Development of the U.S. Pharmacopeia’s Monograph on Ginkgo Biloba

It is amazing that the Ginkgo tree, Ginkgo biloba L., the last remaining member of the Ginkgoaceae family has suddenly engaged the interest of Western medicine. Worldwide use of Ginkgo leaf extracts is reported to be on the rise in view of the reported beneficial effects of the Ginkgo as an antiasthmatic and on the circulatory system. Is it more appropriate to say that Western medicine has at last considered the medicinal properties of Ginkgo biloba whose fruits and seeds have been valued in China for their medicinal properties for the last several hundred years? Though the leaves of Ginkgo biloba were sparsely used, in comparison to the fruits and seeds, in ancient medicines in the East, the Western medicine as practiced extensively in Europe uses the leaves.

Senna leaves are used directly in the preparation of tea, but traditionally extracts of Ginkgo leaves are used directly in the Western medicine preparations. United States Pharmacopeia’s (USP) interest and involvement in the development and establishment of public standards began in 1820 with the publication of its first edition. The first edition of the United States Pharmacopeia included monographs on botanicals. Thereafter for the next 120 years or so the USP Committee of Revision were busy adding more botanicals monographs to the United States Pharmacopeia. At the turn of the 20th century, USP had included monographs for over 600 botanicals and their preparations. However, with the arrival of isolated active ingredients, and later of synthetic organic medicinal substances, the focus of USP Committee of Revision turned to the more well-defined and easily characterized single organic chemicals. Also the use of tablets as a precise and convenient dosage form made botanical extracts less appealing. As a result gradually over the next 45 years or so the USP Committee of Revision replaced the older monographs on botanicals with those of newer synthetic organic medicinal substances and purified active principles of botanicals and their dosage forms. The result is that today at the time of writing this chapter the current edition of U.S. Pharmacopeia (USP 23-NF 18 1995) has only about 30 or so monographs on botanicals and their preparations out of more than 3500 monographs (e.g. Rauwolfia serpentina, Digitalis, Psyllium husk, Senna leaves etc.).

The recent resurgence of interest in the use of botanicals by the American public is contributing to the rapid change in the healthcare scene in the United States of America. Those who are disillusioned with modern healthcare which uses modern synthetic medicines are seeking alternative therapies. These alternative therapies include botanical preparations. The U.S. Pharmacopeial Convention, in response to this evolving change in the healthcare system, adopted a resolution at its March 1995 Convention which encouraged the U.S. Pharmacopeia to explore the feasibility and advisability of developing and establishing public standards for dietary supplements. Even though the resolution did not specifically mention botanicals, in the context of the definition provided in the Dietary Supplement Health Education Act of 1994, dietary supplements include botanicals. The United States Pharmacopeia thus is going back to its roots.

Considerations In The Selection Of Botanicals For Development Work.

In regard to the selection and prioritization of botanicals for compendial standards development work due consideration should be given to those botanicals widely used in the U.S.A. and which are also significant in international usage. This is immediately followed by other considerations such as harmonization of standards with other major pharmacopeias such as the European Pharmacopeia and Japanese Pharmacopeia. In following the above, the USP Committee of Revision prioritized a list of about 20 botanicals which includes Ginkgo biloba. Irrespective of whether all the active constituents of a botanical have been identified (and established), the strategy of the USP Committee of Revision is to develop a definition of the plant part. Since this book is exclusively devoted to a discussion of Ginkgo this chapter will deal with issues involved and considered in the development of pharmacopeial standards for Ginkgo biloba (leaves).

