Cognitive and other behavioral effects of EGb 761 in animal models.

Extensive pre-clinical and clinical studies conducted over more than three decades have established that EGb 761 (definition see editorial) represents a polyvalent therapeutic principle that is useful in the therapy of mildly to moderately severe dementia and other cognitive disorders. Besides cognition, other emotional and affective aspects of brain function also seem to benefit from EGb 761 treatment. Extensive behavioural studies in experimental animals are generally in line with clinical data since cognition improvement, stress protection, and antidepressive effects have been identified with this extract in proper animal models. While individual effects in all areas have been reported for adult animals and acute dosing, more pronounced effects are usually seen in aged animals and after subchronic treatment. Specifically, for the cognition improving properties pronounced beneficial effects are mainly present in those situations where cognition was impaired by aging or other noxious stimuli. Since all these conditions are associated with mitochondrial dysfunction, the stabilizing or even protecting effect of EGb 761 on mitochondrial function seems to be a major mechanism associated with many of EGb 761's behavioural effects. Bilobalide is most important in this respect. Moreover, bilobalide and the ginkgolides have recently been shown to affect chloride conductance by interfering with the function of membrane proteins related to receptor-gated chloride channels. These mechanisms are probably associated with behavioural effects requiring acute changes of neuronal activity, but might indirectly also improve mitochondrial function.

Structure-activity studies with Ginkgo biloba extract constituents as receptor-gated chloride channel blockers and modulators.

The constituents of Ginkgo biloba leaf extract, ginkgolides A, B, C and J are known as effective antagonists of platelet-activating factor (PAF). Here, we will demonstrate that these substances are also effective blockers of glycine-activated chloride channels in the hippocampal neurons of rat. As examined in several other voltage- and ligand-operated channels, this ginkgolide action is selective. The blocking action of all tested ginkgolides is use-dependent--they block open glycine-activated channels. The IC (50) values for saturating blocking action of ginkgolides B and C are 0.273 microM and 0.267 microM, respectively, while ginkgolides A and J are less effective--IC (50) values are 1.97 microM and 2.0 microM. Corresponding dose-response relationships are close to single-site binding isotherms. Another constituent of EGb 761, bilobalide, is a weak inhibitor of NMDA receptor-activated current. Its synthetic analogue, NV-31, demonstrates a weak facilitatory action on Gly-activated conductance. Novel findings have indicated the possibility that the unique modulating activity profiles of the EGb 761 (definition see editorial) constituents examined are due to their effects on the anion homeostasis of central neurons.

St John's wort (Hypericum perforatum) modulates evoked potentials in guinea pig hippocampal slices via AMPA and GABA receptors.

The effects of an ethanolic extract of the plant Hypericum perforatum L. (St John's wort) (HYP) and its hydrosoluble fraction (HYPWS) on electrically evoked population spikes and fEPSP were investigated in this study. Concentration dependent (10(-6) to 10(-4) g/l) excitatory effects were found. Above concentrations of 10(-3) g/l, HYP reduced the evoked responses, whereas HYPWS further increased them. Paired pulse facilitation was unaffected with HYPWS (10(-4) to 10(-2) g/l). The excitatory effects of HYPWS were amplified by the GABA(A) and GABA(B) receptor antagonists bicuculline and phaclofen, respectively. These excitations were antagonised by the AMPA receptor antagonist CNQX. Excitations caused by hypericum were not antagonised by the NMDA receptor antagonists D-APV and MK801, the metabotropic glutamate receptor (type I and II) antagonist MCPG, or the L-type calcium channel blocker verapamil. Hypericin and hyperforin, two components of H. perforatum, were found not to be responsible for the excitatory effects of the extract.

Stimulation of glutamate, aspartate and gamma-aminobutyric acid release from synaptosomes by hyperforin.

