A critical review of ginkgolic acids in Ginkgo biloba leaf extract (EGb): toxicity and technologies to remove ginkgolic acids and their promising bioactivities.

Ginkgo biloba leaf extract (EGb) is high in bioactive components (over 170), which are used in food additives, medicine, cosmetics, health products, and other sectors. Nonetheless, ginkgolic acids (GAs) in Ginkgo biloba (GB) have been identified as the primary source of EGb's adverse effects such as embryotoxicity, cytotoxicity, neurotoxicity, and inhibition of enzyme systems. As a result, the Chinese, European, and United States pharmacopeias all mandate that the GA concentration in EGb be less than 5 µg g-1. This review looked at the toxicity of ginkgolic acid (from in vitro and in vitro trials) as well as the technologies (such as adsorption/desorption, enzymatic degradation, counter-current chromatography, liquid-liquid microextraction, dual-frequency ultrasonic-solvent extraction, deep eutectic solvent, etc.) used to lower the GA to the desired concentration. These technologies' advantages, disadvantages, viability, and future trends were compared. In addition, several pharmacological significances of GA extraction, such as anti-microbial, anti-inflammatory, anti-tumor, etc., were discussed, as well as future directions.

The metabolism and hepatotoxicity of ginkgolic acid (17 : 1) in vitro.

Pharmacological activities and adverse side effects of ginkgolic acids (GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA (17 : 1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA (17 : 1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA (17 : 1) metabolism were human CYP1A2, CYP3A4, UGT1A6, UGT1A9, and UGT2B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA (17 : 1) in HepG2 cells occurred in a time- and dose-dependent manner. Further investigation showed that GA (17 : 1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1A- and CYP3A-mediated metabolism.

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.

Salicylate-Induced Hearing Loss Trigger Structural Synaptic Modifications in the Ventral Cochlear Nucleus of Rats via Medial Olivocochlear (MOC) Feedback Circuit.

Lesion-induced cochlear damage can result in synaptic outgrowth in the ventral cochlear nucleus (VCN). Tinnitus may be associated with the synaptic outgrowth and hyperactivity in the VCN. However, it remains unclear how hearing loss triggers structural synaptic modifications in the VCN of rats subjected to salicylate-induced tinnitus. To address this issue, we evaluated tinnitus-like behavior in rats after salicylate treatment and compared the amplitude of the distortion product evoked otoacoustic emission (DPOAE) and auditory brainstem response (ABR) between control and treated rats. Moreover, we observed the changes in the synaptic ultrastructure and in the expression levels of growth-associated protein (GAP-43), brain-derived neurotrophic factor (BDNF), the microglial marker Iba-1 and glial fibrillary acidic protein (GFAP) in the VCN. After salicylate treatment (300 mg/kg/day for 4 and 8 days), analysis of the gap prepulse inhibition of the acoustic startle showed that the rats were experiencing tinnitus. The changes in the DPOAE and ABR amplitude indicated an improvement in cochlear sensitivity and a reduction in auditory input following salicylate treatment. The treated rats displayed more synaptic vesicles and longer postsynaptic density in the VCN than the control rats. We observed that the GAP-43 expression, predominantly from medial olivocochlear (MOC) neurons, was significantly up-regulated, and that BDNF- and Iba-1-immunoreactive cells were persistently decreased after salicylate administration. Furthermore, GFAP-immunoreactive astrocytes, which is associated with synaptic regrowth, was significantly increased in the treated groups. Our study revealed that reduced auditory nerve activity triggers synaptic outgrowth and hyperactivity in the VCN via a MOC neural feedback circuit. Structural synaptic modifications may be a reflexive process that compensates for the reduced auditory input after salicylate administration. However, massive increases in excitatory synapses in the VCN may represent a detrimental process that causes central hyperactivity, leading to tinnitus.

In vitro antitumor activities of the lichen compounds olivetoric, physodic and psoromic acid in rat neuron and glioblastoma cells.