Issues In The Standardization

Pharmacopeial monographs imply shelf-life standards. In other words the product purported to conform to the United States Pharmacopeia must be in conformity with the Pharmacopeial specifications when analyzed by the methods specified therein till the last date of expiry claimed on the product label. It should be kept in mind that pharmacopeial standards are not routine quality control standards but are referee standards. The General Notices of the U.S. Pharmacopeia (USP 23-NF 18 1995) allow use of alternate methods i.e. determination of compliance with the pharmacopeia may be done by the use of alternate methods which are chosen for advantages in accuracy, sensitivity, precision, selectivity or adaptability to automation provided these alternate procedures are validated. However since pharmacopeial standards and procedures are interrelated, where a difference appears or in the event of dispute only the result obtained by the procedure given in the Pharmacopeia is conclusive. Therefore one can expect the standards setting organization to choose only those methods that would definitively determine the extent of decomposition of the botanical article under test. In developing a pharmacopeial monograph for articles of botanical origin considerations should be given to the way the botanical parts have been used in the past or moving in the international commerce. In reviewing the pharmacopeial monographs of yesteryear one can see clearly three categories namely (1) plant parts as a whole (e.g. subterranean parts, leaves, flowers etc.) or their powdered forms, (2) simple extracts of the plant parts derived from simple percolation procedures or (3) extracts of the plant parts that have undergone extensive processing. The standards for the above would obviously differ considerably from each other.

The purpose of this chapter is to highlight the issues and considerations in the development of pharmacopeial standards for botanical material as it relates to Ginkgo biloba leaf. Since a draft monograph on Ginkgo biloba leaf prepared by the USP Committee of Revision has just been published for public comments, references are made to the draft throughout this chapter (Ginkgo biloba leaf, 1997). It is to be understood that any or all aspects of the proposed draft monograph may undergo further changes as comments received from interested parties are reviewed by the members of the Committee of Revision.

Having said the above, the initial draft monograph on Ginkgo biloba developed by the USP Committee of Revision, whom the author has the privilege to work with, defines the Ginkgo leaves. Even though Ginkgo leaves as such are not used like Senna leaves, pharmacopeial practice of defining the raw material used in the preparation of their extracts or infusions or powders is a sine qua non for the quality assurance of the dosage forms derived therefrom. A future focus of the pharmacopeias is likely to be development of monographs on plant extracts and dosage forms where the issues involved are somewhat more complex.

Definition

A pharmacopeial monograph on a botanical has to be very specific in its definition by specifying the genus and species thereby indicating that the standards apply to that particular plant part of the stated genus and species. The draft monograph defines Ginkgo Leaf as consisting of the dried leaf of Ginkgo biloba belonging to the family Ginkgoaceae. In keeping with the customary practice of the U.S. Pharmacopeia to state, after the name of the species, the name of the botanist, Linnaeus, who first described the species (or the current commercially dominant description), is specified in the definition.

Packaging, Storage and Labeling

As stated above since pharmacopeial specifications are shelf-life specifications, the type of container in which the article is to be stored as well as the temperature conditions of storage need to be specified in the monograph. Under the stated conditions the pharmacopeial articles are expected to retain their stated potency till the expiry date claimed on the product label.

The name in Latin binomial form is the only recourse we have at present to save the public from the prevailing hopeless situation in the market place where manufacturers and distributors of botanicals use several different names on the product thus causing confusion in the minds of the consumers. The product label of a pharmacopeial article is expected to specify, in addition to the common name Ginkgo, the name in Latin binomial form as given in the definition. The insistence of the name in Latin binomial form is both a regulatory issue and a consumer issue. Thus when a product labeled as Ginkgo USP or NF (National Formulary) (assuming the monograph on Ginkgo would be adopted officially by the USP in the near future) is seized by regulatory authorities for determination of compliance, all that would be needed is the product label and the United States Pharmacopeia book. The label enables the consumers to compare the product purchased to other species of botanicals they have been purchasing.

Identification

A Pharmacopeial monograph specifies Identification tests to ensure confirmation that the identity of the article under examination is in agreement with what is stated in the definition of the article. Histology and morphology alone are not sufficient for positive identification of botanicals. This does not mean magnifying lenses and microscopes, which were the only tools available for the 19th and early 20th century scientists for positive identification of botanicals, have no place in modern pharmacopeias. In addition to the microscopic examination, pharmacopeial monographs would employ two other types of identification tests. For chemical methods of identifying the active ingredients or marker substances either as a class (e.g. alkaloids, terpenes) or by their functional group (e.g. ketones, phenolic hydroxyl), color tests or spectrophotometric techniques such as UV, IR are specified. Use of separation techniques such as thin-layer chromatography, high pressure liquid chromatography etc. permit not only identification of a marker or active ingredient but can detect contamination with other foreign substances. The use of the last two identification tests above have evolved during the last fifty years and are powerful, reliable and critical sources for positive identification.