Hyperforin is one pharmacologically active constituent of the medicinal herb Hypericum perforatum. The mechanism of its antidepressant-like activity is currently considered to be the inhibition of synaptic reuptake of neurotransmitters. Here, we will demonstrate that it also stimulates the release of glutamate from rat cortical synaptosomes, and that this effect is preceded by an increase in their free calcium [Ca2+]i levels. These hyperforin-related effects were also observed in the absence of Ca2+ in the medium. Although we noted enhanced glutamate, aspartate and GABA release under the influence of hyperforin, the release of various other amino acids was not enhanced. In contrast, reserpine did not influence the release of any of the amino acids studied. Adding hyperforin to synaptosomal suspension decreased their pHi, which returned to basal levels under certain incubation conditions. It also prevented the generation of ATP-induced pH gradients of isolated synaptic vesicles, and preformed pH-gradients were reversed by it. We will discuss the implications of our studies in understanding the mechanisms of hyperforin activity in relation to current findings on its pharmacological activity profile. Our observations suggest that neurotransmitter release stimulation from synaptosomes and the previously reported reuptake inhibitory properties of hyperforin are consequences of its effects on synaptosomal ionic homeostasis, and that it is not a reserpine-like agent.

Hypericum extract and hyperforin: memory-enhancing properties in rodents.

Effects of a Hypericum extract in therapeutic use and hyperforin sodium salt were evaluated in rat and mouse avoidance tests. In a conditioned avoidance response (CAR) test on the rat, oral daily administration of hyperforin (1.25 mg/kg/day) or of the extract (50 mg/kg/day) before the training sessions considerably improved learning ability from the second day onwards until the day 7. In addition, the memory of the learned responses acquired during 7 consecutive days of administration and training was largely retained even after 9 days without further treatment or training. The observations made using different doses indicate that these learning-facilitating and/or memory-consolidating effects by the agents follow inverse U-shaped dose-response curves in dose ranges lower than (for hyperforin) or equal to (for Hypericum extract) their effective dose in the behavioral despair test for antidepressants. In a passive avoidance response test on the mouse, a single oral dose (1.25 mg/kg) of hyperforin not only improved memory acquisition and consolidation, but also almost completely reversed scopolamine-induced amnesia. The single Hypericum extract dose tested (25 mg/kg) did not reveal any significant effects in the passive avoidance response (PAR) test on the mouse. These observations suggest that the Hypericum extract could be a novel type of antidepressant with memory enhancing properties, and indicate that hyperforin is involved in its cognitive effects. Pure hyperforin seems to be a more potent antidementia agent than an antidepressant.

Hyperforin stimulates intracellular calcium mobilisation and enhances extracellular acidification in DDT1-MF2 smooth muscle cells.

Hyperforin, an acylphloroglucinol derivative, is a major constituent of St. John's wort extract (Hypericum perforatum L.), which is used in treating depressive disorders. Hyperforin has been demonstrated as a modulator of several neuronal ion channels, and inhibits smooth-muscle contraction induced by various neurotransmitters. To evaluate the spasmolytic properties of hyperforin in more detail, we performed studies on the hamster vas deferens smooth muscle cell line DDT1-MF2. In a first series of experiments, we determined the effect of hyperforin on intracellular Ca2+ concentration ([Ca2+]i) using the fluorochrome fura-2. These investigations were supplemented in a second series of assays, where the effects on cellular metabolism were analysed by measuring the rate of extracellular release of acidic metabolites with the help of a microphysiometer. Hyperforin (0.3-10 microg/ml) caused a concentration-dependent elevation of [Ca2+]i and extracellular acidification rate (ECAR). Both of these effects were independent of extracellular Ca2+. To elucidate whether the increase of [Ca2+]i by hyperforin causes or results from its ECAR-stimulating properties, we used various pharmacological tools to reveal the sequence of events and the molecular mechanisms involved. Our results suggest that hyperforin induces release of Ca2+ from as yet unidentified sources. Since the ECAR stimulation was inhibited to a different extent by the intracellular Ca2+ chelator BAPTA as well as by inhibitors of plasmalemmal and mitochondrial Na+/Ca2+ exchange, but not by inhibitors of Na+/H+ antiport, the intracellular Ca2+ increase seems to be essential for this hyperforin effect. However, further studies are needed to establish the exact mode of action, and to deduce whether this aspect of hyperforin activity contributes to its antidepressant and neuroprotective effects.

