Preclinical Studies
One overview of the prolific pharmacological research on Ginkgo extract postulates that its therapeutic efficacy as due to the dynamic and "self-propagating" interactions of 3 basic components of the body: vasculature, blood and other bodily fluids, and the metabolic and neurohormonal regulatory mechanisms of injured tissue. Each component is integral to the pathology of ischemic vascular diseases:. In the case of cerebral disease, the latter would refer to brain tissue. It is theorized that any disturbance in either one of these components would trigger the process of the interplay between them and result in a multifactorial action appropriate to the multifactorial nature of ischemic vascular disease (Chatterjee, 1985).
Generally, the flavonoids in Ginkgo leaf act as antioxidants by scavenging free radicals in the body which result from oxidative processes, while the ginkgolides antagonize activities of inflammation and blood clotting associated with platelet-activating factor (PAF) (Chang and Chang, 1997). Most in vitro pharmacological studies on Ginkgo leaf have been performed using a specific extract known as EGb 761 (DeFeudis, 1991).
Cardiovascular and Circulatory Functions
Ginkgo extract influences many components of the cardiovascular system, including arteries, veins, capillaries, blood components (e.g. erythrocytes, platelets), arterial blood flow, capillary perfusion, and venous return. Multiple actions are due to the heterogeneous nature of Ginkgo extracts. Therefore, data interpretation is more difficult than in the case of a single chemical entity (DeFeudis, 1991).
In vitro studies by DeFeudis (1991) on isolated arterial preparations (rabbit aorta) indicated that Ginkgo extract EGb 761 caused a reproducible concentration-dependent contraction of rabbit aortic strips involving catecholamine receptors. These studies further indicated that low concentrations of EGb 761 might influence catecholaminergic systems by an indirect mechanism; an action resembling that of tyramine more than norepinephrine. In addition, EGb 761 can relax rabbit aorta, probably via potentiation of the effects of endothelium-derived relaxing factor(s) (EDRF), which is spontaneously produced. Collectively, the experiments on the effect of EGb 761 on isolated veins, platelets, and arterial preparations support the theory of "trivasoregulation," and that cardiovascular diseases that have multifactorial origins are best treated with polyvalent agents. DeFeudis commented that with its actions of enzyme-inhibition and free radical scavenging (antioxidant), Ginkgo extract may therefore be useful.
Cheung et al. (1999) examined the effects of Ginkgo on nitric oxide (NO) in human endothelial cells (ECV3C4, American Type Culture Collection) that express both calcium-independent and calcium-dependent forms of nitric oxide synthase (NOS) activities: inducible nitric oxide synthase (iNOS) and constitutive nitric oxide synthase (cNOS). A standardized Ginkgo extract (Shanghai Luyuan Industry Company Ltd., 50 µg/mL) caused a significant reduction in the production of NO (30%), which was attended by a reduction in iNOS expression. Tests showed that the effect was not calcium-dependent, that calcium-dependent activity of NOS was essentially unchanged, and that the effect of the Ginkgo extract on iNOS activity was not attributable to a direct action on NOS enzyme. The research showed that the Ginkgo extract did not affect endothelial cell DNA synthesis, although it did cause levels of iNOS protein to decrease by 27% and by 43% at 100 µg/mL. As the authors explained, the significance of these results is found in the fact that NO (cNOS-derived) at normal levels in the body causes blood vessels to relax, inhibits the oxidation of lipoproteins, and decreases the aggregation of platelets and the adhesion of monocytes to vessel walls. With excess production, NO and more so iNOS, is believed to be toxic to vessel walls through oxidative mechanisms, and higher levels of NO have been found in atherosclerosis and in Alzheimer’s disease. Researchers in the field of cardiovascular drug development have made numerous attempts to find iNOS inhibitors that will leave cNOS intact so as not to interfere with its vasodilating role in helping to maintain vessel tone. While the active constituent(s) involved have yet to be identified, the research by Cheung and coworkers indicates that Ginkgo extract holds just such a substance.
In vivo studies have generally mirrored or confirmed the results obtained from in vitro studies. Ginkgo extract EGb 761 exhibits in vitro antioxidant activities (which may be relevant to its relationship with EDRF), and ginkgolides have been shown to exhibit PAF-antagonist activity, potentially supporting the use of Ginkgo extract in treating hypertensive states. EGb 761 has shown anti-ischemic activity in the CNS (central nervous system), and it is thought that ginkgolides, flavonoids, and bilobalide may all be involved. EGb 761 also exhibits effects on central neurotransmitter systems and behavior. Due to the possible involvement of several plant constituents, it is important for therapeutic purposes that the total extract is used (DeFeudis, 1991).
