Industrial Quality Control of Ginkgo Products

The marketing of pharmaceutical products in Europe requires an authorisation, according to the directives 65/65 EEC and 75/318 EEC. Phytomedicines are included in this general legislation, and are subjected to the same treatment as medicinal products in general. Thus, evidence of the product’s safety, efficacy and quality has to be submitted before a marketing authorisation will be granted.

In this chapter the focus will be on the parameters and the analytical methods to evaluate the quality of Ginkgo biloba leaves extracts and finished products. Earlier experience gained at the University of Lausanne (Camponovo) and at Pharmaton for the standardisation of GK 501 Ginkgo biloba extract Pharmaton and for the finished product Gincosan and Memfit Pharmaton, plays an important role in this.

Control of Starting Material (Ginkgo Biloba Leaves)

A consistent quality for products of vegetable origin can only be guaranteed if the starting materials are rigorously defined. But what does a good quality of Ginkgo leaves mean? So far, there are no pharmacopoeia monographs on Ginkgo biloba leaf to which one can refer. Each producer supplies its own monograph with some specifications that are more or less detailed and dependent on the technical capacity of the supplier.

From a medical point of view, one can assume that the quality of Ginkgo biloba leaves, and generally of all vegetal raw materials, depend on the content of markers/ active substances and on the absence of contaminants and toxic secondary metabolites. In Ginkgo biloba, the active and toxic substances are well known and as will be seen, the requirements for high quality leaves are well defined and accepted by the whole scientific community. The secondary metabolite content of Ginkgo leaves, depends on the site of collection as well as the time of harvesting and the stage of growth of the plant. Little changes on these parameters can drastically influence and modify the ratio of compounds present in the plant and consequently change the final quality of the extract. Thus, a main point to assure the consistency of the quality of the vegetal material, is the standardisation of these parameters.

According to the literature the terpene trilactones show the biggest variations between the two main classes of active compounds. Van Beek et al. (1991) have found that the content of single ginkgolides can vary by a factor of more than 100 between leaves having a different origin. The flavonoids, an other class of active compounds, have shown a smaller variation. Hasler et al. (1990) investigated the flavonol glycoside content in different batches of leaves obtained from the market and they found a content varying from 0.5 to 1% w/w. Therefore, to avoid serious problems with the standardisation of the final extract, control of the amounts of these compounds in Ginkgo leaves is an important step before starting the extraction procedure. However, from experience it is known that if the growth parameters are well controlled and the leaves are harvested yearly from cultivated plants in a specific place, the variation in concentration of active substances is not a critical issue. Usually, Ginkgo biloba leaves are harvested from July to September, when the leaves are completely developed and just before the green colour begins to turn yellow.

In 1991, Pharmaton investigated the variation of the content of terpene trilactones and flavonoids, during the harvesting period, in leaves of male and female trees. The leaves used for the study were collected from four male and female trees at Peace Memorial Park in Hiroshima by Professor K.Yamasaki.

This means that even the sex of the trees has to be taken into account during the collection of the leaves for the manufacturing of the extract. In the leaves collected from Ginkgo trees of the same gender, however, only little variation was observed during the harvesting period from July to September.

Identification

The authentication of the vegetal raw material is an important step, therefore, in addition to the macroscopic and microscopic examination, a chromatographic identification of characteristic substances has to be performed. The relevant macroscopic and histological characteristics are described in the USP pharmacopoeia monograph preview on Ginkgo Leaf (1997).

The terpene trilactones and the main Ginkgo flavonol glycosides can be used as characteristic substances for a chromatographic identification of the drug. The identification can be performed by means of published TLC or HPLC methods.

Quantitative Determination of Markers/Active Substances

The identification of the drug is followed by a quantitative determination of the known active constituents. In the case of Ginkgo biloba leaves, the total amount of flavonoids, ginkgolides and bilobalide has to be quantified. The analytical methods that can be used will be briefly discussed in the section dealing with the analysis of extracts.

Foreign Matter

The plant material should neither present any pathogenic organism, nor any possible extraneous materials, e.g. micro-organisms, undesirable parts of plants, insects, animal excreta and so on. The presence of extraneous materials can be checked according to Ph. Eur. III, 2.8.2.

Determination of Ash

The aim of this test is the determination of the amount of material present after ignition. This remaining material is partially derived from the plant tissue itself but also, possibly from extraneous matters (e.g. sand and soil).

For Ginkgo leaves the sulphated ash can be determined according to the method of Ph.Eur. III, 2.4.14.

Loss on Drying

This test determines both water and volatile matters. According to the USP pharmacopoeia preview on Ginkgo leaf (1997) it can be determined by weight after 2 hours at 105°C.

Test for Aflatoxins

This test is designated to detect the possible presence of aflatoxins Bl5 B2, Gt and G2, which are highly toxic contaminants. The analytical method is described in the section reporting on the analysis of the intermediate product (the extract of leaves). The test on the plant material is very important, as any deteriorated raw material can be eliminated before starting the manufacturing procedure, thus avoiding a waste of time and money.

