Comparison of phytochemical composition of Ginkgo biloba extracts using a combination of non-targeted and targeted analytical approaches

Abstract

Ginkgo biloba extract (GbE) is a dietary supplement derived from an ethanolic extract of Ginkgo biloba leaves. Unfinished bulk GbE is used to make finished products that are sold as dietary supplements. The variable, complex composition of GbE makes it difficult to obtain consistent toxicological assessments of potential risk. The National Toxicology Program (NTP) observed hepatotoxicity in its rodent studies of a commercially available, unfinished GbE product, but the application of these results to the broader GbE supplement market is unclear. Here, we use a combination of non-targeted and targeted chromatographic and spectrophotometric methods to obtain profiles of 24 commercially available finished GbE products and unfinished standardized and unstandardized extracts with and without hydrolysis, then used principal component analysis to group unfinished products according to their similarity to each other and to National Institute of Standards and Technology (NIST) standard reference materials (SRM), and the finished products. Unfinished products were grouped into those that were characteristic and uncharacteristic of standardized GbE. Our work demonstrates that different analytical approaches produced similar classifications of characteristic and uncharacteristic products in unhydrolyzed samples, but the distinctions largely disappeared once the samples were hydrolyzed. Using our approach, the NTP GbE was most similar to two unfinished GbE products classified as characteristic, finished products, and the NIST GbE SRM. We propose that a simple analysis for the presence, absence, or amounts of compounds unique to GbE in unhydrolyzed samples could be sufficient to determine a sample's authenticity.Graphical abstract.

Keywords: Dietary supplements; Ginkgo biloba; Natural products; Non-targeted analysis; Phytochemical characterization.

Fig. 1

Chromatograms of unhydrolyzed GbE unfinished samples (GbE A–T), commercial GbE finished products (GbE W–Z), NTP lots (1, 1F), and NIST standard extract (GbE U) and tablet (GbE V)

Fig. 2

Chromatograms of hydrolyzed GbE unfinished samples (GbE A–T), commercial GbE finished products (GbE W–Z), NTP lots (1, 1F), and NIST standard extract (GbE U) and tablet (GbE V), Peak at ~ 58 min in GbE J chromatogram is due to electronic noise in the detector

Fig. 3

Chromatograms of GbE constituent standards bilobalide, ginkgolide B, ginkgolic acids I and II, GbE flavonol aglycone and terpene trilactone mixtures, and NIST standard extract (GbE U)

Fig. 4

PCA score plots for chromatograms of a unhydrolyzed and b hydrolyzed samples. In a, the clusters are color coded: cluster A (samples U, V, W, X, Y, Z, 1, and 1F), cluster B (samples I, P, Q, and R), cluster C (samples J, M, N, O, and S), cluster D (samples K, L, and T), and cluster E (samples D and E). In b, the color codes from a are used to illustrate the change in relative composition of the samples

Fig. 5

An overlay of the NMR spectra of GbE U (NIST standard, black line) versus GbE A (green line). Green asterisks indicate rutin peaks

Fig. 6

Unsupervised analysis of the NMR spectra of GbE. a Dendrogram analysis of the similarities of NMR spectra. b Constellation plot to visualize the hierarchical clustering results

Fig. 7

High-performance thin layer chromatogram showing: (a) GbE P, Q, R, S, T, U, W, and 1A (Lanes 1–8); Ginkgo biloba leaf (Lanes 9 and 10); Sophora japonica flower (Lanes 11 and 12), S. japonica fruit (Lanes 13 and 14); NIST standard GbE SRM 3247 (Lane 15); genistein (Lane 16); cffeic acid, hyperoside, rutin, and chlorogenic acid (Lanes 17 and 18). Mobile phase (System 1): ethyl acetate: acetic acid: formic acid: water (10/1.1/1.1/2.6) @35–40% humidity. Visualized at 365 nm (b) Lane assignments same as in (a). Visualized with Natural Product Reagent + polyethylene glycol @365 nm. (c) Lane assignments for 1–15 same as in (a) except quercetin, genistin, and genistein (Lane 16); isorhamnetin, caffeic acid, rutin, hyperoside, chlorogenic acid (Lane 17), kaempferol (Lane 18). Mobile phase (System 2): toluene:ethyl acetate: formic acid: (7/3/1) @35–40% humidity. Visualized at 365 nm. (d) Lane assignments as in (c). Visualized with Natural Product Reagent + Polyethylene glycol @365 nm

Fig. 8

PCA analyses of measured constituents. Panels a and b show the scores plot of the first 2 principal component analysis of the concentration of the measured constituents by HPLC and NMR, respectively. Panels c and d show the loading plots from the concentration of the measured constituents by HPLC and NMR, respectively. 95% confidence intervals are shown with ellipses based on the color groups in panels a and b