenolic (5.69%). The larvicidal bioassay showed that extracts of ginkgo exocarp have LC50 of 18.6, 12.7, and 25.0 mg/liter for deltamethrin-susceptible, deltamethrin-resistant, and field strains, respectively. The acute toxicity concentrations of the ginkgo extracts that killed 50% (LD50) of Wistar rats within 2 wk and young carp within 96 h were 4947.2 mg/kg and 557.9 mg/liter, respectively. These results are promising in creating new, effective, and affordable approaches to mosquito control.

Amikacin ototoxicity enhanced by Ginkgo biloba extract (EGb 761).

An animal study was realized to investigate the possible beneficial effect of EGb 761 as an antioxidant agent on amikacin ototoxicity by measuring distortion product otoacoustic emissions (DPOAEs). Twenty-eight adult rats were grouped equally as follows. GROUP AMIKACIN: rats received amikacin 600 mg/kg/day intramuscularly between postnatal days (PND) 30 and PND44. Group amikacin/EGb 761: rats received amikacin 600 mg/kg/day intramuscularly between PND30 and PND44 and EGb 761 100 mg/kg/day orally between PND30 and PND50. Group EGb 761: rats received equivolume saline intramuscularly between PND30 and PND44 and EGb 761 100 mg/kg/day orally between PND30 and PND50. NO TREATMENT GROUP: rats received nothing. Group amikacin was found to be affected only on the last measurement day of study (PND57). The frequencies greater than 2002 Hz were significantly reduced compared with the amplitudes of PND30 (P<0.05). Group amikacin/EGb 761 was most and earliest affected by amikacin-induced ototoxicity. DPOAE amplitudes were found in this group to be decreased at 2-6 kHz starting on PND50. The results of Group EGb 761 and No treatment group were not significantly changed. For the DPOAE input/output amplitude thresholds, Group amikacin (P<0.05) and Group amikacin/EGb 761 (P<0.01) had significantly elevated thresholds on PND57, except at 5 kHz for Group amikacin (P=0,06). According to the results of the study, EGb 761 may be regarded as a facilitating drug for the development of amikacin ototoxicity. The results of the present study may warn against concomitant use of aminoglycosides, specifically amikacin, with EGb 761.

Bleeding complications precipitated by unrecognized Gingko biloba use after liver transplantation.

Because of its neurocognitive enhancing effects, Gingko biloba has emerged as amongst the most commonly used herbal products. We report a liver transplant recipient with potentially life-threatening toxicity resulting from Gingko biloba use. Seven days after a second liver transplantation for recurrent hepatitisB virus infection, subphrenic hematoma was documented in a 59-year-old Korean patient. Failure to control bleeding with CT-guided drainage necessitated exploratory laparotomy for the evacuation of a large subphrenic hematoma. Three weeks later, an episode of vitreous hemorrhage was documented. Unbeknownst to his care providers, the patient had been consuming Gingko biloba throughout the postoperative period. No further bleeding episodes occurred after the cessation of Gingko biloba use. Unrecognized use of herbal products may be associated with serious side effects and adverse clinical sequelae in transplant recipients. Given their increasing popularity, the use of herbal products should be routinely sought as part of the history in transplant recipients.

Evidence for toxic effects of alkylphenols from Ginkgo biloba in the hen's egg test (HET).