Thrombosis, hemostasis, and embolism
Ginkgolides A, B, and C were shown to be potent inhibitors of platelet activating factor (PAF) by Braquet et al. (1985). PAF antagonism is the most notable single activity of Ginkgo, attributed solely to the ginkgolides. In fact, ginkgolides, as natural PAF antagonists, opened up a completely new area of understanding for the pharmacological basis of several other botanicals, including kadsurenone and futoxine from Piper futokadsura, swietemahonin A and E from Swietenia mahogani, and L-652,496 from Tussilago farfara (Braquet and Hosford, 1991).
Neurological, Psychological,
and Behavioral Functions
Neurodegenerative disorders
Retinopathies
Studies by Baudouin et al. (1999) have shown that Ginkgo can significantly ameliorate free radical-induced vitreoretinopathy (xanthine-xanthine oxidase-induced) in an experimental model of intraocular inflammation in rabbits. Ginkgo significantly decreased the incidence of retinal detachment and the degree of vitreoretinal proliferation when administered 7 days prior to vitreoretinpathy or 24 hours after (100 mg/kg/day p.o.). The benefits of Ginkgo were most evident in the complete absence of neovascularization versus an incidence in the control group of 38%; an incidence of retinal detachment/folding of 50% versus 12.5% to 25% in the Ginkgo group; and of epiretinal membrane development in 12.5% to 25% of the Ginkgo-treated rabbits versus 75% of the control group (Baudouin et al., 1999).
Grosche et al. (1995) studied the effect of Ginkgo on the expression of the proto-oncogene Bcl-2 protein in Müller (glial) cells of male albino rats. This protein shows increased expression in hereditary rod photoreceptor cell degeneration, ganglion cell degeneration (experimentally induced), and in retinal neuronal damage. As the rats aged, the Müller cells showed a correspondingly greater expression of Bcl-2 protein, which the authors found also increased in light-damaged rat retinas. Two groups of retinal light-damaged rats aged 16 months (hereditary retinal dystropy-free), were assigned to a control group or a group administered Ginkgo (EGb 761, 40 mg/kg in drinking water, 30 mL/day) for 8 months Approximately 50% of the photoreceptors of their eyes were absent due to light damage. At 24 months, when the treatment period ended, the number of photoreceptors lost, reduced glial-specific enzyme GS expression, and the transformed morphology of Müller cells were the same in both groups. However, Ginkgo-treated rats showed no expression of the Bcl-2 protein in Müller cells whereas it was significantly expressed in Müller cells of 16- and 24-month-old rats, although barely at all in young control rats. The Bcl-2 protein is known to act in preventing apoptosis and necrosis mediated by free radicals. The authors proposed that Ginkgo, acting as a free radical scavenger and having shown protective effects against free radical damage to the retina, may act in such a way that it takes out the "trigger" that allows Bcl-2 expression.
De Kozak et al. (1994) examined the effect of Ginkgo extract (EGb 761) and the platelet activating factor (PAF) antagonist ginkgolide BN 50730 (Institut Henri Beaufour, Courtaboeuf, France) in experimental autoimmune uveoretinitis in pigmented and albino rats as a model of severe acute inflammation largely affecting the choroid and retina. Daily administration of EGb 761 (100 mg/kg/day p.o.) or BN 50730 (50 mg/kg/day p.o.) one week prior to induced uveoretinitis and until death resulted in little inhibition of whole ocular inflammation. Yet, both EGb 761 and ginkgolide BN 50730 significantly reduced tissue damage to the outer layers of the retina by 50% (p<0.0004 and p<0.001, respectively); albeit, only in the albino rats.
Remé et al. (1992) suggested that PAF was involved in light- (400-450 lux white fluorescent light) and lithium-induced (2.6 g/kg rat chow) rod outer segment (ROS) lesion development of rats when administration of ginkgolide BN 50730 (25 mg/kg p.o.) beforehand caused significantly fewer ROS lesions. Doly et al. (1992) found results suggesting the involvement of PAF in vincristine-induced retinopathy when, compared to rats receiving vincristine alone, retinal damage was significantly reduced after pretreatment of the animals with ginkgolide BN 50730 (10 mg/kg p.o. daily X 10).
