Elsevier

Journal of Chromatography A

Volume 1216, Issue 11, 13 March 2009, Pages 2002-2032
Journal of Chromatography A

Review
Chemical analysis and quality control of Ginkgo biloba leaves, extracts, and phytopharmaceuticals

https://doi.org/10.1016/j.chroma.2009.01.013Get rights and content

Abstract

The chemical analysis and quality control of Ginkgo leaves, extracts, phytopharmaceuticals and some herbal supplements is comprehensively reviewed. The review is an update of a similar, earlier review in this journal [T.A. van Beek, J. Chromatogr. A 967 (2002) 21–55]. Since 2001 over 3000 papers on Ginkgo biloba have appeared, and about 400 of them pertain to chemical analysis in a broad sense and are cited herein. The more important ones are discussed and, where relevant, compared with the best methods published prior to 2002. In the same period over 2500 patents were filed on Ginkgo and the very few related to analysis are mentioned as well. Important constituents include terpene trilactones, i.e. ginkgolide A, B, C, J and bilobalide, flavonol glycosides, biflavones, proanthocyanidins, alkylphenols, simple phenolic acids, 6-hydroxykynurenic acid, 4-O-methylpyridoxine and polyprenols. In the most common so-called “standardised” Ginkgo extracts and phytopharmaceuticals several of these classes are no longer present. About 130 new papers deal with the analysis of the terpene trilactones. They are mostly extracted with methanol or water or mixtures thereof. Supercritical fluid extraction and pressurised water extraction are also possible. Sample clean-up is mostly by liquid–liquid extraction with ethyl acetate although no sample clean-up at all in combination with LC/MS/MS is gaining in importance. Separation and detection can be routinely carried out by RP-HPLC with ELSD, RI or MS, or by GC/FID or GC/MS after silylation. Hydrolysis followed by LC/MS allows the simultaneous analysis of terpene trilactones and flavonol aglycones. No quantitative procedure for all major flavonol glycosides has yet been published because they are not commercially available. The quantitation of a few available glycosides has been carried out but does not serve a real purpose. After acidic hydrolysis to the aglycones quercetin, kaempferol and isorhamnetin and separation by HPLC, quantitation is straightforward and yields by recalculation an estimation of the original total flavonol glycoside content. A profile of the genuine flavonol glycosides can detect poor storage or adulteration. Although the toxicity of Ginkgo alkylphenols upon oral administration has never been undoubtedly proven, most suppliers limit their content in extracts to 5 ppm and dozens of papers on their analysis were published. One procedure in which a methanolic extract is directly injected on a C8 HPLC column appears superior in terms of sensitivity (<5 ppm), separation, simplicity and validation and will be incorporated in the European Pharmacopoeia. Alternatively GC/MS and ELISA methods can be used. A sharp contrast to the plethora of papers on terpene trilactones, flavonol glycosides, and ginkgolic acids forms the low number of papers on biflavones, proanthocyanidins, simple phenolics, simple acids, and other constituents that make up the remaining 70% of Ginkgo standardised extracts. More research in this direction is clearly needed. For the analysis of Ginkgo proanthocyanidins (7%) for instance, no reliable assays are yet existing. Finally the growing literature on pharmacokinetic and fingerprinting studies of Ginkgo is briefly summarised.

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

References (589)

  • T.A. van Beek

    J. Chromatogr. A

    (2002)
  • G. Xie et al.

    Xibei Yaoxue Zazhi

    (2005)
  • B. Singh et al.

    Fitoterapia

    (2008)
  • H. Huang et al.

    Protein Peptide Lett.

    (2001)
  • G. Sarijeva et al.

    J. Plant Physiol.

    (2007)
  • J.L. Garrido et al.

    J. Chromatogr. A

    (2003)
  • Y. Liu et al.

    Shipin Keji

    (2007)
  • S. Mongrand et al.

    Phytochemistry

    (2001)
  • M.M.C. Ritter et al.

    J. Food Compos. Anal.

    (2007)
  • Z. Xiaoshan et al.

    Atmos. Environ.

    (2000)
  • J.L. Gu

    Guangpu Shiyanshi

    (2007)
  • W. Li et al.

    Phytomedicine

    (2002)
  • E. Malinowska et al.

    Food Chem. Toxicol.

    (2008)
  • E.D. Caldas et al.

    Food Chem. Toxicol.

    (2004)
  • P. Del Tredici

    Arnoldia-Boston

    (1991)
  • P. Del Tredici
  • V. Melzheimer et al.
  • H.-L. Li

    Morris Arboretum Bull.

    (1956)
  • K. Drieu et al.
  • M.R. Farlow et al.

    Dement. Geriatr. Cogn. Disord.

    (2008)
  • D.B. Hogan

    Neuropsychiatr. Dis. Treat.

    (2007)
  • O. Napryeyenko et al.

    Arzneim. -Forsch. (Drug Res.)

    (2007)
  • S.T. DeKosky et al.

    J. Am. Med. Assoc.

    (2008)
  • L.S. Schneider

    J. Am. Med. Assoc.

    (2008)
  • E. Bombardelli, G. Mustich, M. Bertani, New extracts of Ginkgo biloba and their methods of preparation, European Patent...
  • J. O’Reilly
  • J. O’Reilly, H. Jaggy, Active component concentrates and new active component combinations from Ginkgo biloba leaves,...
  • R. Bauer

    Drug Inf. J.

    (1998)
  • F.F. Camponovo et al.
  • K.-H. Stumpf, in: Organizing Committee for ‘97 International Seminar on Ginkgo (Ed.), Proceedings of ‘97 International...
  • O. Sticher

    Planta Med.

    (1993)
  • T.A. van Beek, in: Organizing Committee for ‘97 International Seminar on Ginkgo (Ed.), Proceedings of ‘97 International...
  • T.A. van Beek et al.

    Fitoterapia

    (1998)
  • P. Xie

    Zhongguo Zhong Yao Za Zhi

    (1999)
  • S. Kressmann

    Dtsche Apoth. Ztg.

    (2003)
  • S. Uchida et al.

    Kosutoresu Shokuhin no Kaihatsu to Tenbo

    (2006)
  • P.C. Chan et al.

    J. Environ. Sci. Health

    (2007)
  • S. Mahadevan et al.

    J. Food Sci.

    (2008)
  • L. Fang

    Zhongguo Yaofang

    (2008)
  • X.-J. Chen et al.

    Shengwuzhi Huaxue Gongcheng

    (2008)
  • Q. Deng et al.

    Yingyang Xuebao

    (2006)
  • Q. Deng et al.

    Zhongcaoyao

    (2007)
  • B. Carratù et al.

    J. Food Sci.

    (2008)
  • Y. Sawano et al.

    Biol. Chem.

    (2007)
  • W. Niu et al.

    Xibei Zhiwu Xuebao

    (2003)
  • T. Miyakawa et al.

    Acta Crystallogr.

    (2007)
  • W. Huang et al.

    Yingyang Xuebao

    (2003)
  • P. Zhu

    Huagong Shengchan Yu Jishu

    (2006)
  • A. Skribanek et al.

    Physiol. Plant

    (2008)
  • B. Sha et al.

    Guangxi Zhiwu

    (2007)
  • Cited by (501)

    View all citing articles on Scopus
    View full text