Published online: 2021-04-16 Original Papers A Detailed View on the Proanthocyanidins in Ginkgo Extract EGb 761 Authors Žarko Kulić 1*, Thomas Ritter2, Birgit Röck 1, Jens Elsäßer 1, Heike Schneider 1,StefanGermer2* Affiliations ABSTRACT 1 Department of Preclinical Research and Development, The Ginkgo extract EGb 761® manufactured with leaves of Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany Ginkgo biloba has been continuously produced over decades 2 Department of Analytical Development, Dr. Willmar at a large scale and is used as a clinically proven remedy for, Schwabe GmbH & Co. KG, Karlsruhe, Germany among other things, the improvement of age-associated cognitive impairment and quality of life in patients with mild Key words dementia. It belongs to the class of extracts addressed as EGb 761, quality, Ginkgo biloba, Ginkgoaceae, quantified extracts according to the European Pharmacopeia. proanthocyanidins, HPLC Accordingly, several compounds (e.g., flavone glycosides and terpene trilactones) are acknowledged to contribute to its received July 21, 2020 clinical efficacy. Covering only about 30% of the mass bal- accepted after revision January 31, 2021 ance, these characterized compounds are accompanied by a published online larger fraction of additional compounds, which might also contribute to the clinical efficacy and safety of the extract. Bibliography As part of our systematic research to fully characterize the Planta Med 2021 constituents of Ginkgo extract EGb 761, we focus on the DOI 10.1055/a-1379-4553 structural class of proanthocyanidins in the present study. ISSN 0032‑0943 Structural insights into the proanthocyanidins present in EGb © 2021. The Author(s). 761 and a quantitative method for their determination using This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying HPLC are shown. The proanthocyanidins were found to be of and reproduction so long as the original work is given appropriate credit. Contents oligomeric to polymeric structure, which yield delphinidin and may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/) cyanidin as main building blocks after acidic hydrolysis. A val- Georg Thieme Verlag KG, Rüdigerstraße 14, idated HPLC method for quantification of the anthocyanidins 70469 Stuttgart, Germany was developed in which delphinidin and cyanidin were de- tected after hydrolysis of the proanthocyanidins. The content Correspondence of proanthocyanidins in Ginkgo extract EGb 761 was found to Dr. Stefan Germer be approximately 7%. Department of Analytical Development, Dr. Willmar Schwabe GmbH & Co. KG Willmar-Schwabe-Straße 4, 76227 Karlsruhe, Germany Phone: + 497214005445, Fax: + 4972140058445 [email protected] ment of age-associated cognitive impairment and quality of life in ABBREVIATIONS mild dementia, with proven clinical efficacy [2–4]. The Commit- PACs proanthocyanidins tee on Herbal Medicinal Products of the European Medicines Ph. Eur. European Pharmacopeia Agency has published a monograph on G. biloba folium [5]. EGb VIS visual light detection 761 is registered as a drug in many countries worldwide. The herbal drug “Ginkgo leaf” is described in the monograph of the European Pharmacopeia [6] together with the monograph, Ginkgo Introduction Dry Extract, Refined and Quantified [7]. In accordance with the The special Ginkgo extract EGb 761 [1] manufactured with leaves of Ginkgo biloba L. (Ginkgoaceae) is widely used for the improve- * These authors contributed equally to this work. KulićŽet al. A Detailed View … Planta Med | © 2021. The author(s). Original Papers monograph, Herbal Drug Extracts, of the European Pharmacopeia chemical complexity of this authentic PACs fraction, we derived a [8], the classification as a quantified extract considers the fact response factor for recalculation of the content of PACs in this frac- that the specified active markers are likely to contribute to the tion and further calculation of PACs in Ginkgo extract EGb 761. clinical efficacy. However, in contrast to constituents with known therapeutic activity in standardized extracts, these active markers Results in quantified extracts cannot explain the clinical efficacy on their own [9]. In the case of the cited monograph of the European Phar- For molecular characterization and as the quantitation reference macopeia for quantified Ginkgo extract [7], the active markers are standard, a representative fraction of the PACs was purified by re- flavone glycosides, specified at 22.0 to 27.0%, and terpene trilac- peated column chromatography. The purified fraction was charac- tones, specified at 2.6 to 3.2% for bilobalide and 2.8 to 3.4% for terized by 1H‑NMR, and the distribution of the molecular weight