Droy-Lefaix et al. (1992) studied the ocular-protective effect of Ginkgo extract (EGb 761) in rats with experimental chloroquine-induced retinopathy. Given that the retina is highly sensitive to peroxidation and that retinal function is greatly affected by PAF, it lends well to measuring effects of oxygenated free radicals and radical scavengers. Droy-Lafaix et al. used these factors to measure the effect of Ginkgo in rats exposed to chloroquine, a widely used antimalarial drug with various impairing effects on the retina; probably owing to localized, immunologically mediated inflammatory reactions. Electroretinogram (ERG) measurements showed deviations from normal in nerve connections and metabolism of the retina in all the chronic chloroquine-treatment rats (75 mg/kg p.o daily for 20 days). Rats pretreated with a high dosage of Ginkgo extract (100 mg/kg p.o. daily) for 10 days prior to chronic chloroquine treatment showed practically no deviation from normal ERG readings. Tests also showed that there was no alteration in ERG measurements in rats treated with the Ginkgo extract alone compared to the controls. The authors found it remarkable that the preventive use of EGb 761 could reduce retinal damage from chloroquine and concluded that it may be useful in preventing chloroquine-induced retinopathy and probably damage to the retina caused by other kinds of agents (Droy-Lefaix et al., 1992).
Baudouin et al. (1992) assessed the possible ocular-protective effect of Ginkgo extract (EGb 761) in rabbits. A model of periretinal proliferation was used to simulate retinal detachment by means of an immunologically-mediated inflammatory reaction, which in turn stimulated intra-ocular growth of vitreous stands. Their test followed observations of a possible immunologically-mediated activation of cell proliferation which they observed in specimens from human cases of periretinal proliferative disorders, which are the main cause of retinal-origin blindness. With half the rabbits serving as an untreated control group, the other half received a high dosage of Ginkgo extract (100 mg/kg p.o. 3 X/day for 4-5 weeks), at the same time as induced periretinal proliferation. Both groups showed intraocular cellular proliferation; however, in the Ginkgo-treated group development of cellular proliferation was delayed, less dense, and localized tractional retinal folding was found in only one eye versus all eyes in the untreated group. Furthermore, resolution of the proliferation was more rapid in the Ginkgo group and by the third week, vitreous membranes showed a progressive decrease. By the fourth and fifth weeks, the vitreous membranes had completely disappeared, accompanied by complete retinal reapplication, including the single incidence of tractional retinal detachment. These results contrasted sharply with those found in the control group, in which retinal detachment persisted along with preretinal membranes. The researchers also found evidence of intravitreal macrophages and lymphocytes in the rabbits, indicating an immune-mediated inflammatory response involved in the pathological state induced, and hypothesized in the etiology of proliferative retinal diseases in humans (Baudouin et al., 1992).
Szabo et al. (1990) demonstrated that EGb 761 acts as a protective agent against free radicals in the retina. Bazan et al. (1987) suggested the involvement of PAF in the inflammatory activity found in an in vivo animal model of corneal injury induced by alkali burn. Drops of a PAF-antagonist ginkgolide (1% suspension of ginkgolide B, then codified BN 52021) to the injured eye specifically inhibited the production of hydroxyderivates and prostaglandins released during the inflammatory response.
Psychological and behavioral functions
Cognitive functions - Memory
In a preliminary data-seeking study of Ginkgo biloba (form uncharacterized) for possible memory-enhancing effects, Gajewski and Hensch (1999) administered two female and 3 male experimentally naive mice the leaves dissolved in water (12 mg/200 mL) in place of normal drinking water. Four phases using a different maze each time were employed in a total of 16 trials. Between each phase the mice received plain water for 4 days. Compared to plain water, the results showed that when the mice received Ginkgo there was a decrease in wrong turns and a relative decrease in errors. In the first phase, the wrong turn incidence decreased 8-60%. Among the 80% of mice showing improved performance scores, an improvement of 5-26% was evident when Ginkgo was reintroduced as a water replacement, raising the possibility of an extended effect beyond the 4-day plain water period. The effect was seen again in phase 4 of the study. The results showed that when mice received Ginkgo they made less errors, indicating a memory-enhancing effect.
Winter (1998) examined the cognitive behavioral and longevity effects of orally self-administered Ginkgo extract (EGb 761, 50 mg/kg) in male rats, using a delayed, nonmatching-to-position task (DNMTP task) and continuous learning tasks. At this dose, there was a tendency towards fewer errors and fewer sessions before attaining criterion performance compared to the controls. A second set of experiments (DNMTP task) in male rats aged 20 months allowed varying doses of the extract immediately prior to one set of tests and 30 min. before another, with control sessions before each dose. Errors were significantly reduced, indicating a dose-dependent effect of Ginkgo (100 mg/kg, p<0.002; 200 mg/kg, p<0.003). At a specific dose of 200 mg/kg, rats aged 26 months showed a significant decrease (p<0.04) in proactive (nonmnemonic) and retroactive (mnemonic) errors in the radial maze compared to controls. An unexpected finding was that the Ginkgo-treated rats lived significantly longer (p<0.05), yet weighed the same as control mice at death.