Pesticide Residues

The analysis of the pesticide residues is another important test to perform on the vegetal material before starting the manufacturing procedure. The analytical method used, and the classes of pesticides analysed, will be described later, in the section dealing with the analysis of the intermediate product. Experience has shown that the amount of pesticides found in Ginkgo leaves is always very low. This is not surprising because due to the extreme resistance of Ginkgo trees, the cultivators do not need to use large amounts of pesticides to protect the plant from insects, moulds or fungi. Furthermore, the leaves in contrast to other organs change every year and the risk to have an accumulation of contaminants is very low. Other tests, such as heavy metals and microbiological contamination, can be performed but if the aim is the production of an extract it is more convenient to perform these tests directly on the extract. In fact, during the manufacturing procedure, some contaminants are removed and the possible microbiological contamination is eliminated by the organic solvents used for the extractions.

Control Tests on Intermediate Product

(extract of Ginkgo biloba leaves)

Only one official monograph on a standardised extract of Ginkgo biloba leaves is available so far. It was drawn up in 1994 by the German Commission E (“Bundesanzeiger Nr. 133”, Vol. 46, July 1994 (pages 413-415). This monograph contains only a few analytical specifications and these are practically the same as those of Ginkgo Biloba Extract 761 and GK 501 extracts:

  • 22-24% of Ginkgo flavonol glycosides
  • 5-7% of terpene trilactones (of which 2.8-3.4% of ginkgolides A, B, C and
  • 2.6-3.2% of bilobalide)
  • <5 ppm of ginkgolic acid

The routine methods for the quality assessment of the extract have to be chosen according to new developments in analytical chemistry but should also consider routine laboratory feasibility.

In this chapter the methods that according to experience should be adopted for the quality control of extracts and the requirements for the release of products will be briefly described.

Control Tests on Finished Product

The finished phytopharmaceutical product (galenic form) should comply with general requirements for particular dosage forms. Product specifications and tests for release have to be established in conformity with the requirements of the pharmacopoeia monographs. A method of identification and quantification of the plant extract in the finished product should be developed and validated. Usually, the analytical methods can be similar to those for intermediate product but they have to be adapted to eliminate the interference of excipients. The quantification of the standardised GK 501 Ginkgo biloba extract in the finished product Gincosan, for example, can be performed by the same HPLC method described above for the extract, but the sample preparation has to be modified. The finished product, in this case a hard gelatine capsule, should also comply with the other tests, specific for this galenic form (disintegration, microbiological purity, water absorption). Furthermore, all the excipients and the permitted colouring agent used during the manufacturing procedure have to be identified. The preservatives have to be quantified and their antimicrobial activity should be demonstrated.

Recently a comparative study of the terpene trilactones content in some finished products containing Ginkgo biloba extract was performed. The results have shown that only the administration of Tanakene and Gincosan at the recommended dosages can provide a daily intake of terpene trilactones similar to that used in the published clinical studies. Terpene trilactones were detected in all other phytoparmaceuticals investigated, albeit at a much lower, and therefore not therapeutic dosage.

Before any drug can enter the market, the stability characteristics of the finished product have to be studied according to the ICH guidelines Q1A (1993). For this purpose, accelerated and long-term stability tests on batches of the same dosage form and in the containers proposed for the marketing have to be performed. The result enables one to establish recommended storage conditions and the shelf life of the finished product. The range of testing should not only cover chemical, physical, biological and microbiological stability but also particular properties of the dosage form (e.g. disintegration time for oral solid dosage form).

In 2010, a meta-analysis of clinical trials has shown Ginkgo to be moderately effective in improving cognition in dementia patients.

Industrial Quality Control of Ginkgo Products: Conclusion

Consumers, regulating agencies as well as the pharmaceutical industry are all interested in some level of phytopharmaceutical regulation. In some countries adulterated or low potency phytopharmaceuticals have been put on the market and there are also problems with contaminants like pesticides. The level of product standardisation should not be determined by its scientific feasibility, but rather by the need for safety and the therapeutic relevance of the chemical components of the natural products.

In the case of Ginkgo biloba extracts, recommended limits of active and toxic substances have been published by the German Authorities. The quality of an extract is not only a matter of control of finished or intermediate products. It already starts during the selection of (cultivated) plants. With chromatographic methods such as HPLC and GC one can determine active (Gingko flavonoids, terpene trilactones) and toxic (ginkgolic acid) constituents as well as contaminants (pesticides, aflatoxins). The validation of the analytical methods is essential, and international guidelines like the ICH Q2A (1994) and ICH Q2B (1996) should be followed.

 

Fabrizio F.Camponovo and F.Soldati “Industrial Quality Control of Ginkgo Products” (2006)