Botanical characteristics

Macroscopical examination of fresh or dried whole Ginkgo leaf shows it to be typically fan-shaped but occasionally sub-reniform and sides somewhat concave thus distinguishing it from any adulterant botanical. Histology of the Ginkgo leaf reveals numerous lignified elements derived from the lamina and petiole including xylem vessels with annular thickening, tracheids and vessels with bordered pits. The extent of lignification, particularly in the petiole, increases with age of the leaf and mature leaves may show the presence of very rare polygonal to circular starch granules approximately 20μm in diameter with a central hilium and exhibiting a marked maltese cross under crossed-polaroids. While macroscopic and microscopic examination results in rich information enabling positive identification, most modern analytical laboratories do not have analysts trained in histological examination. Thus the necessity for other tests for positive identification remains.

Chemical methods

Analytical methods involving chemical reactions contribute to a more unequivocal identification of botanicals. However since Ginkgo leaves contain a variety of compounds such as isoprenoids (sterols, terpene trilactones), aliphatic alcohols and ketones, organic acids, polysaccharides, flavonol glycosides etc., a single chemical reaction yielding a bright color characteristic of flavonol glycosides or terpene trilactones does not appear to be promising. Hence the best course for a pharmacopeial monograph would be to specify methods involving separation techniques.

Separation techniques

Constituents responsible for the beneficial properties of Ginkgo leaves are reported to be flavonol glycosides and terpene trilactones: the sesquiterpene bilobalide and diterpenic ginkgolides. These compounds are reported to show effects on vascular and cerebral metabolic processes and ginkgolides inhibit platelet activating factor (PAF). Quantitative determinations of these by liquid chromatographic techniques have been reported in the literature. It is therefore logical that pharmacopeial identification tests should focus on tests that would positively identify the presence of the above compounds in Ginkgo leaves. It could be argued that biflavones present in Ginkgo leaves are also characteristic marker substances but they do not lend themselves for standardization at least at this point of time though there are literature reports of attempted quantification of these compounds.

The critical aspect of identification of botanical materials by separation techniques is the use of reference standards since it provides an unambiguous calibration at the time of use. The reference standards used in pharmacopeial monographs are invariably those that are present in the material under test. When individual reference standards are difficult to develop and/or are prohibitively expensive, other substances which display suitable characteristics for the intended purpose can be occasionally employed. With regard to the separation technique for the positive identification of flavonol glycosides and terpene trilactones in Ginkgo leaves the natural choice is thin-layer chromatography (TLC) since it is the most simple and widely used technique available at sources of small scale manufacturers and ports of imports. There is a need to use two different solvent systems for the identification of flavonol glycosides and terpene trilactones because of the different nature of functional groups present in these compounds. Accordingly the TLC system chosen for the identification of flavonol glycosides in the draft monograph is practically identical to the one that is under consideration for the European Pharmacopeia. This will achieve the objective of harmonization as well. The test involves comparison of the TLC of the test solution, prepared by methanol extraction of the Ginkgo leaves, with the chromatogram of the standard solution containing rutin and chlorogenic acid run in a developing solvent consisting of a mixture of ethyl acetate, water, anhydrous formic acid and glacial acetic acid (67.5:17.5:7.5:7.5). The spray reagents used are a methanolic solution of diphenylboryloxyethylamine and a methanolic solution of polyethylene glycol 400. Visualization is under ultraviolet light (365nm). Though neither rutin nor chlorogenic acid is present in Ginkgo leaves, their spots provide a reference point for easy location of the spots due to the major flavonol glycosides.