Extracts from the leaves of the Gingko tree (Ginkgo biloba L.) are therapeutically used for the treatment of peripheral and cerebral vascular disorders as well as multi-infarct or Alzheimer-type dementia. As constituents with potential contact allergenic and toxic properties in crude Ginkgo extracts a group of alkylphenols (e.g., ginkgolic acids, ginkgol, bilobol) has been described. Thus, for reasons of drug safety a maximal concentration (< or = 5 ppm) of ginkgolic acids is requested by the Monograph of the Commission E of the former German Federal Health Agency (Bundesgesundheitsamt, BGA). During production of the standardized Ginkgo extract EGb 761, alkylphenols are largely eliminated as water insoluble compounds (decanter sludge) from the primary acetone extract. To further assess the adverse properties of alkylphenols, different fractions derived from the decanter sludge were evaluated for their embryotoxic effects in the hen's egg test (HET). A fraction enriched for ginkgolic acids (16%) and biflavones (6.7%) was found to induce death of 50% of the chick embryos (LD50) at a dose of 1.8 mg/egg (approximately/= 33 ppm). A similar strong lethal effect (LD50: 3.5 mg/egg; 64 ppm) was oberserved for a fraction which contained 58% ginkgolic acids but less than 0.02% biflavones. In contrast, an extreme low toxic potential (LD50: 250 mg/egg or 4540 ppm) was established for a fraction containing 16% biflavones and 1% ginkgolic acids. Thus, the present investigations confirm the high toxic potential of ginkgolic acids, although it can not be excluded that biflavones or some other constituents in the different fractions may amplify the adverse effect of these substances. Since no contribution of alkylphenols to the therapeutic efficacy of Ginkgo extracts has been confirmed and their elimination during the manufacturing process does not cause technical problems, these results further support the requirement for the completest possible removal of these compounds under toxicological considerations.

Biosynthesis of vitamin B6 and structurally related derivatives.

In spite of the rather simple structure of pyridoxal 5'-phosphate (I), a member of the vitamin B6 group, the elucidation of its de novo biosynthesis remained largely unexplored until recently. Experiments designed to investigate the formation of the vitamin B6 pyridine nucleus mainly concentrated on Escherichia coli. The results of tracer experiments with radioactive and stable isotopes, feeding experiments, and molecular biological studies led to the prediction that 4-hydroxy-L-threonine (VIII, R = H) and 1-deoxy-D-xylulose (VII, R = H) are precursors which are assembled to yield the carbon-nitrogen skeleton of vitamin B6. At this point, the involvement of the phosphorylated forms of these precursors in this assembly seems quite clear. However, vitamin B6 biosynthesis in organisms other than E. coli remains largely unknown. Toxic derivatives of vitamin B6, such as ginkgotoxin, occurring in higher plants may be suitable targets to gain further insight into this tricky problem.

Evidence for immunotoxic effects of crude Ginkgo biloba L. leaf extracts using the popliteal lymph node assay in the mouse.

Allergic reactions due to contact with different parts of the ancient tree Ginkgo biloba L. have repeatedly been reported. Provocation tests in patients and animal experiments have identified alkylphenols such as ginkgolic acids as causative constituents. Leaf extracts from Ginkgo are widely used to treat peripheral or cerebral circulatory disorders and Alzheimer's disease. Since alkylphenols are also present in leaves, potential allergic and other immunological hazards of such preparations have to be carefully controlled. Thus, we have evaluated if the popliteal lymph node assay (PLNA) in the mouse may represent a suitable model for the detection of constituents with immunotoxic properties in a complex mixture of biologically active agents such as plant extracts. Subplantar injection (2 mg) of a crude aqueous-ethanolic extract from Ginkgo leaves caused a significant lymphoproliferative reaction (LPR) in the ipsilateral popliteal lymph node. PLNA-active compounds in this extract could be enriched in the lipophilic phase by liquid-liquid partition between heptane and water. Chemical analysis of the heptane extract revealed the presence of a high concentration of alkylphenols (approx. 30%) and further subfractionation indicated that the enlargement of the popliteal lymph node was mainly due to the content of ginkgolic acids. This presumption was corroborated by observing a similar LPR following injection of a purified mixture of ginkgolic or hydroginkgolic acids. Thus, our experiments confirm that Ginkgo leaf extracts may contain constituents with immunotoxic properties, underlining the need to apply adequate production procedures to guarantee the completest possible removal of these compounds. The PLNA appears to represent a simple test model for the detection, characterisation and control of ingredients with potential immunotoxic side effects in complex herbal drugs.