ReviewChemical analysis and quality control of Ginkgo biloba leaves, extracts, and phytopharmaceuticals
Introduction
Ginkgo biloba is the sole surviving species of the once large plant division Ginkgophyta. All other species have become extinct. Fossils of plants quite similar to G. biloba date back to 180 million years ago and thus it has been called a living fossil [1], [2], [3]. In spite of unfounded claims, Ginkgo's introduction in Traditional Chinese Medicine is relatively recent. It does not occur in the oldest Chinese Herbal, Shen Nong Ben Cao Jing (2800 BC) [1] and the use of seeds against cough, asthma, enuresis, alcohol misuse, pyogenic skin infections and wurm infections in the intestinal tract is first mentioned in the great herbal Pen Ts’ao Kang Mu of 1596 by Li Shih-chen [4]. The prescription of leaf extracts for the improvement of the blood circulation, both peripherally and centrally, started in the 1960s in Germany [5]. Currently the medical indications are intermittent claudication, decreased mental vitality at old age (forgetfulness, early dementia, concentration problems) and tinnitus. Ginkgo extracts such as EGb761 are also used as alternative therapy against Alzheimer's disease [6], [7], [8]. However a recent, large (3069 persons), double blind clinical trial of long duration (6.1 years) (“GEM study”) showed no effect of a standardised EGb761 extract in preventing dementia of any type [9], [10]. Nevertheless G. biloba is currently among the most sold and most studied medicinal plants. Accurate and recent worldwide sales figures are not available but in the previous review an indicative figure of US$ 1 billion was cited and DeKosky et al. cite an annual sales figure in the USA of a quarter of a billion $ [9]. Research figures are easier to obtain and reliable. According to a SciFinder search not less than 1930 papers and patents were published or filed in 2006–2007 on Ginkgo, i.e. more than 2 each day.
Two types of leaf extracts can be distinguished: full extracts and standardised extracts. The former are usually prepared with alcohol and contain all constituents soluble in alcohol. The latter extracts are more commonly encountered and contain 6% of terpene trilactones (TTLs), 24% of flavonol glycosides and less than 5 ppm of ginkgolic acids. Such extracts are prepared in a multi-step process which may vary from manufacturer to manufacturer [11], [12], [13]. During this process some compounds are enriched (TTLs, flavonoids) while others (biflavones, ginkgolic acids) are removed. By blending of batches with high and low contents of flavonol glycosides and TTLs a constant quality can be achieved [14].
All serious manufacturers perform extensive quality control on their extracts. The concentration of the major classes of chemicals present in the first standardised Ginkgo extract (EGb761) is presented in Table 1. An example of a limited list of extract specifications is given in Table 2. Sometimes in addition to those demands, the proanthocyanidin content, organic acid content, individual content of bilobalide and ginkgolides A, B, C and J, solubility, qualitative fingerprints for TTLs, flavonol glycosides and organic acids, a 13C NMR fingerprint, sulphated ash, total residual organic solvents, separate residual ethanol and chlorinated solvents, microbiological contamination, aflatoxins, presence of phosphorous and chlorine containing pesticides, positive reaction in test tube assays for the presence of specific functional groups, pH-value, various metal species, and particle size are checked. Many of those tests are well known, described in Pharmacopoeias and not specific for Ginkgo. Therefore they will not be discussed in this review although some references are given in Table 3. The reader is further referred to an overview on industrial quality control by Camponovo and Soldati [15]. Monographs on Ginkgo folium and standardised leaf extracts have appeared or are under construction for various pharmacopoeias.
This review is an updated version of an earlier review published in this journal in 2002 [17]. Like its predecessor, it mainly focuses on the quantitative chemical analysis of the main secondary metabolites occurring in G. biloba leaves and extracts, i.e. terpene trilactones, flavonol glycosides, biflavones, proanthocyanidins, alkylphenols, simple phenolic acids, 6-hydroxykynurenic acid, 4-O-methylpyridoxine and polyprenols. In the last 6 years the interest in the quality control and analysis of Ginkgo has greatly increased (Fig. 1). Especially the number of Chinese papers has risen tremendously in comparison with the review of 2002. All papers are cited; however only the more interesting ones are discussed. Various analytical approaches are compared and suggestions are given. New in this review is the discussion of fingerprinting. Also the patent literature was screened. Around 3200 patents have been filed on Ginkgo and its constituents. Almost all of them do not relate to quality control. Relevant patents that appeared in the period 2001–2008 are mentioned in the appropriate sections. Papers related to topics or groups of compounds other than the above such as preparative isolations, quantitative results without methodology, spectroscopy or synthesis of Ginkgo constituents, extraneous constituents in phytopharmaceuticals, constituents from other parts of the Ginkgo tree than leaves, plant cell biotechnology, finished drugs, or leaf constituents not related to the medicinal activity are not discussed. However in Table 3, Table 10, Table 14, Table 17 short overviews with references of these non-discussed papers are presented. Smaller reviews on the analysis and quality control of Ginkgo leaves and extracts published before 2002 can be found in [18], [19], [20], [21]. More recent reviews on various aspects of Ginkgo were published in [22], [23], [24], [25], [26], [27], [28], [29].
Section snippets
Terpene trilactones
Of all the compound classes present in G. biloba, the TTLs have received by far the most attention. Since the previous review [17] not less than 130 papers dealing with the analysis of these compounds appeared and are thus cited and/or discussed herein. The three major TTLs with the trivial names ginkgolides A, B, C (further abbreviated as G-A, G-B, and G-C) were first isolated in 1932 [131] and independently identified in 1967 by Nakanishi [132] and Sakabe et al. [133]. A minor ginkgolide
Fingerprints
In recent years with the growing interest in Traditional Chinese Medicine (TCM) and systems biology, the “fingerprinting” technique has witnessed a revival. Quality control through quantitative analyses of one or more bioactive constituents or marker compounds remains of prime importance but a constant quality cannot be guaranteed by merely looking at a few compounds. More and more it becomes clear that synergism between constituents in phytomedicines or multiple active analytes interacting
Conclusion
What is very different in this review covering the period 2001–2008 relative to the previous review covering 1975–2001, is the huge increase in the number of Chinese papers and patents. About two-thirds of all recent patents are Chinese. This reflects the rapid economic, scientific and social development of China and of course G. biloba is originally a tree native to China and Ginkgo seeds have been used for hundreds of years in TCM. Currently the main medicinal interest and cultivation concern
Acknowledgements
We are highly indebted to Prof. Pei-shan Xie (Zhuhai, China) who sent us hundreds of Chinese papers as pdf files and translated several of them. We are grateful to Prof. Bo Chen (Changsha, China) for providing us with photocopies of several Chinese papers and Dr. Qiang Zhang (Wageningen, The Netherlands) for translating a number of Chinese papers. We wish to thank Prof. Arnold Vlietinck (Antwerpen, Belgium) and Prof. Wieslav Oleszek (Lublin, Poland) for providing us with photocopies of several
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