Free radical scavenging effect

The important role of oxidative stress and ROS in aging and AD has been previously discussed in section 1.1.8. One of the highly proposed mechanisms of action of EGb 761^® is its free radical scavenging effect. Several researchers have linked the neuroprotective effect of EGb 761^® to its anti-oxidative effect.

Previous studies have highlighted the potential of EGb 761^® and its components as scavengers of free radicals such as OH^., O2

- and NO in both in vitro acellular and cellular studies, as well as in vivo studies (Defeudis 1998).

For instance, Smith and Luo have demonstrated that a pre-treatment with EGb 761^® significantly lowered ROS levels both in a cellular model stably expressing an AD associated double mutation and in C.elegans AD model (Smith and Luo 2003). EGb 761^® and α-tocopherol exhibited similar inhibitory effects in vitro on lipid peroxidation in rat brain synaptosomes and human lymphocytes (Sram et al. 1993).

In post-mortem tissue from AD patients’ it was shown that EGb 761^® prevented lipid peroxidation induced by the pro-oxidant system H2O2/Fe²⁺

(Ramassamy et al. 1999). Moreover, intracellular and mitochondrial ROS decreased when hippocampal neurons were treated for 24 hours with EGb 761^® (Bastianetto et al. 2000a).

Not only in vitro studies linked the anti-oxidative effects of EGb 761^® to its neuroprotective effects, but a number of in vivo studies supported this fact.

Treating rats with 50 or 100 mg/kg body weight for 12 weeks resulted in decrease of lipid peroxidation in the cerebral cortex, and only the higher

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concentration [100 mg/kg body weight] was able to decrease lipid peroxidation in the hippocampus (Sram et al. 1993).

Another in vivo study by Holgado et al in 1995 showed very interesting results after treating 4 and 33 months old rats with 100 mg/kg body weight EGb 761^® daily for 3 months. In the younger rats EGb 761^® was able to decrease the rates of O2

- with no effect on superoxide dismutase [SOD], catalase and glutathione peroxidase. However in the older rats EGb 761^® treatment was able to decrease the rate of O2

generation as well as increase the scavenging activities of the 3 mentioned enzymes. Other results were also able to confirm the ability of EGb 761^® to increase the activities of anti-oxidant enzymes, for example glutathione in mouse liver (Sasaki et al. 2002) as well as SOD and catalase (Bridi et al. 2001) in rats.

A more recent study showed that both Ginkgo biloba extract and vitamin E reduced the oxidative stress resulting from senile plaques in vivo as monitored with intracranial imaging (Garcia-Alloza et al. 2006).

With all the above mentioned evidence it seems that the anti-oxidant property of EGb 761^® plays a significant role in its therapeutic effects, the question that arises is clearly which of EGb 761^® constituents is responsible for these effects.

Many researchers have addressed this question and the majority links the radical scavenging activity of EGb 761^® to the flavonoid fraction. This is partly due to their chemical structure.

In acellular models and test-tube reactions EGb 761^® scavenges hydroxyl radicals and superoxide radicals efficiently, while the terpenoid fractions fails (Pietri et al. 1997).

Ramassamy et al were able to show that the flavonoid fraction of EGb 761^® has higher ability to prevent oxidative-induced damages than the terpene fraction (Ramassamy et al. 1993). In another study, the anti-lipoperoxidative

effect of the terpenoids and the flavonoids were determined in isolated hepatocytes and the terpenoid fraction showed no effect while the flavoniods were effective (Joyeux et al. 1995). Moreover, the flavonoid fraction [CP 205], but not the terpenoid constituents [CP 160 and BN 52021], was also able to decrease the Aß-induced ROS production in hippocampal cells (Bastianetto et al. 2000a).

Although many studies assert the anti-oxidative effect of EGb 761^® to the flavonoids, it seems that the terpenoid fraction also play a role here.

In an acellular model it was shown that BB, GB, GC and GJ but not GA have radical scavenging activities (Scholtyssek et al. 1997) when using DMSO as a solvent instead of water.

In vitro, Zhou et al were also able to show that BB can protect neurons against oxidative stress (Zhou and Zhu 2000). Some studies showed that the flavonoid fraction alone was not able to exert the same effect as the whole extract. For example, the H2O2-induced toxicity in hippocampal cells was attenuated by a co-treatment with EGb 761^®; however the flavonoid fraction CP 205 was not effective (Bastianetto et al. 2000a).

Therefore, the primary assumption that the anti-oxidative properties of EGb 761^® are only due to the presence of its flavonoid fraction is imprecise.

Concluding, it seems that EGb 761^® has indeed free radical scavenging and anti-oxidant properties, regardless of the exact constituents that are implicated. It appears that the flavonoid fraction exert a direct radical scavenging activity which could be partly explained by its chemical structure, and that the terpenoids seem to somehow decrease the generation of free radicals.

Although EGb 761^® is known to act as a vasodilator, and can influence the endothelial nitric oxide synthase induced NO production (Li et al. 2001), it is

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synthase could be controlled by EGb 761^®. The deleterious effects of excessive NO have also been previously discussed [section 1.1.8] and fortunately EGb 761^® shows not only anti-oxidative properties but also can protect cells from nitrosative stress. In 1994, EGb 761^® was described as an NO scavenger in acellular system (Marcocci et al. 1994). Later on other in vitro studies showed the ability of EGb 761^® to protect cells against NO damage in PC-12 cells and hippocampal neurons (Eckert et al.

2005;Bastianetto et al. 2000b).

As for the EGb 761^® constituents, in PC 12 cells BB was able to inhibit the NO induced toxicity, on the other hand in hippocampal neurons the flavonoid fraction was most effective and the terpenoid and GB were not effective (Song et al. 2000;Bastianetto et al. 2000b).

To sum up EGb 761^® and its components are capable of scavenging reactive oxygen species and reactive nitrogen species, preventing their damage on cells. Which constituent/ constituents play a part here is highly debatable. In order to reach such conclusions all components should be evaluated under the same conditions. Meaning the method used, cell model, experimental procedures and all influencing factors should be identical.