ginkgolides A, B, and C (calculated on the dried extract). In sum, was determined by analytical gel permeation chromatography and these compounds yield approximately 30% of the dry extract. As HRMS (▶ Fig. 1). The NMR spectrum featured broad signals that are part of our systematic research on constituents of the extract EGb characteristic for polymers in general and PACs in particular 761 from leaves of G. biloba, we are working on elucidation of (▶ Fig. 1a). Besides a considerable amount of water observed as a compounds not covered by the monograph. In this study, we fo- characteristic resonance at 3.3 ppm in DMSO‑d6, only minor sharp cus on the class of structures usually addressed as PACs. peaks of small-molecule impurities were observable, which were PACs are complex oligomeric and polymeric compounds in negligible in comparison to the integrals of the broad polymer sig- plants [10]. Depending on their degree of polymerization and nals. The water content was determined to be 9.19% by Karl Fischer the occurrence of different building blocks, fractions containing titration using common standard procedures. In gel permeation PACs show a multitude of compounds. Quantitative analytical ap- chromatography, a large fraction of the proanthocyanidins ap- proaches mainly applied classical methods such as color reactions peared in the void volume, indicating a high polymerization degree. and hydrolysis by hydrochloric acid followed by photometric as- However, a considerable fraction appeared as a broad peak with a says [11] or used more modern methods like the (dimethyl- retention volume, which would correspond to tetra- to about hex- amino)cinnamaldehyde (DMAC) assay [12]. However, due to the americ procyanidins (▶ Fig. 1b), referred to as oligomers in the fol- manifold combination of the building blocks, linkage-types, lowing. The molecular weights of these oligomers could be ob- stereochemical configuration, and polymerization degree, PACs served up to pentamers in HRMS spectra (▶ Fig. 1c). The observed are a highly challenging compound class that often require tai- mass patterns correspond to pure prodelphinidins or prodelphini- lored methods for characterization [13]. The PACs in Ginkgo dins with 1 procyanidin building block (mass difference of 16 amu), leaves were extensively studied by Qaʼdan et al. [14] and were all with mainly B-type linkages and with A-type linkages only ob- found to consist of B-type linkages (4β → 6or4β → 8) of mainly served for dimers. Due to the mass range limitation of the Orbitrap (epi-)gallocatechins and some minor amount of (epi-)catechins. detector and low signal-to-noise ratio, higher polymers could not be However, structures and quantity of PACs in Gingko extracts used detected. However, the detected oligomers were not stable in solu- for finished products like EGb 761 were not studied in detail. tion. This was observed in gel permeation chromatography, where In the present study, an authentic fraction of PACs in Ginkgo ex- the oligomer peak disappeared after storage times of > 24 h in solu- tract EGb 761 was purified and subjected to a phytochemical char- tion, yielding only polymers eluting in the void volume (▶ Fig. 1d). acterization to establish a robust quantification method for PACs in This behavior indicates that the PACs undergo a transformation in Ginkgo extract EGb 761 through liquid chromatography and pho- solution, which might be oxygen-induced polymerization or non- tometric detection that could be applied for routine analysis. In the covalent aggregation. Other tested protocols for characterization native fraction of PACs in Ginkgo extract EGb 761, the multitude of of the PACs such as Diol-phase separation [15] did not yield PAC clus- compounds does not allow a good peak separation. Thus, we de- ters as distinct peaks as expected from literature data. This observa- cided to use an approach where PACs are hydrolyzed and quantifi- tion may be due to the nature of the Ginkgo PACs, which seem to be cation is done using the resulting peaks of defined anthocyanidins. prone to polymerization in solution and are mainly composed of As anthocyanidins are known to exhibit low stability as reference prodelphinidins instead of procyanidins, the latter being the class substances, we used procyanidin B2 as a reference substance for of compounds applied to Diol-phase separation in literature [15]. calibration purposes. This compound can be handled easily and is Due to the negligible impurity signals in NMR spectra and a chro- known to be sufficiently stable. Additionally, this compound is matogram that is characteristic for oligomers and polymers with commercially available. For calibration