Proanthocyanidins from Ginkgo extract EGb 761® improve bioenergetics and stimulate neurite outgrowth in vitro

Abstract

EGb 761^® is a proprietary extract from Ginkgo biloba leaves approved as an herbal medication for the treatment of dementia and its related disorders. Preclinical studies highlight antioxidant, ROS scavenging, mitochondria-stabilizing, and neuroplastic properties as some of the reported pharmacological activities. Efficacy is traditionally ascribed to terpene lactones and flavone glycosides. However, these quantified known active compounds in EGb 761^® only cover approximately 30% of the mass balance, and there is the possibility that additional compounds from the residual 70% may enhance the activity of the quantified extract EGb 761^®. Proanthocyanidins (PACs) are a quantitatively relevant component in EGb 761^®, and some pharmacological activity has been reported for PACs from Ginkgo and other herbal sources. In this study, we focused on the effects of EGb 761^® and its isolated PACs on mitochondrial bioenergetics and neuroplasticity in the human neuroblastoma cell line SH-SY5Y. We successfully demonstrated positive effects of EGb 761^® and its isolated PACs on several mitochondrial characteristics and neurite outgrowth. As PACs exhibited similar effects compared to the respective extract concentration, they can be considered a pharmacologically relevant component of EGb 761^®.

Keywords: EGb 761®; bioenergetics; mitochondria; neurite outgrowth; proanthocyanidins.

FIGURE 1

EGb 761^® and PACs decreased mitochondrial superoxide (A) and increased mitochondrial membrane potential (B) and ATP levels (C). Values represent the mean ± SEM of three independent experiments for (A) and five independent experiments for (B, C). Each open circle represents one replicate. Values were normalized to 100% of untreated CTRL cells. Statistical analysis was performed with ANOVA and post hoc Dunnett’s multiple comparisons test. *P < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.001 compared to control condition. CTRL, control; PACs, proanthocyanidins-fraction; EGb, EGb 761^® extract.

FIGURE 2

EGb 761^® and PACs increase metabolic activity (A) and mitochondrial (B) mass, while modulating the expression of PGC1-α (C) in SH-SY5Y cells. Optimal effects on metabolic activity and mitochondrial mass were achieved under the conditions of EGb 761^® at 10 μg/mL and PACs at 1 μg/mL. Consequently, gene expression was evaluated under these respective conditions. Values represent the mean ± SEM of five independent experiments for (A) and three independent experiments for (B, C). Each open circle represents one replicate. Values were normalized to 100% of untreated CTRL cells. Statistical analysis was performed with ANOVA and post hoc Dunnett’s multiple comparisons test. *P < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.001 compared to the control condition. CTRL, control; PACs, proanthocyanidins-fraction; EGb, EGb 761^® extract.

FIGURE 3

EGb 761^® and PACs increase the oxygen consumption rate (OCR) (A) and extracellular acidification rate (ECAR) (B) in human neuroblastoma cells. Treatment with PACs at 1 μg/mL and EGb 761^® at 10 μg/mL led to a more energetic state (C). Values represent the mean ± SEM of three independent experiments and six replicates per condition. Each replicate is measured in technical quadruplicates. Statistical analysis was performed with the unpaired t-test. *P < 0.05, **p < 0.01, and ****p < 0.001 compared to control condition. CTRL, control; PACs, proanthocyanidins-fraction; EGb, EGb 761^® extract.

FIGURE 4

EGb 761^® and PACs increased the neurite outgrowth in the human neuroblastoma cells. In total, approximately 10,000 cells and 57,000–101,000 neurites were evaluated. Images were taken with the Cytation 3 cell imaging multi-mode reader and analyzed with the ImageJ neurophology software to evaluate parameters of neuroplasticity. Values represent the mean ± SEM of three independent experiments and were normalized to the cell count. Data are presented as % normalized to the CTRL group. Per condition, 3,400–11,300 cells were analyzed in total. Each open circle represents one image. One way ANOVA and post hoc Dunnett’s multiple comparisons versus CTRL. *P < 0.05, **p < 0.01, and ***p < 0.001. CTRL, control; PACs, proanthocyanidins-fraction; EGB, EGb 761^® extract.