Context Since methods utilised in the treatment of glioblastoma multiforme (GBM) are inadequate and have too many side effects, usage of herbal products in the treatment process comes into prominence. Lichens are symbiotic organisms used for medicinal purposes for many years. There are various anticancer treatments about components of two lichen species used in the present study. Objective Antitumor potential of three lichen secondary metabolites including olivetoric acid (OLA) and physodic acid (PHA) isolated from Pseudevernia furfuracea (L.) Zopf (Parmeliaceae) and psoromic acid (PSA) isolated from Rhizoplaca melanophthalma (DC.) Leuckert (Lecanoraceae) were investigated on human U87MG-GBM cell lines and primary rat cerebral cortex (PRCC) cells for the first time. Materials and methods PRCC cells used as healthy brain cells were obtained from Sprague-Dawley rats. The treatments were carried out on the cells cultured for 48 h. Cytotoxic effects of different concentrations (2.5, 5, 10, 20 and 40 mg/L) of metabolites on the cells were determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) analyses. Total antioxidant capacity (TAC) and total oxidant status (TOS) parameters were used for assessing oxidative alterations. Oxidative DNA damage potentials of metabolites were investigated via evaluating 8-hydroxy-2'-deoxyguanosine (8-OH-dG) levels. Results Median inhibitory concentration (IC50) values of OLA, PHA and PSA were 125.71, 698.19 and 79.40 mg/L for PRCC cells and 17.55, 410.72 and 56.22 mg/L for U87MG cells, respectively. It was revealed that cytotoxic effects of these metabolites showed positive correlation with concentration, LDH activity and oxidative DNA damage. Discussion and conclusion The present findings obtained in this study revealed that primarily OLA and then PSA had high potential for use in the treatment of GBM.

RIFM fragrance ingredient safety assessment, isoamyl salicylate, CAS registry number 87-20-7.

The use of this material under current use conditions is supported by the existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity, skin sensitization potential, as well as, environmental safety. Repeated dose toxicity was determined using to have the most conservative systemic exposure derived NOAEL of 47 mg/kg/day. A dietary 13-week subchronic toxicity study conducted in rats on a suitable read across analog resulted in a MOE of 2350 while considering 10.3% absorption from skin contact and 100% from inhalation. A MOE of >100 is deemed acceptable.

Evaluation of the cytotoxic and mutagenic potential of three ginkgolic acids.

Ginkgolic acids (GAs) are alkylphenols which can be found in the fruits and leaves of Ginkgo biloba L. (Ginkgoaceae) used in herbal teas, drugs and food supplements. Standardized leaf extracts of G. biloba are widely used in the therapy of cognitive decline including Alzheimer's diseases. However, GAs are known to have cytotoxic and allergenic potential and are suspected to possess genotoxic properties. Therefore, we examined in this study the cytotoxicity and mutagenicity of three major GAs with different alkyl or alkenyl groups (13:0, 15:1, 17:1). Cytotoxicity was assessed in male Chinese hamster lung fibroblasts (V79 cells) using the resazurin reduction assay. The substances showed concentration dependent cytotoxic effects after 24h of incubation at concentrations of 50µM and higher. Mutagenicity was determined by using the Ames fluctuation assay in different Salmonella typhimurium strains (TA97a, TA98, TA100 and TA102) with and without exogenous metabolic activation (S9 mix). Furthermore, we analyzed the mutagenic potency of the three major GAs in V79 cells by performing the hypoxanthine phosphoribosyl transferase (HPRT) assay which detects gene mutations at the HPRT locus. None of the mutagenic assays showed any increase in mutagenicity above background. Therefore, these data provide evidence that the GAs tested have some cytotoxic potency but are not mutagenic. Thus, our findings contribute to the risk assessment of preparations containing plant extracts from G. biloba.

Evaluation of the pharmacological properties of salicylic acid-derivative organoselenium: 2-hydroxy-5-selenocyanatobenzoic acid as an anti-inflammatory and antinociceptive compound.

The present study evaluated the antinociceptive and anti-inflammatory effects of per oral (p.o.) administration of salicylic acid-derivative organoselenium compounds in chemical models of nociception in mice. The compounds (50 mg/kg; p.o.) were administered 30 and 60 min before the nociceptive behavior and compared to the positive-control, acetylsalicylic acid (ASA; 200 mg/kg; p.o.). In addition, a dose-response curve (25-100 mg/kg) for compounds was carried out in the formalin test. When assessed in the chemical models, acetic acid-induced writhing behavior, formalin and glutamate tests, the compounds showed the following antinociceptive profile 1B>2B>1A>2A, suggesting a chemical structure-dependent relationship. Then, the anti-inflammatory properties and toxicological potential of compound 1B were investigated. Compound 1B, similar to the positive-control, ASA, diminished the edema formation and decreased the myeloperoxidase activity induced by croton oil (2.5%) in the ear tissue. The results also indicate that a single oral administration of 1B caused neither signs of acute toxicity nor those of gastrointestinal injury. The administration of 1B did not alter the water and food intakes, plasma alanine and aspartate aminotransferase activities or urea levels and cerebral or hepatic δ-aminolevulinate dehydratase activity. Salicylic acid-derivative organoseleniums, mainly compound 1B, have been found to be novel compounds with antinociceptive/anti-inflammatory properties; nevertheless, more studies are required to examine their therapeutic potential for pain treatment.