Modulation of ion channels in rat neurons by the constituents of Hypericum perforatum.

Despite almost forty years of widespread use of antidepressant drugs, their mode of action is still unknown. Hyperforin, a phloroglucinol derivative, is a major pharmacologically and therapeutically active constituent of Hypericum perforatum extract that is widely used as an herbal antidepressant drug. However, the mechanism or mechanisms of action of these naturally abundant, non-toxic extracts remain unclear. Enzymatically isolated patch-clamped rat central and peripheral neurons exposed to rapid changes in the composition of external medium (concentration clamp) were used in our experiments to investigate the modulation of the various voltage- and ligand-gated channels by hyperforin, as well as by other constituents of Hypericum perforatum. At nanomolar concentrations, hyperforin induced significant inhibition of various ion channels. In the case of P-type Ca2+ channels, we established that hyperforin acts via interaction with calmodulin or through calmodulin-activated pathways involving at least one second messenger. The results presented here indicate that multiple mechanisms and extract constituents may be involved in the antidepressant action of Hypericum extracts, and that they could also possess neuroprotective and analgesic effects.

Effects of Hypericum perforatum L. on evoked potentials in guinea pig hippocampal slices.

Therapeutic uses of Hypericum extracts have been demonstrated as safe and effective in treating mild to moderate depression in numerous clinical trials. To date, however, no definitive statements on their mode of action can be made, and little information on their electrophysiological effects is available. The present communication summarises the results of our efforts directed towards clarifying the effects of an ethanolic Hypericum extract (HYP) and its hydrosoluble fraction (HYPWS), and two of its constituents hypericin and hyperforin on electrically evoked population spikes in guinea pig hippocampal slices. In higher concentrations (>10 microM), the two extract constituents tested revealed inhibitory effects only, whereas concentration-dependent (between 10(-6) to 10(-4) g/l) excitatory effects were observed for HYP and HYPWS. The excitatory effects were strongly amplified by the GABA(B) antagonist phaclofen, whereas the effects of bicucullin, a GABA(A) antagonist, were marginal. The excitations were completely blocked by the AMPA antagonist CNQX, but not by the NMDA antagonists APV and MK801 or the L-type calcium-channel blocker verapamil. This kind of excitatory effect on the hippocampus is unknown in other antidepressants and; indeed, many of the latter reduce neuronal excitability. We conclude, therefore, that the mechanisms involved in the antidepressant activity of Hypericum extracts are different from those of conventional antidepressants, and that identifying their excitatory components may facilitate their more rational standardisation.

Effect of Ginkgo biloba extract (EGb761) on glucose metabolism-related markers in streptozotocin-damaged rat brain.

To reveal whether an extract of Ginkgo biloba (EGb761) may affect streptozotocin (STZ)-induced impairments in brain glucose metabolism, autoradiographies of [3H]cytochalasin-B binding to the total population of glucose transporters, [125I]insulin binding to insulin receptors, [3H]glyburide binding to sulfonylurea receptors, and radioactive in situ hybridization for GLUT3 mRNA were carried out in hippocampal brain sections of adult rats that have additionally been divided into good performers (GP) and poor performers (PP) by behavioural tests before the experiments. The STZ-induced increases in hippocampal [3H]cytochalasin-B binding to (total) glucose transporters returned to almost normal values following EGb761 treatment, regardless of the experimental animal group (GP or PP) tested. Similarly, the STZ-mediated enhancements in hippocampal insulin receptor binding of GP rats were partially compensated by the treatment with EGb761. The data suggest beneficial effects of EGb671 on impaired brain glucose metabolism, at least under the experimental conditions used in the study presented.

