Quercetin Potentiates the NGF-Induced Effects in Cultured PC 12 Cells: Identification by HerboChips Showing a Binding with NGF

Abstract

Dementia is a persistent disorder of the mental processes and is strongly related to depression. However, the performance of current antidepression medicine is far from satisfactory. Herbal extract provides an excellent source to identify compounds for possible drug development against depression. Here, HerboChips were employed to search herbal compounds that could bind nerve growth factor (NGF). By screening over 500 types of herbal extracts, the water extract of Ginkgo Folium, the leaf of Ginkgo biloba, showed a strong binding to NGF. The herbal fractions showing NGF binding were further isolated and enriched. By using LC-MS/MS analysis, one of the NGF binding fractions was enriched, which was further identified as quercetin, a major flavonoid in Ginkgo Folium. Quercetin, similar to Ginkgo Folium extract, could enhance the effect of NGF in cultured PC 12 cells, including potentiation of neurite outgrowth and phosphorylation of Erk-1/2. This is the first report of discovering an NGF binding compound by using HerboChips from herbal extracts, which could be further developed for antidepression application.

Figure 1

Extract of Ginkgo Folium was fractionated by HPLC and then dotted and fixed on a chip. The chip was incubated with biotinylated protein target (NGF) and then probed with streptavidin-Cy5 before fluorescence detection. (a) Adopted from Huang et al., 2015, showing the design of HerboChip, the quantifier and qualifier controls were included. (b) Scanning result of HerboChip using Ginkgo Folium extract and probed with biotinylated NGF (left) and probed with incubation buffer only (right). Representative scanned images were shown, n = 5. (c) Superimposition of the read-out of HerboChip screening and HPLC chromatogram. HPLC chromatograms (solid line) and quantified HerboChip screening read-out (dotted line with shaded area) of the extract of Ginkgo Folium were superimposed. A will resolved peak at retention time ~67 min was labelled as D1 and submitted for further analysis.

Figure 2

(a) Under full scanning mode, the molecular weight of the enriched fraction D1 was shown by LC-MS/MS. (b) ¹H-NMR spectra of enriched fraction D1. (c) ¹³C-NMR spectra of enriched fraction D1. Corresponding chemical structures were indicated according to their ppm in different NMR spectrum. The spectrum was submitted to the database, Biological Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu) for characterization. (a) HPLC chromatograms using the HPLC condition described in the section of Materials and Methods were shown: quercetin at 1 mM. Chemical structure of the enriched fraction from the extract of Ginkgo Folium (D1), that is, quercetin, was shown. (b) Extract of Ginkgo Folium (100 mg/mL) and (c) extract of Ginkgo Folium (50 mg/mL) spiked with 0.5 mM quercetin. The position of quercetin was being indicated in (b) and (c).

Figure 3

Cultured PC12 cells were treated with control, NGF (0.5 ng/mL or 50 ng/mL), and enriched fraction from the extract of Ginkgo Folium (quercetin) with or without NGF at 0.5 ng/mL for 48 h, and then the neurite outgrowth was examined under microscope. (a) Microscopic image of PC12 cells treated with buffer alone or treated with NGF in 50 ng/mL. Representative images were shown, n = 3. Bar = 50 μm. (b) To quantify the differentiation effect, the percentage of differentiated cell numbers and length of neurites were counted by the methods described in Materials and Methods section. Data were expressed as % of cells in 100 counted cells, mean ± SEM, n = 3. ^∗∗∗P < 0.001 verse control.

Figure 4

Serum-starved cultured PC12 cells were treated with control, NGF (0.5 ng/mL or 50 ng/mL), or quercetin (0.01–1.0 μM) with or without NGF at 0.5 ng/mL, or the extract of Ginkgo Folium (0.1–10 μg/mL) with or without NGF at 0.5 ng/mL for 48 hours, and then the neurite outgrowth was examined under microscope. To quantify the differentiation effect, the percentage of differentiated cell numbers and length of neurites were counted by the methods described in Material and Methods section. Data were expressed as % of cells in 100 counted cells, Mean ± SEM, n = 3. ^∗∗∗P < 0.001 verse control.

Figure 5

PC12 cells were serum-starved for 3 hours before treatment of control, 0.5 ng/mL, or 50 ng/mL of NGF and quercetin and cotreatment of quercetin with NGF at 0.5 ng/mL. Cell lysates (20 μg) were subjected to western blotting for phosphorylation analysis. Only 50 ng/mL NGF and the cotreated quercetin with 0.5 ng/mL NGF induced phosphorylation of Erk-1/2 (P-Erk-1/2 at ~42 and ~44 kDa) at 5 min after treatments, no notable changes in phosphorylation were observed in other groups, n = 4. The representative gels were shown here.