Lichenic extracts and metabolites as UV filters.

Three lichen extracts and ten lichenic compounds have been screened for their photoprotective activities. The determination of their Sun Protection Factor (SPF) and Protection Factor-UVA (PF-UVA) values was done in vitro. Among them, a Lasallia pustulata extract and gyrophoric acid exhibited SPF values over 5, which is better than Homosalate (SPF≈4). Their photoprotective properties are only slightly modified after a 2-hours period of irradiation. Salazinic acid and L. pustulata presented characteristics of a UVA booster like the butyl-methoxydibenzoylmethane (Avobenzone) (PF-UVA≈2 vs. 2.8 for Avobenzone). Salazinic acid was a better anion superoxide scavenger than ascorbic acid and none of them exhibited a photosensitizing cytotoxicity by exposing them on HaCaT cells to UVA radiations (photo-irritancy factor PIF<5).

[Advance in study of ginkgolic acid contained in Ginkgo biloba preparations].

Ginkgo biloba has a very high medicinal value. The flavonol glycosides and terpene lactones contained in G. biloba extract (GBE) have such pharmacological effects as antioxidant, anti-platelet aggregation and memory improvement, enhancement of immune function. However, the ginkgolic acid (GA) contained in GBE is proved to be highly allergenic and cytotoxic, even minimal residual could also cause severe adverse effects. To minimize the potential safety hazards of ginkgo leaf preparations, this study focuses on GA's chemical structure, adverse effects, toxicity and genesis mechanism, desorption and attenuation in the hope of providing a new thought for studies on safety of Ginkgo biloba preparations.

Effect of laser phototherapy on the release of TNF-alpha and MMP-1 by endodontic sealer-stimulated macrophages.

OBJECTIVE: Our aim was to analyze the effect of laser phototherapy on the secretory activity of macrophages activated by interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS), and stimulated by substances leached from an epoxy resin-based sealer (AH-Plus) and a calcium hydroxide-based sealer (Sealapex). BACKGROUND DATA: Laser phototherapy can modulate the inflammatory process, improving wound healing. This type of therapy could be useful for modulating postoperative symptoms seen after endodontic treatment. MATERIALS AND METHODS: Cytotoxicity was indirectly assessed by measuring mitochondrial activity. Macrophages were stimulated by the leached substances or not (controls), and the groups were then irradiated or not. The secretion of pro-inflammatory cytokines (TNF-alpha and MMP-1) was analyzed using ELISA. Two irradiations at 6-h intervals were done with an As-Ga-Al diode laser (780 nm, 70 mW, spot size 4.0 mm(2), 3 J/cm(2), for 1.5 sec) in contact mode. RESULTS: The sealers were non-cytotoxic to macrophages. The production of TNF-alpha was significantly decreased by laser phototherapy, regardless of experimental group. The level of secretion of MMP-1 was similar in all groups. CONCLUSION: Based on the conditions of this study we concluded that in activated macrophages, laser phototherapy impairs the secretion of the pro-inflammatory cytokine TNF-alpha, but has no influence on MMP-1 secretion.

Effect of three commercial mouth rinses on cultured human gingival fibroblast: an in vitro study.

AIM: To examine the effect of three commercial mouth rinses (Hexidine 0.2%, Listerine Cool Mint, Betadine 1%) upon cultured human gingival fibroblast proliferation. MATERIALS AND METHODS: Human gingival fibroblasts were cultured and incubated in Dulbecco's Minimum Eagle's Medium containing Chlorhexidine, Listerine, Povidone-Iodine at varying concentrations (1%, 2%, 5%, 10%, 20% and 100% of the given solution) at 37 degrees C for 1, 5 and 15 min. Control cells received an equal volume of Dulbecco's Minimum Eagle's Medium without adding mouth rinses, for similar duration of exposure at 37 degrees C. Following incubation the media were removed, cells were washed twice with medium, supplemented with 10% Fetal Bovine Serum, and fibroblasts in the test and control group were allowed to recover in the same media for 24 h. RESULTS: In all the three groups, the proliferation inhibition was dependent on the concentration of solublized mouth rinses in the cell culture but independent of the duration of exposure to all three mouth rinses. The results showed that all three solutions were toxic to cultured human gingival fibroblasts, Chlorhexidine being the most cytotoxic. It was seen that at dilute concentrations (1% and 2% of given solutions) Listerine was more cytotoxic than Chlorhexidine and Povidone-Iodine. CONCLUSION: These results suggest that Chlorhexidine, Listerine and Povidone-Iodine are capable of inducing a dose-dependent reduction in cellular proliferation of fibroblasts. The results presented are interesting, but to know the clinical significance, further studies are needed.

