25 July, 2001

Ginkgo biloba leave extracts have been used as remedies for a variety of physiological symptoms for 6 millennia. Constituent compounds are being developed for novel treatment, ginkgo-flavonol glycosides for neurodegenerative damage caused by free radicals, ginkgolide B (terpene trilactone) for asthma, and bilobalide (sesquiterpene) for ischemia-induced damage. Ginkgo biloba compounds should be valuable therapeutics in increasingly elder population as well as additives to enhance quality of healthcare products for daily health maintenance.

Chinese tree Ginkgo biloba is referred to as "maiden hair tree", and extracts from its dry leaves have been used as remedies for a variety of physiological symptoms for 6 millennia. These extracts were commercialized initially such as Tanakan, Tebonin, Rokin and Kaveri (LI 3170) but a well-defined extract now designated "EGb 761" has been developed as a standardized extract of dried leaves of Ginkgo biloba that contains 24% ginkgo-flavonol glycosides and 6% terpene lactones. Ginkgolic acid that causes skin irritation should be less than 10 ppm, preferably less than 3 ppm in the standard extract. Ginkgo-flavonol glycosides are a mixture of biflavones and flavonol glycosides, while ginkgo terpenes are trilactones and a mixture of ginkgolides and bilobalide that constitutes about 0.1% in dry Ginkgo biloba leaves. Ginkgolides are further separated into several components A, B, C, J and M, respectively having differential levels of pharmacological activities.

EGb761 is used mainly in the treatment of peripheral and cerebral circulation disorders, and also as a remedy against asthma, coughs and bladder inflammation. From its broad spectrum of pharmacological activities, 4 primary concepts of action have been proposed to explain the therapeutic benefits of EGb761, namely, vasoregulatory, cognition-enhancing, stress-alleviating, and gene-regulatory. These actions are believed to be realized through the principal active ingredients, flavonoids (ginkgo-flavonol glycosides) and terpenoids (ginkgolides and bilobalide), acting simultaneously in concert, combination and synergy, in what is so-called ﾒpolyvalent actionﾓ. For instance, EGb761 is used for numerous pathological requirements (hemodynamic, metabolic) which occur in cerebral, retinal, cardiac or peripheral ischemia. In fact, one of the major therapeutic applications is "cerebral insufficiency" that is supposed to comprise 12 different symptons in the elderly (Curtis-Prior P et al, 1999: uid=10411212) , arising from cerebral disorders, neurosensory problems and peripheral circulatory disturbances. Well-controlled clinical studies, in Europe and in USA, have revealed that EGb 761 is an effective therapy for early cognitive decline in senile dementias (including Alzheimer's disease) and in improvement for memory loss, attention, and vigilance. In particular, recent studies have shown the efficacy and safety of EGb 761 in patients with mild to severe Alzheimer's disease and multi-infarct dementia, and progression of symptoms was delayed by approximately 6 months (Clostre F, 1999: uid=10481350).

Some evidence obtained with animal models indicates that EGb 761 can interact with nearly all reactive oxygen species as a free radical-scavenger and inhibits lipid peroxidation (Clostre F, 1999: uid=10481350). Moreover, EGb 761 regulates ionic balance in damaged cells and exerts a specific and potent antagonist activity to platelet-activating factor receptor. Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and its receptor are known to play important roles in modulating neuronal plasticity and inflammatory responses, particularly during neuronal injury. PAF receptors are widespread in different brain regions, on the cell surface as well as in intracellular membrane compartments. The direct effects of EGb 761 against necrosis and apoptosis of neurons and on the improvement in neural plasticity go well along with its anti-PAF receptor activity.

The reference should be made here to bioavailability of some constituents of EGb 761. The bioavailability of ginkgolides A, B and bilobalide was studied in rats after single oral administration of 30, 55 and 100 mg/kg Ginkgo extract EGb 761. The plasma levels of the terpene lactones were measured by a specific GC/MS method. The pharmacokinetics of the mentioned substances were found to be dose-linear. For the lowest dose, maximum concentrations were 68, 40 and 159 ng/ml and half-lives 1.7, 2.0 and 2.2 h for ginkgolide A, B and bilobalide, respectively. Clearance values ranged from 24.2 to 37.6 ml/min/kg (Biber A et al, 1999: uid=10193211).

What are then the active components in EGb 761?

Flavonoids (ginkgo-flavonol glycosides, a mixture of biflavones and flavonol glycosides)
An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders, and the hippocampus is a brain area particularly susceptible to neurodegenerative damage. In a test system where free radical accumulation and decrease in cell survival were induced in rat primary mixed hippocampal cell cultures by a NO generator, sodium nitroprusside (100 uM) or 3-morpholinosydnonimine, EGb 761 (10-100 uM/ml) and its flavonoid fraction CP 205 (25 uM/ml) blocked these induced events (Bastianetto S et al, 2000: uid=10820186), showing that the flavonoid fraction CP 205 of EGb 761 has efficacy against toxicity induced by the NO generators. In addition to potential anti-neuronal damage, the flavonoid fraction is already used in healthcare supplement to resist lipid oxidation, prevent skin consenescence and promote blood recycle, as cosmetic ingredient and a hair growth agent.

