The standard extract of Ginkgo biloba leaves (EGb761) is being clinically used in Europe for the treatment of impaired cerebral function in primary degenerative disorders such as Alzheimer disease (AD) and vascular dementia. The abnormal production and aggregation of amyloid β peptide (Aβ) and the deposition of fibrils in the brain is considered as key steps in the onset of AD. For this reason, the inhibition of Aβ aggregation and the destabilization of preformed fibrils have been identified as effective approaches for the prevention and treatment of AD. EGb761 mainly contains three categories of components: flavonol glycosides (FGs), terpene trilactones (TTLs), and catechins and procyanidins. Among them, only TTLs have been evaluated for the inhibition towards Aβ aggregation, and the effects were much weaker than that of the extract. It was suggested that EGb761 should contain several other compounds that are responsible for this activity.In the current study, we have conducted a comprehensive investigation of the generally chemical and bioactive properties of the compounds belonging to all three of the major component categories present in EGb761. We aimed to identify the compounds responsible for the inhibitory activity exhibited by EGb761 towards Aβ42 aggregation and the destabilization of preformed fibrils. The results can be summarized as follows.Seven major FGs were isolated from EGb761 and determined to be quercetin 3-O-α-(6′′′-p-coumaroyl glucopyranosyl-β-1,2-rhamnopyranoside (1), kaempferol 3-O-α-(6′′′-p-coumaroyl glucopyranosyl-β-1,2-rhamnopyranoside) (2), quercetin 3-O-β-D-rutinoside (3), quercetin 3-O-α-L-(β-D-glucopyranosyl)-(1,2)-rhamnopyranoside (4), isorhamnetin 3-O-β-D-rutinoside (5), kaempferol 3-O-β-D-rutinoside (6), and kaempferol 3-O-α-L-(β-D-glucopyranosyl)-(1,2)-rhamnopyranoside (7) by structural analysis. Four catechins and two procyanidins were also found in EGb761 and identified as catechin, epicatechin, gallocatechin, epigallocatechin, procyanidins B1 and B3.The inhibitory activities of these compounds and four major TTLs (i.e., ginkgolides A, B, and C and bilobalide) towards Aβ42 aggregation were evaluated using a thioflavin T fluorescence assay. The results revealed that catechins and procyanidins compounds exhibited significant inhibitory activities with IC50 values in the range of 3-16 μM, and those of the procyanidins were stronger. Three of the FGs (FG1, FG3 and FG4) showed moderate inhibitory activities, with IC50 values in the range of 30-70 μM, whereas the other four FGs (i.e., FG2, FG5, FG6 and FG7) and the four TTLs showed much weaker activity. The catechins and procyanidins also showed potent activities towards the destabilization of preformed Aβ fibrils. Based on these results, we established several structure-activity relationships (SARs) that specific structural groups, as well as the number and position of hydroxyl groups, the linking sequences of the sugar moieties, and the three dimensional conformations of the compounds were important to their inhibitory activity. We also established quantitative analysis of all these tested compounds and simultaneously determined their contents in the Ginkgo extracts. In addition, total FGs, total TTLs, and total catechins and procyanidins were prepared by column chromatography. Similarly, they were also quantitatively analyzed and their inhibitory activities towards Aβ42 aggregation were evaluated. The results showed that total catechins and procyanidins exerted much stronger activity than total FGs and total TTLs. Comprehensive assessment of their activities and contents in the extract indicated that the contribution of the FGs was almost twice that of the catechins and procyanidins, which indicated that they played an important role in the effect of the extract. TTLs alone could barely contribute to the EGb’ effect and probably exerted their influence through the synergistic effect with FGs, which was speculated from the study.In conclusion, the current study has shown that the catechins and procyanidins present in EGb761 possessed potent ability to inhibit Aβ aggregation and destabilize preformed fibrils. FGs made a significant contribution to EGb761’s inhibitory activity towards Aβ aggregation and its neuroprotective effects. Furthermore, the SAR study provide evidences for further research and development of Ginkgo products and drugs designed to target Aβ aggregation or the destabilization of preformed fibrils. Despite their low contents in EGb761 and related products, catechins and procyanidins, and even proanthocyanidins deserve further study because of their potent effects towards Aβ aggregation and the destabilization of preformed fibrils, which could have a significant impact on the quality control of Ginkgo leaves and Ginkgo products.
| Date of Award | 8 Aug 2014 |
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| Original language | English |
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| Supervisor | Zhongzhen ZHAO (Supervisor) |
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