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.

Bilobalide, a constituent of Ginkgo biloba, inhibits NMDA-induced phospholipase A2 activation and phospholipid breakdown in rat hippocampus.

In rat hippocampal slices superfused with magnesium-free buffer, glutamate (1 mM) caused the release of large amounts of choline due to phospholipid breakdown. This phenomenon was mimicked by N-methyl-D-aspartate (NMDA) in a calcium-sensitive manner and was blocked by NMDA receptor antagonists such as MK-801 and 7-chlorokynurenate. The NMDA-induced release of choline was not caused by activation of phospholipase D but was mediated by phospholipase A2 (PLA2) activation as the release of choline was accompanied by the formation of lyso-phosphatidylcholine (lyso-PC) and glycerophospho-choline (GPCh) and was blocked by 5-[2-(2-carboxyethyl)-4-dodecanoyl-3,5-dimethylpyrrol-1-yl]pentano ic acid, a PLA2 inhibitor. Bilobalide, a constituent of Ginkgo biloba, inhibited the NMDA-induced efflux of choline with an IC50 value of 2.3 microM and also prevented the formation of lyso-PC and GPCh. NMDA also caused a release of choline in vivo when infused into the hippocampus of freely moving rats by retrograde dialysis. Again, the effect was completely inhibited by bilobalide which was administered systemically (20 mg/kg i.p.). Interestingly, convulsions which were observed in the NMDA-treated rats were almost totally suppressed by bilobalide. We conclude that release of choline is a sensitive marker for NMDA-induced phospholipase A2 activation and phospholipid breakdown. Bilobalide inhibited the glutamatergic excitotoxic membrane breakdown both in vitro and in vivo, an effect which may be beneficial in the treatment of brain hypoxia and/or neuronal hyperactivity.

Damaged neuronal energy metabolism and behavior are improved by Ginkgo biloba extract (EGb 761).

The standardized extract EGb 761 from the dried green leaves of Ginkgo biloba is a complex mixture of ingredients with an uniquely broad spectrum of pharmacological activities on the central nervous system e.g. in memory enhancing properties and in the regulation of cerebral glucose/energy metabolism. To test these effects on both behavioral and metabolic brain parameters, the animal model of intracerebroventricular (i.c.v.) streptozotocin (STZ) treatment was used. To quantify the experimental data more precisely, animals that were good performers were separated from poor performers by means of the holeboard test before i.c.v. administration of STZ. Good performers only were considered for the study. After a 1-week training period on the holeboard improvement was seen in all animals in learning, memory and cognition, and the improvement was maintained over the investigation period of 12 weeks in the control group. In this group, the energy pool in the cerebral parietotemporal cortex was found to be large and the energy turnover high. After triplicate i.c.v. STZ injection, working memory (WM), reference memory (RM), and passive avoidance (PA) behavior fell off and continued to deteriorate throughout the investigation period. Otherwise there were no significant differences in locomotor activity, excluding the possibility that activity per se might have contributed to the behavioral abnormalities. These were accompanied by a permanent deficit in cerebral energy metabolism. The ongoing deterioration in behavior and the maintained deficit in cerebral energy metabolism occurring after a triplicate i.c.v. STZ injection were significantly slowed down by EGb761. The deficits in learning, memory and cognition were partially compensated, and the disturbances in cerebral energy metabolism returned to almost completely normal values. These findings underscore the beneficial effect of EGb761 that had been reported in dementia disorders.

Hyperforin as a possible antidepressant component of hypericum extracts.