Ginkgo terpenoids (terpene trilactones) constitute 6% of EGb 761 and are a mixture of ginkgolides and bilobalide. By what is called supercritical fluids crystallization technology (Jian Zhang, 2001: personal communication), ginkgo terpenes may be increased from 6% to above 50%, by re-extraction to be almost pure, and at the same time ginkgolides and bilobalide can be separated.

Ginkgolides:
As mentioned above, ginkgolides are highly active antagonists to PAF receptor, and ginkgolide B is most active and ginkgolides A, C, M follow in that order. Numerous studies have shown that PAF induces endotoxin-induced leukocyte adherence, and is important in the pathogenesis of asthma. Ginkgolides were found to reduce the pathogenesis of asthma and the order of anti-PAF receptor activity among ginkgolides is similar to that of the anti-asthma effect. In addition, as PAF is associated with ischemia/reperfusion injury (I/R) after lung transplantation, ginkgolide B (BN 52021) helps optimize pulmonary function, with the reduction of PAF levels, in the early post-ischemic period (Wittwer T et al, 2001: uid=11257563). Further, ginkgolides A and B, at 5 ug/ml, showed cardioprotective and anti-oxidant effects in control perfusion, low-flow ischemia of rat hearts and recovered post-ischemic functions (Liebgott T et al, 2000: uid=11099163). This finding indicates that PAF is also responsible for myocardial ischemia.

Bilobalide:
Bilobalide is a sesquiterpene. Bilobalide derivatives are used for treating neuropathy, edema, encephalopathy, spinal cord disease and senile dementia. What is the mechanism of effect of these compounds on neuronal diseases? Some of the recent findings are summarized here.

Ischemia is a common feature of most vascular diseases. In liver and in brain, the decrease in respiratory activity induced by ischemia could be prevented by bilobalide, and this effect appears to arise from the protection of mitochondrial complex I and probably complex III activities (Janssens D et al, 2000: uid=10895958). By protecting complex I and III activities, bilobalide allows mitochondria to maintain respiratory activity under ischemic conditions as long as some oxygen is present and keeps the energy pool, namely cellular ATP content of tissues, thus delaying the onset of ischemia-induced damage. This mechanism provides a possible explanation for the anti-ischemic properties of bilobalide and of Ginkgo biloba extract in therapeutic interventions (Janssens D et al, 1999: uid=10403524).

Bilobalide protect cultured neurons from hypoxia- and glutamate-induced damage. Apoptosis was induced in cultured chick embryonic neurons as well as in mixed cultures of neurons and astrocytes from neonatal rat hippocampus by staurosporine (200 nM). The treatment with staurosporine for 24 h led from control 12% to 74% apoptotic chick neurons. Bilobalide (10 uM) decreased apoptotic damage induced by staurosporine treatment nearly to the control level, and Bilobalide is the most potent among ginkgo terpenoids (Ahlemeyer B et al, 1999: uid=10079019). An additional interesting finding is the anti-convulsant effect of bilobalide. Bilobalide inhibited the NMDA-induced efflux of choline with an IC50 value of 2.3 uM. The convulsions which were observed in the NMDA-treated rats were almost totally suppressed by bilobalide (Weichel O et al, 1999: uid=10619176). The release of choline is a sensitive marker for NMDA-induced phospholipase A2 activation and phospholipid breakdown. The anticonvulsant effect of bilobalide may also be due to elevation of GABA levels, possibly through potentiation of glutamic acid decarboxylase activity and enhancement of the protein amount of 67 kDa glutamic acid decarboxylase by bilobalide (Sasaki K et al, 1999: uid=10078989). Thus bilobalide inhibits 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. Finally, it should be added that a stable compound, 4-hydroxy-4-tert-butyl-2,3,5,6-tetrahydrothiopyran-1-oxide (NV-31), was synthesized to mimic the neuroprotective and antioxidative profile of bilobalide and promote neuronal survival in neuronal cultures at 10-100 nM, or in mice at 10-20 mg/kg (Ahlemeyer B et al, 2001: uid=11164801)

From brief summary of the recent findings on therapeutic efficacies of EGb 761 and its cmonstutients, flavonoids and terpenoids, it is clear that the mechanistic study has just begun and much more remains to be done to define specific effect of each compound at the molecular level. Because of neuronal effects and anti-asthma activity, Ginkgo biloba compounds should be valuable therapeutics in increasingly elder population as well as additives to enhance quality of healthcare products for daily health maintenance.