Anxiety and Stress Response
Porsolt et al. (1990) examined the effects of Ginkgo extract in rats (EGb 761, 25 and 50 mg/kg p.o. twice daily X 5) using models of learned helplessness, behavioral despair (forced swimming test), shock-suppressed exploration, emotional hypophagia ("food consumption in a novel situation"), spontaneous exploration, the Vogel conflict test ("shock suppressed licking"), and memory using the passive avoidance test. Ginkgo extract was effective as a preventive in the test for unavoidable shock as a model of learned helplessness, significantly reducing avoidance deficits compared to the untreated controls (50 mg/kg, p<0.05), and more effectively than diazepam (4 mg/kg p.o. daily). Administered at the same time as the test, Ginkgo-treated mice also showed reduced avoidance deficits, although not by a statistically significant amount compared to the control. Also, mice administered Ginkgo (100 mg/kg p.o.) consumed significantly more food in the hypophagia test when the dosing was conducted prior to the test, but not after, at which diazepam produced an equally significant effect (p<0.01). In all the other tests, Ginkgo was not significantly effective given before or after the stress-inducing event. Porsolt and colleagues concluded that Ginkgo extract appeared to ameliorate the effects of unavoidable stress in a way that could not be easily ascribed to either classical antidepressant or anxiolytic activity.
Receptor and neurotransmitter mediated functions
Huguet et al. (1994) investigated the effects of Ginkgo extract (EGb 761, 50 mg/kg i.p. daily X 21) compared to placebo on the age-related decrease of serotonin receptor binding in the cerebral cortex of aged (24 months old) and young male rats (4 months old). In young rats, chronic Ginkgo treatment produced no alteration in binding of 5-hydroxytryptamine (5-HT1A) to 5-HT1A receptors in the cerebral cortex membrane. In aged rats that had displayed a significant 22% reduction in the number of maximal binding sites as a result of age, binding density increased 33%. According to the authors, in senescent rats Ginkgo may have a restorative effect on 5-HT1A receptor binding in the cerebral cortex which had decreased as a result of aging. This age-related decrease has also been shown in the cerebral cortex of humans. The evidence suggested that the increase in 5-HT1A receptors was not the result of modified synaptic serotonergic activity. A more likely explanation offered by the authors was a stimulation receptor synthesis by Ginkgo.
Ramassamy et al. (1992), using considerably higher doses of EGb 761 than Huguet et al. (1994), examined the neuromodulating effects of Ginkgo on cerebral cortex synaptosomes of mice. A significant increase in [3H]5-HT uptake (+25%, p<0.01) was evident in cortex synaptosomes from mice administered EGb 761 semi-chronically in a very high dosage (1000 mg/kg p.o. twice daily X 4). In vitro studies with synaptosomes showed that in the presence of clomipramine, a 5-HT uptake inhibitor, there was no increase in [3H]5-HT from EGb 761. Attempting to identify the active fractions, the researchers determined that whereas a quercetin- and a flavonoid-free form of EGb 761 were inactive as [3H]5-HT uptake-increasing constituents, a terpene-free form of EGb 761 containing mostly flavonoids, was active. The authors also found that [3H]dopamine uptake in synaptosomes from striatum of mice showed no increase from EGb 761 in vitro.
Immune Functions; Inflammation and Disease
Cancer
Antiproliferative activity
Ten phenolics from the sarcotestas (fleshy part of the seeds) of Ginkgo have shown inhibitory activity against phosphotidylinositol-specific phospholipase C gamma-1 (PI-PLC gamma-1) in vitro (the most potent being phenolic acid C17:1 or 6-[10’ (Z)-heptadecenyl]salicylic acid with an IC50 value of 0.83 µg/mL) (Lee et al., 1998). PI-PLC gamma-1 is the critical enzyme acting in the signal transduction of hormones, growth factors and neurotransmitters. The enzyme became a target for new antitumor agents since the discovery of increased PI-PLC gamma-1 activity in human cancers (Noh et al., 1994), including human glial tumors (Haas et al., 1991). This enzyme also aids in the progression and proliferation of human cancer (Noh et al., 1994; Hill et al., 1994).