With regard to the method for the identification of terpene trilactones, the lack of a suitable UV-absorbing functional group and their very low concentrations (0.01-0.1%) along with a host of other compounds in Ginkgo leaves make the analysis of these terpenes trilactones very difficult. Although all the five terpene trilactones in Ginkgo leaves have been separated by HPLC using a refractive index detector it is not a practical procedure for a quick identification check because of extensive clean-up steps involved in the procedure. Thus the best alternative to HPLC is TLC. Several papers have appeared in the literature on the thin-layer separation of terpene trilactones of Ginkgo biloba leaves but unfortunately a clear separation and subsequent detection of ginkgolides A and B and ginkgolides C and J on a thin-layer chromatographic plate can be seen as a major problem in these publications. The best published TLC separation of all relevant terpene trilactones appears to be the one involving the use of silica gel plates impregnated with sodium acetate. Use of a silica gel impregnated with sodium acetate as reported by van Beek and Lelyveld gives an excellent separation of all major terpene trilactones in Ginkgo leaves using the solvent system ethyl acetate and hexane (9:1) contained in a non-saturated chamber or by using methyl acetate as the solvent in a saturated chamber. The detection of all the terpene trilactones is achieved by spraying the plate with acetic anhydride followed by heating at 105° and viewing under ultraviolet light at 254nm and 366nm. The method is reported to detect all the four ginkgolides and bilobalide in quantities less than 1µg. The above procedure does not involve any known toxic solvent and uses readily available chemicals. Since the United States Pharmacopeia tends to use such simple procedures with advantages at all fronts, the above procedure was considered for adoption in the draft for public comments.

Microbial Limits

It is well known that medicinal plant materials generally are contaminated with a great number of bacteria and moulds arising from the soil. Additional contamination is normally caused by practices of harvesting, handling and processing. An index of good harvesting practices and good manufacturing practices can be expected to come from a determination of E. coli and yeast and moulds counts. One would expect the botanical materials to be decontaminated by post-harvest methods (e.g. chemical treatment, gamma irradiation) at the source of export (e.g. in the field, customs shed). However this does not assure that the material would arrive at the port of import free from any contamination. The General Notices of the USP 23-NF 18 under Foreign substances and impurities states “it is manifestly impossible to include in each monograph a test for every impurity, contaminant, or adulterant that might be present, including microbial contamination” (USP 23-NF 18, 1995). In view of this, United States Pharmacopeia monographs would not be expected to specify tests for every known microorganism.

No one would disagree on the pharmacopeial requirements of the absence of pathogens such as Salmonella in the articles of commerce. However in regard to the total aerobic count, mould count and E. coli limits, the end use should dictate these rather than a single set of values for all botanicals. One can tolerate rather generous limits for the above organisms for botanicals that will undergo extensive processing such as boiling followed by extraction with solvents since the processing can be expected to reduce the bio-burden. Conversely where no processing other than boiling with water (e.g. senna leaves for tea preparation) is involved, relatively tighter limits may be called for. In regard to the determination of moulds count there are divergent views and there are arguments that microbiological methods alone are not satisfactory. Instrumental methods for the determination of aflatoxins (e.g. TLC, HPLC) are a sine qua non for testing botanicals since aflatoxins present in plant materials can cause health hazards if absorbed even in very small amounts. A more practical approach for the testing of aflatoxins would be to require it depending on the parts of the plant material used; for example subterranean organs such as rhizomes, roots, stolons are the ones more likely to be contaminated with aflatoxins than the aerial parts such as leaves, or flowers. Alternatively requirements of an aflatoxin test may be specified if the mould counts exceed certain limits irrespective of the plant parts used. In this regard Grady’s comments are noteworthy “One possibility for testing for moulds would be a screening test based on a serological response to Aspergillus antigen, which could be done by contract laboratories and then, only if this were positive, one could look for aflatoxins”.

Given the fact that pharmacopeias have been giving due consideration to harmonization of standards for articles moving in the international commerce, the USP draft monograph on Ginkgo leaves proposed the following limits for microbial contaminants. In turn these limits have been proposed by the World Health Organization: total combined yeasts and moulds counts does not exceed 100μg, total bacterial count does not exceed 10,000μg, and meets the requirements for the absence of Salmonella, E. coli, and Staphylococcus aureus.