We demonstrate that the phloroglucinol derivative hyperforin is not only the major lipophilic chemical constituent of the medicinal plant Hypericum perforatum (St. John's wort) but also a potent uptake inhibitor of serotonin (5-HT), dopamine (DA), noradrenaline (NA), GABA and L-Glutamate with IC50 values of about 0.05-0.10 microg/ml (5-HT, NA, DA, GABA) and about 0.5 microg/ml (L-glutamate) in synaptosomal preparations. Furthermore, potencies of two different hypericum extracts in two conventional pharmacological paradigms useful for the detection of antidepressants (behavioral despair, learned helplessness), closely correlate with their hyperforin contents. In addition, most till now known neuropharmacological properties of the clinically used hypericum extracts can also be demonstrated with pure hyperforin. It appears, therefore, that this non-nitrogenous constituent is a possible major active principle responsible for the observed clinical efficacies of the extract as an antidepressant and that it could also be a starting point for drug discovery projects engaged in the search of psychoactive drugs with novel mode of action.

Antidepressant activity of hypericum perforatum and hyperforin: the neglected possibility.

Efforts leading to the identification of hyperforin as an antidepressive component of therapeutically used alcoholic hypericum extracts are described and discussed. Initially, the effects of this unique and major constituent of the herb were detected in peripheral organs using in vitro models and an extract was obtained by supercritical extraction of the herb by carbon dioxide. These extracts are highly enriched in hyperforin (38.8%) and are devoid of hypericines and numerous other components of alcoholic extracts. Studies with such an extract and with isolated hyperforin indicated that this acylphloroglucinol derivative can inhibit serotonin-induced responses and uptake of this neurotransmitter in peritoneal cells. Assuming that the effects of hyperforin were due to its actions on serotoninergic 5-HT3/5-HT4 receptors, further studies were conducted to investigate its effects on the CNS. These efforts revealed its antidepressant activity in the behavioral despair test and led to the working hypothesis that hyperforin and serotoninergic mechanisms are involved in the antidepressant activities of alcoholic hypericum extracts. The observations made during this study also indicate that hyperforin is the major, but not the only antidepressive component of alcoholic extracts.

Activity profiles of two hyperforin-containing hypericum extracts in behavioral models.

The behavioral activity profile of a therapeutically used alcoholic hypericum extract containing hyperforin (4.5%) in rodent models was compared with that of an experimental CO2 extract devoid of hypericines but highly enriched in hyperforin (38.8%). The antidepressant activities of 50, 150 and 300 mg/ kg/day of the alcoholic extract were similar to those of 5, 15 and 30 mg/kg/day respectively of the CO2 extract. The ethanol extract in the same dose range potentiated dopaminergic behavioral responses, whereas these effects were either absent or less pronounced in the CO2 extract treated groups. By contrast, serotoninergic effects of the CO2 extract were more pronounced than those of the alcoholic extract. These and various other observations made during the study confirm that although the antidepressant action of hypericum extracts depends on their hyperforin contents, their spectrums of central activity are due to other component(s). Our working hypothesis that hyperforin and serotoninergic mechanisms are involved in the therapeutically observed antidepressant activities of hypericum extracts is in agreement with these observations.

Oral bioavailability of hyperforin from hypericum extracts in rats and human volunteers.

Validated analytical methods suitable for determining hyperforin in plasma after administration of alcoholic Hypericum perforatum extracts containing hyperforin are described. After oral administration of 300 mg/kg Hypericum extract (WS 5572, containing 5% hyperforin) to rats maximum plasma levels of approximately 370 ng/ml (approx. 690 nM) were reached after 3 h, as quantified by a HPLC and UV detection method. Estimated half-life and clearance values were 6 h and 70 ml/min/kg respectively. Since therapeutic doses of Hypericum extracts are much lower than that used in rats, a more sensitive LC/MS/MS method was developed. The lower limit of quantification of this method was 1 ng/ml. Using this method, plasma levels of hyperforin could be followed for up to 24 h in healthy volunteers after administration of film coated tablets containing 300 mg hypericum extracts representing 14.8 mg hyperforin. The maximum plasma levels of approximately 150 ng/ml (approx. 280 nM) were reached 3.5 h after administration. Half-life and mean residence time were 9 and 12 h respectively. Hyperforin pharmacokinetics were linear up to 600 mg of the extract. Increasing the doses to 900 or 1200 mg of extract resulted in lower Cmax and AUC values than those expected from linear extrapolation of data from lower doses. Plasma concentration curves in volunteers fitted well in an open two-compartment model. In a repeated dose study, no accumulation of hyperforin in plasma was observed. Using the observed AUC values from the repeated dose study, the estimated steady state plasma concentrations of hyperforin after 3 x 300 mg/day of the extract, i.e., after normal therapeutic dose regimen, was approximately 100 ng/ml (approx. 180 nM).