All 10 phenolic compounds also showed in vitro growth-inhibitory activity against human tumor cell lines, whether ovary cancer cells (SKOV-3), cancer cells of the lung (A-549), breast (MCF-7), bladder (HT-1197), or colon (HCT-15). The activity of the most active phenolic acid against normal colon cells (CCD-18-Co) was less cytotoxic than t in the colon cancer cell line (Lee et al., 1998).
Itakawa et al. (1987) reported remarkable antitumor activity from a methanolic extract of the sarcotestas of Ginkgo against sarcoma 180 ascites in mice. Subsequent research identified cardenol and bilobol compounds as the most active constituents (each 40 mg/kg i.p.), inhibiting sarcoma 180 ascites by as much as 99.6% and 100%, respectively, following tumor cell implantation.
Metabolic and Nutritional Functions
Antioxidant activity
Çeliköz et al., (1998) reported that in rats the area of necrosis of surgical skin flaps inhibited by pre-treatments with Ginkgo extract (EGb 761, 100 mg/kg i.p.) or deferoxamine (150 mg/kg i.p.) was equally significant compared to the control group. Each was superior to vitamin E (20 mg/kg i.m.) and vitamin C (340 mg/kg i.p.) pretreatments as compared to the control. Rapin et al. (1998) demonstrated that male rats administered Ginkgo extract (EGb 761, 50 mg/kg p.o. daily X 8) showed significantly increased cell viability and significantly decreased numbers of peroxyl radical-induced and spontaneously occurring apoptoses in hippocampal nerve cells ex vivo. They found similar results from the constituent ginkgolide B (2 mg/kg/day p.o. daily X 8) at a dosage/concentration/rat oral bioavailability representative of the percentage found in Ginkgo extract. However, there was no activity from the constituent bilobalide in the same representative dosage. These results verified previous in vitro tests by Rapin et al. (1998); cell viability reached higher levels in cells treated with ginkgolide B at 0.4 µg/mL than untreated control cells.
Çeliköz et al. (1997) compared the antioxidant deferoxamine and G. biloba extract (EGb 761) in a rat groin island skin flap model to show rates of free radical scavenging activity and inhibition of skin flap necrosis. Rats were assigned to 3 groups: a Ginkgo group (TebokanTM, 5 mg/kg p.o. twice daily X 11); a deferoxamine group (DesferalTM, 20 mg/kg/day i.p. for 11 days); and a nontreatment control group. The rats began treatment one day before flap harvesting. At the tenth day, the area of skin necrosis in the deferoxamine (445.0±55.9 mm2) and the Ginkgo groups (284.4±39.9 mm2) was significantly less (p<0.001) than that of the control group (755±75.8 mm2). The Ginkgo group showed significantly less skin necrosis than the deferoxamine group (p<0.05). Antioxidant activity in the skin flap biopsies from the 3 groups as measured with estimates of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px). Significantly higher levels of activity were found in the two active treatment groups compared to the control group (p<0.001), while the antioxidant activity of the Ginkgo group was significantly less than that of the deferoxamine group (p±0.05). Electron microscopic examination of the tissue structures of the 3 groups revealed a normal appearance in the two active treatment groups compared to the control group; however, ultrastructure of the dermis in the deferoxamine group was deteriorated, showing loss of the dermo-epidermal border and preserved collagen showed a patchy distribution. The Ginkgo group faired best, showing the dermo-epidermal junction, epidermis and dermis were little different than normal, and with normal distribution of preserved collagen. The researchers noted that injury induced by ischemia-reperfusion causes skin flap failure and organ damage in a number of clinical interventions (e.g., stroke, organ transplantation, heart attack, hemorrhagic shock, and sepsis), and that reactive oxygen species are implicated in this tissue injury. Çeliköz and colleagues concluded that Ginkgo extract is a preferable agent for administration post- and pre-operatively for elective operations involving flap transfer operations.
Barth et al. (1991) demonstrated that Ginkgo extract (Schwabe, Tebonin®) could inhibit the cyclosporine A-induced increase in malondialdehyde (MDA) in human liver cells in vitro. Total inhibition of CsA-induced lipid peroxidation occurred at a concentration of Ginkgo extract of 50 µg/mL, while CsA-induced MDA was significantly and dose-dependently inhibited by Ginkgo, starting even at a low concentration of the extract (15 µg/mL). The addition of free iron (FeCl3) to CsA resulted in a diminished ability of the Ginkgo extract to inhibit the free radical production induced, though not completely.