Quantitative Determination of “Active” Ingredients

Pharmacopeial monographs of synthetic organic medicinal substances or purified active principles of plant extracts, whose pharmacological activity is predictable, always include a quantitative determination for the active ingredient under Assay. The United States Pharmacopeia tends to use stability-indicating liquid chromatographic procedures involving comparison with a well-defined Reference Standard of known purity (usually 100%) which provide a stable accuracy system transferable to routine quality control work. Pharmacopeial Assay procedures, prior to adoption, are subject to extensive validation requirements-accuracy, specificity, precision, limit of detection, limit of quantitation, linearity, and range-outlined in the chapter Validation of Compendial methods (USP 23-NF 18, 1995). Such validation parameters have been and continue to be successfully used in the validation of pharmacopeial procedures containing multiple active ingredients (perhaps up to five) which are well-defined substances. In dealing with botanicals such as Ginkgo biloba leaves that contain multitudes of compounds — a mixture of flavonol glycosides, terpene trilactones, polysaccharides, etc. — a different approach is called for. This is necessitated by the fact that in many of the botanicals it is not known with certainty what the active ingredients responsible for the therapeutic properties are. Historical data on these have shown in most cases it is not a single compound that is responsible for the biological properties but a group of compounds — some of them may be known and others unknown — due perhaps to synergistic effects. Under these circumstances perhaps the best option open for a pharmacopeial monograph on Ginkgo biloba is to specify a procedure for the quantitative determination of the Content of flavonol glycosides and terpene trilactones since available evidence suggests the assignment of the pharmacological properties of Ginkgo to these groups of compounds. A more practical approach, albeit less desirable, here would be not to place too much emphasis on validation of the chosen procedure for all the parameters mentioned above that are a sine qua non for the Assay of synthetic organic medicinal substances. It is hoped that tests such as identification by either TLC, HPLC, or gas chromatography and colorimetric procedures would identify any decomposed and decomposition products thereby providing good quality control of the material under test.

In view of the different nature of the flavonol glycosides and terpene trilactones, two separate analytical procedures are called for. It is clear from studies reported in the literature that the Ginkgo leaves collected in the spring season contain the highest concentration of flavonol glycosides and a very low concentration of terpene trilactones. Leaves collected in the early fall season contain the lowest concentration of flavonol glycosides while the concentration of the terpene trilactones is the highest. Pharmacopeial monographs would be expected to specify a minimum content for the ingredients which are serious candidates for the observed pharmacological properties and in some cases a minimum-maximum content where dose responses are known.

Content of flavonol glycosides

The general procedure, followed in the past, for the determination of flavonol glycosides in plant materials, involves hydrolysis of the glycosides to free aglycones which are subsequently complexed with aluminium chloride before spectrophotometric measurements. However this spectrophotometric procedure is not specific and quantitation of individual glycosides is not possible. It is reported that the spectrophotometric procedure when applied to Ginkgo biloba leaves was not reproducible, possibly due to the high concentration of proanthocyanidins. This necessitated the search for a reproducible and specific procedure for the quantitative determination of at least the major glycosides — kaempferol, quercetin, and isorhamnetin in Ginkgo biloba leaves. In this connection the liquid chromatographic procedure validated and reported by Sticher et al. appeared to be the most promising one. It was thought this procedure with suitable modifications could serve the compendial requirement of an appropriate procedure and eventually be adopted in the draft. The procedure involves hydrolysis of glycosides to free aglycones which are injected into the HPLC equipped with a 370 nm UV detector and a column containing a packing of octadecylsilane chemically bonded to porous silica or ceramic micro-particles. Quantitation of kaempferol, quercetin and isorhamnetin in the sample under test is achieved by using quercetin as the reference standard. The choice of quercetin as the external reference standard is dictated by closeness of its molar absorptivity to those of the other two. Further quercetin can be expected to be made available for worldwide distribution by a USP Reference Standard program.