[In vitro and in vivo studies on the cardioprotective action of oligomeric procyanidins in a Crataegus extract of leaves and blooms]. / In-vitro- und In-vivo-Untersuchungen zur kardioprotektiven Wirkung von oligomeren Procyanidinen in einem Crataegus-Extrakt aus Blättern mit Blüten.

Cardioprotective effects of a standardized extract from leaves with flowers of Crataegus (WS-1442; content of oligomeric procyandins [OPC]: 18.75%) have recently been demonstrated in an ischemia-reperfusion model in rats. Further studies were now conducted to clarify the mechanism of action and to identify active constituents involved in these effects of WS-1442. Exhausting partitioning between ethyl acetate/water and successive ultrafiltration of the aqueous layer led to the quantitative recovery of three fractions, which were tested for their in vitro radical scavenging (RS) and human neutrophil elastase (HNE) inhibitory activity. The lipophilic ethylacetate-soluble fraction A, enriched in flavone derivatives and constituting 14.9% of WS-1442, was as active as WS-1442 in inhibiting HNE. However, its RS activity was only about half that of the primary extract. Although 67.9% of WS-1442 was recovered in a water-soluble low molecular weight fraction B, this fraction displayed only weak RS and HNE inhibiting activity. In contrast, the RS and HNE inhibiting potencies of an essentially flavone-free and OPC-rich fraction C (21.3% of WS-1442) were significantly higher (inhibition of lipid peroxidation: IC50 0.3 microgram/ml; inhibition of HNE: IC50 0.84 microgram/ml) as those of WS-1442. The RS and HNE inhibitory activities of the extract and those of its fractions correlated well with their OPC-content but not with their concentration of flavonols. These results demonstrate that OPCs of Crataegus extracts possess stronger radical scavenging activities than flavone derivatives or other constituents. In addition, the oligomeric components are potent inhibitors of HNE. Oral administration of 20 mg/kg/d of the OPC-rich fraction C to rats afforded similar protection against ischemia-reperfusion induced pathologies as treatment with WS-1442 at a dose of 100 mg/kg/d. These observations indicate that radical scavenging and elastase inhibitory activities could indeed be involved in the observed cardioprotective effects of WS-1442, and demonstrate that OPCs are major orally active constituents of WS-1442. Thus, Crataegus extracts used therapeutically for cardiovascular diseases should be analyzed and standardized for their OPC-content.

Phospholipid breakdown and choline release under hypoxic conditions: inhibition by bilobalide, a constituent of Ginkgo biloba.

A marked increase of choline release from rat hippocampal slices was observed when the slices were superfused with oxygen-free buffer, indicating hypoxia-induced hydrolysis of choline-containing phospholipids. This increase of choline release was suppressed by bilobalide, an ingredient of Ginkgo biloba, but not by a mixture of ginkgolides. The EC50 value for bilobalide was 0.38 microM. In ex vivo experiments, bilobalide also inhibited hypoxia-induced choline release when given p.o. in doses of 2-20 mg/kg 1 h prior to slice preparation. The half-maximum effect was observed with 6 mg/kg bilobalide. A similar effect was noted after p.o. administration of 200 mg/kg EGb 761, a ginkgo extract containing approximately 3% of bilobalide. We conclude that ginkgo extracts can suppress hypoxia-induced membrane breakdown in the brain, and that bilobalide is the active constituent for this effect.