[Preparation of five ginkgolic acid monomers and their molluscicidal effects against Oncomelania hupensis].

OBJECTIVE: To investigate the molluscicidal activities of the ginkgolic acid(GA) monomers isolated and purified from GAs. METHODS: Five monomers of GAs from the sarcotesta of Ginkgo biloba. were extracted by petrol ether, separated by silica gel column chromatography, purified by semi-prepared reversed-phased HPLC, and identified by LC-MS analysis. The molluscicidal activities of GAs and their monomers against Oncomelania hupensis were determined as referring to the WHO guidelines for laboratory molluscicidal test. RESULTS: The five purified ginkgolic acid monomers were GA(13:0), GA(15:0), GA(15:1), GA(17:1) and GA(17:2), with a side chain of 13, 15, 17 alkyl or ethylenic radicals res pectively on their benzene loop. The five monomer proportions to the total GAs were 17.6%, 3.2%, 52.3%, 23.3% and 3.6% respectively. The order of molluscicidal activities for the five monomers was as follows: GA(13:0)>GA(15:1)>GA(15:0)>GA(17:1)>GA(17:2), and their LC50 for snails was 20.79 mg/L, 22.28 mg/L, 33.76 mg/L, 51.89 mg/L, and 59.10 mg/L respectively after immersion for 24 hours. Two monomers, GA(13:0), and GA(15:1) inhibited the snails' climbing up significantly. CONCLUSION: The molluscicidal activities of GAs may be dependent on the monomer's structure with different number of carbon molecules and double-bonds on the side carbon-chain. The two monomers, GA(13:0) and GA(15:1), are mainly responsible for the molluscicidal activities of GAs and both effectively inhibit snails' climbing up as well. GA(15:0) also shows certain molluscicidal activity.

Cochlear NMDA receptor blockade prevents salicylate-induced tinnitus.

Large doses of aspirin produce reversible hearing loss and tinnitus. These effects have been attributed to the salicylate ion, the active component of aspirin. Salicylate acts as a competitive antagonist at the anion-binding site of prestin, the motor protein of sensory outer hair cells. This provides an explanation for the hearing loss induced by aspirin. However, the molecular mechanism of salicylate-induced tinnitus remains obscure. One physiological explanation is that salicylate ototoxicity is likely to originate in an alteration to arachidonic acid metabolism. Arachidonic acid potentiates NMDA receptor currents. We therefore tested the involvement of cochlear NMDA receptors in the occurrence of tinnitus. Tinnitus was assessed with a behavioural test based on an active avoidance paradigm. Results showed that the tinnitus induced by salicylate may be suppressed by the introduction of NMDA antagonists into the cochlear fluids. To determine if the activation of NMDA receptors was linked to cyclooxygenase inhibition, we investigated the effect of mefenamate (a potent cyclooxygenase inhibitor). Since NMDA antagonists also blocked mefenamate-induced tinnitus, we suggest that salicylate-induced tinnitus is mediated by cochlear NMDA receptors through the inhibition of cyclooxygenase activity. Target cochlear NMDA receptors may therefore present a therapeutic strategy for the treatment of tinnitus.

Calcium oxalate crystals and methyl salicylate as toxic principles of the fresh leaves from Palicourea longiflora, an endemic species in the Amazonas state.

The species of the genus Palicourea (Rubiaceae family) is well-known for its toxicity towards animals, particularly livestock. This work reports the occurrence of skin irritation during the manipulation of Palicourea longiflora, considering the prevalence of the monofluoracetic acid (MFAA) and another toxic compound: methyl salicylate. The MFAA was identified by 19F-NMR and methyl salicylate by gas chromatography linked to mass spectrometry (GC/MS) analysis. Additionally, an anatomical study of leaves had been used to explain the mechanism of penetration of the toxic principles.

Acute methyl salicylate toxicity complicating herbal skin treatment for psoriasis.

