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Reviewed: Wohlmuth H, Savage K, Dowell A, Mouatt P. Adulteration of Ginkgo biloba products and a simple
method to improve its detection. Phytomedicine.
2014. doi: 10.1016/j.phymed.2014.01.010.
There is growing concern in the global botanical industry regarding
the adulteration of ginkgo (Ginkgo biloba)
leaf concentrated extracts with exogenously derived (from other plants) free
flavonols (also known as flavonol aglycones) or flavonol glycosides.1,2
Accordingly, several methods have been developed to deal with the adulteration
of ginkgo extracts with admixtures of flavonols or flavonol glycosides. Since
the official monographs (e.g., the methods of the United States Pharmacopeia-National Formulary [USP-NF] and the European Pharmacopoeia [EP]) for quality
control of ginkgo extracts specify a
flavonoid content of 22-27% measured by high-performance liquid chromatography
(HPLC) after hydrolysis* of the flavonol glycosides, it is relatively easy to
bring a low-quality extract into compliance with the specified levels of
flavonoid content by adding pure rutin, pure quercetin, or flavonol-rich
extracts from other plant material. An overview of the widespread and unethical
practice was published in 2008.3
Despite the increased awareness of the problem in the dietary
supplement industry, both in the United States and elsewhere, the adulteration
of ginkgo extracts has persisted, as evidenced by more recent publications.4,5
Possible solutions for detecting such adulteration are the following methods:
(1) calculation of quercetin (Q) to kaempferol (K) and isorhamnetin (I) ratios
(ginkgo material should have a Q:K ratio between 0.8 and 1.2 and a Q:I ratio ≥
0.1 according to the USP); (2) the use of a HPLC fingerprint of the
non-hydrolyzed extract; or (3) a combination of HPLC fingerprints and
ultraviolet (UV) spectral images analyzed by statistical means.3-6
The newly-published method by Australian researchers Hans Wohlmuth,
PhD, and colleagues of Southern Cross University in Lismore describes a simple
addition to the current pharmacopeial methods. (Dr. Wohlmuth is the research
and development manager at Integria Healthcare in Australia and is a recently
appointed member of the ABC Advisory Board.) The authors analyzed ginkgo leaf
material and commercial extracts before and after hydrolysis using the HPLC
conditions detailed in the USP36-NF31 (the combined 36th edition of the United States Pharmacopeia and the 31st
edition of the National Formulary).6
Pharmacopeial-grade ginkgo extract is made by concentrating ginkgo material by
approximately 50 times, thereby producing the compendially required levels of
ginkgolides and bilobalide (6-7%; these compounds are unique to ginkgo) and the
flavonol levels of 22-27% (these flavonols are relatively ubiquitous in
plants). Adulteration with exogenously obtained flavonols creates the false
impression that the adulterated extract has been concentrated to the level of
the pharmacopeial-grade extract.
Non-hydrolyzed ginkgo leaf raw material contains very little to no
detectable quercetin, kaempferol, or isorhamnetin (even a low-quality
manufacturing process is not expected to yield large amounts of free flavonols,
according to the authors); therefore, substantial amounts of these flavonols
are an indicator of adulteration. The total flavonoid content can be calculated
using the difference between pre- and post-hydrolysis aglycone content. An advantage
of using this approach is the ability to use the same HPLC conditions that are
specified in the pharmacopeial monographs, so a quality control laboratory does
not have to invest in a new analytical instrument or a new HPLC column. Samples
are prepared by sonicating (sonication is a process in which energy in the form of
ultrasonic waves is applied to a material in a solvent to accelerate extraction
or dissolution) 300-1000 mg of commercial product in 50 mL ethanol for 15
minutes.
The authors discovered admixtures of free quercetin and kaempferol in
three of the eight commercial samples that were analyzed, even if the flavonoid
contents were within the specifications set by the USP36-NF31. In addition, the
three adulterated samples contained genistein, an isoflavone that has not been
found in ginkgo leaves but has been reported in one instance in a ginkgo leaf
extract of Chinese provenance.7 The authors hypothesize that the
genistein could come from extracts of the fruit of the Japanese pagoda tree (Styphnolobium japonicum, syn: Sophora japonica), a plant that previously
has been identified as a likely adulterant of ginkgo extracts.8
Comment: The proposed new
method is a quick and easy way to detect the admixture of free flavonols added to
ginkgo extracts and is suggested as an amendment to current pharmacopeial
methods. Large amounts of quercetin, kaempferol, or isorhamnetin in
non-hydrolyzed materials indicate adulteration. One drawback of the method is
that adulteration with pure flavonol glycosides (e.g., pure rutin, or flavonol
glycoside-rich herbal extracts such as those of buckwheat [Fagopyrum esculentum] or Japanese pagoda) will remain undetected.
In such cases, the determination of the Q:K and Q:I ratios outlined in the
USP36-NF31 provides a solution. In addition, the published HPLC fingerprinting
methods4,5 can be used to obtain a definitive answer regarding the
authenticity of the ginkgo extract.
*Hydrolysis is a chemical reaction used in this case to remove the
sugar moieties from the flavonol aglycones quercetin, keampferol, and
isorhamnetin. The result is the reduction of a highly complex chromatographic
profile to a simple HPLC trace consisting of only three major peaks. The total
flavonoid contents are obtained using a mathematical function that converts the
amount of flavonol aglycone to the corresponding glycosides.
—Stefan Gafner, PhD
References
1. Ethical Naturals,
Inc. Ginkgo biloba extract: brand QC
analytical report. 2006.
2. Tawab M, Krzywon
M, Schubert-Zsilavecz M. Nahrungsergänzungsmittel mit Gingko unter der Lupe.
Pharm Ztg Online. 2010;20. Available here.
3. Myers S. Adulteration stifles the Ginkgo biloba market. Nat Prod Insider. October 2008. Available here.
4. Chandra A, Li Y, Rana J, et al. Qualitative categorization of supplement
grade Ginkgo biloba leaf extracts for authenticity. J Funct Foods.
2011;3(2):107-114.
5. Harnly JM, Luthria D, Chen P. Detection
of adulterated Ginkgo biloba supplements using chromatographic and
spectral fingerprints. J AOAC Int.
2012;95(6):1579-1587.
6. United States
Pharmacopeial Convention. Ginkgo biloba. In: USP 36-NF 31. Rockville,
MD: United States Pharmacopeial Convention. 2013.
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