Content of terpene trilactones

Several HPLC procedures have been reported in the literature for the determination of ginkgolides and bilobalide using ultraviolet detection at 220 nm. Some researchers have reported the use of refractive index detectors in LC procedures. A more attractive and perhaps a suitable method for routine quality control work appears to be the gas-chromatographic method that involves derivatization and use of a capillary column and flame-ionization detector. In all these publications, ginkgolides and bilobalide isolated from the leaves are subjected to purification procedures prior to their injection into the chromatograph. Recently Camponovo et al. (1995) reported a quantitative determination of ginkgolides and bilobalide by using a liquid chromatograph coupled with an evaporative light scattering detector and a thermospray-mass spectrometer. All of the above methods use pure reference compounds of ginkgolides and bilobalide which are difficult to obtain commercially, are of limited purity and in addition very expensive and this therefore makes these procedures less attractive for compendial adoption. In this regard a more attractive procedure for the quantitative determination of terpene trilactones appears to be the one which uses nuclear magnetic resonance (NMR). The NMR procedure has the advantage that no reference standards of terpene trilactones are required and is based on the comparison of the integral of each H-12 proton of the five Ginkgo terpene trilactones with that of the olefinic protons of maleic acid used as an internal standard. However a 200 MHz NMR spectrometer is very expensive and therefore is not likely to be a routine quality control instrument at the source of supply. At this point of time, it appears that there is no suitable analytical method, from the compendial perspective for the determination of terpene trilactones that could be considered for adoption. This may be an area, from the pharmacopeial perspective, for researchers in Ginkgo biloba to develop a suitable liquid chromatographic procedure using a refractive index detector and a readily available substance for comparison.

Toxic Impurities In Botanical Monographs And Their Limits

The pharmacopeial approach is to specify limits for any identified toxic impurity or impurities in individual monographs and botanical monographs will be no exception. However the botanicals present a totally different challenge to pharmacopeial committees. Toxic impurities may either be inherently present in the botanical under test or could conceivably come from contamination with other plant materials such as related species or due to treatment with chemicals such as ethylene oxide or pesticides. Risks due to the latter, one would hope, could be minimized to a great extent by following certain good harvesting practices, by minimizing the use of pesticides and herbicides and by appropriate identification — botanical and chemical — tests.

Pesticides Limits

In view of the enormous variety of pesticides used world-wide in the cultivation of botanicals and considering these pesticides are toxic, a limit for pesticides is considered appropriate in all botanical monographs. Here a more practical approach would be to require testing for lists of pesticides depending on the country of origin in addition to those that are in use at the importing country. It is to be kept in mind that analysis of pesticides requires special skills and expensive instrumentation facilities. To minimize the analytical work load, efforts on the development of a list of worldwide banned pesticides and a list of only those pesticides that can be used should be speeded up.

Other Requirements

Pharmacopeial monographs on botanicals are specific in defining the article by stating the part of the plant (e.g. leaves). This therefore necessitates specifying a visual test and limits for excluding contamination with other parts of the plant such as twigs, stems etc. as well as other extraneous matter under Foreign organic matter. In addition other pharmacopeial tests such as Residue on ignition, Heavy metals etc. are essential to monitor contamination during harvesting and/or processing.

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

Development of the U.S. Pharmacopeia’s Monograph on Ginkgo Biloba: Conclusion

Development of modern pharmacopeial monographs for botanicals poses the greatest challenge to the USP Committee of Revision and certainly dwarfs the issues and efforts involved in the development of monographs on modern synthetic organic medicinal compounds. The fact that the USP Committee of Revision could develop a draft monograph on Ginkgo biloba leaf attests to the availability in the literature of research publications on the chemical identification and quantitative determination of certain key chemical constituents present in it. The aim of protecting the public health though development and establishment of a pharmacopeial monograph would be hard to achieve without the contributions of those scientists, who have contributed to our understanding of the profile of Ginkgo biloba leaf, and the work of the USP Committee of Revision in reviewing those publications and arriving at appropriate specifications and test procedures for regulatory control. The task of establishing standards for Ginkgo biloba leaf has just begun with the development of a draft monograph. Since United States Pharmacopeia has a continuous revision policy, as new research evidence surfaces and new analytical techniques are developed for better assessment of the quality of Ginkgo biloba, one can expect the USP Committee of Revision to deal with the situation appropriately.

Development of the U.S. Pharmacopeia’s Monograph on Ginkgo Biloba: Appendix

V.Srini Srinivasan “Considerations in the Development of the U.S. Pharmacopeia’s Monograph on Ginkgo Biloba L.” (2006)