We present an interesting case of salicylism arising from the use of methyl salicylate as part of a herbal skin cream for the treatment of psoriasis. A 40-year-old man became quite suddenly and acutely unwell after receiving treatment from an unregistered naturopath. Methyl salicylate (Oil of Wintergreen) is widely available in many over the counter topical analgesic preparations and Chinese medicated oils. Transcutaneous absorption of the methyl salicylate was enhanced in this case due to the abnormal areas of skin and use of an occlusive dressing. The presence of tinnitus, vomiting, tachypnoea and typical acid/base disturbance allowed a diagnosis of salicylate toxicity to be made. Our patient had decontaminated his skin prior to presentation, limiting the extent of toxicity and was successfully treated with rehydration and establishment of good urine flow.

In vitro evaluation of the cytotoxic potential of alkylphenols from Ginkgo biloba L.

Extracts from the leaves of Ginkgo biloba L. belong to the most widely used phytopharmaceuticals. In crude Ginkgo extracts, ginkgolic acids (GA) and related alkylphenols (e.g. cardanols and cardols) have been recognized as hazardous compounds with suspected cytotoxic, allergenic, mutagenic and carcinogenic properties. To further assess the cytotoxic potential of GA, their effect on the human keratinocyte cell line HaCaT and the rhesus monkey kidney tubular epithelial cell line LLC-MK(2) was investigated. The action of a defined mixture of GA on cell growth, viability and integrity was evaluated by the neutral red uptake assay as well as the release of lactate dehydrogenase (LDH) and acid phosphatase (ACP). Cell morphology was examined by electron microscopy. For comparison, the effect of the standardized Ginkgo extract EGb 761, which contains less than 5 ppm GA, was also investigated. Following incubation of cells with EGb 761, neutral red uptake was half-maximally inhibited at concentrations of 900 mg/l (HaCaT) and 1480 mg/ml (LLC-MK(2)). The corresponding IC(50)-values for the mixture of GA ranged between 22 mg/l (HaCaT) and 4.6 mg/l (LLC-MK(2)), respectively. In parallel to the inhibition of neutral red uptake, a concentration dependent release of LDH was observed when cells were incubated in the presence of GA (1-100 mg/l). In contrast, even at a concentration of 1800 mg/l EGb 761 did not cause release of LDH above controls. Since GA interacted with the assay for ACP, no index of lysosomal damage could be established by this method. Incubation of HaCaT cells with GA for 18 h increased the proportion of apoptotic cells from about 6% (control) to nearly 80% at concentrations of >or=30 mg/l. Electron microscopic analysis of HaCaT cells revealed a drug induced formation of myelinosomes possibly due to the inhibition of lysosomal enzymes, while morphological evaluation of LLC-MK(2) cells indicated that the cytotoxic activity of GA in these cells is primarily mediated by transformation of mitochondria, which is probably induced by uncoupling of oxidative phosphorylation.

Ginkgolic acids induce neuronal death and activate protein phosphatase type-2C.

The standardized extract from Ginkgo biloba (EGb 761) is used for the treatment of dementia. Because of allergenic and genotoxic effects, ginkgolic acids are restricted in EGb 761 to 5 ppm. The question arises whether ginkgolic acids also have neurotoxic effects. In the present study, ginkgolic acids caused death of cultured chick embryonic neurons in a concentration-dependent manner, in the presence and in the absence of serum. Ginkgolic acids-induced death showed features of apoptosis as we observed chromatin condensation, shrinkage of the nucleus and reduction of the damage by the protein synthesis inhibitor cycloheximide, demonstrating an active type of cell death. However, DNA fragmentation detected by the terminal-transferase-mediated ddUTP-digoxigenin nick-end labeling (TUNEL) assay and caspase-3 activation, which are also considered as hallmarks of apoptosis, were not seen after treatment with 150 microM ginkgolic acids in serum-free medium, a dose which increased the percentage of neurons with chromatin condensation and shrunken nuclei to 88% compared with 25% in serum-deprived, vehicle-treated controls. This suggests that ginkgolic acid-induced death showed signs of apoptosis as well as of necrosis. Ginkgolic acids specifically increased the activity of protein phosphatase type-2C, whereas other protein phosphatases such as protein phosphatases 1A, 2A and 2B, tyrosine phosphatase, and unspecific acid- and alkaline phosphatases were inhibited or remained unchanged, suggesting protein phosphatase 2C to play a role in the neurotoxic effect mediated by ginkgolic acids.

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.