Substantial Differences in Proanthocyanidin Contents among Ginkgo biloba Leaf Extracts in Herbal Medicinal Products Obtained from the German Market

Keywords

Herbal medicinal products - EGb 761 - quality - extract characterization - Ginkgo biloba - Ginkgoaceae - Ginkgo biloba leaf extract - proanthocyanidins - PACs - HPLC

Abbreviations

BfArM: Bundesinstitut für Arzneimittel und Medizinprodukte (Federal Institute for Drugs and Medical Devices), The Federal Institute for Drugs and Medical Devices is an independent federal higher authority within the portfolio of the Federal Ministry of Health, Germany.

PACs: proanthocyanidins

Ph. Eur.: European Pharmacopeia
 

Introduction

Ginkgo biloba L. (Ginkgoaceae) leaf dry extracts are active pharmaceutical ingredients in herbal medicinal products in many regions around the globe. Main indications include the improvement of (age-associated) cognitive impairment and quality of life in patients with mild dementia [1], [2], [3], tinnitus [4], and vertigo [5].

The herbal drug “Ginkgo leaf” is described in the monograph of the Ph. Eur. [6] together with the monograph “Ginkgo dry extract, refined and quantified” [7]. The Committee on Herbal Medicinal Products of the European Medicines Agency publishes monographs as a basis for harmonized safety and efficacy assessment of national regulatory authorities in Europe. Such a monograph was published on G. biloba L., folium [8], summarizing the current knowledge about the clinical application of herbal medicinal products containing Ginkgo extracts complying with the Ph. Eur. The concept for classification of extracts in Europe is laid down in the monograph “Herbal Drug Extracts” of the Ph. Eur. [9]. According to the monograph “Ginkgo dry extract, refined and quantified” [7] of the Ph. Eur., Ginkgo extract is classified as a so-called quantified extract where some compounds are acknowledged to contribute to the clinical efficacy of the respective medicinal products and are thus used as active markers for quality control. The clinical efficacy and safety of the extract, which is the actual active ingredient, relies on the overall composition of the extract and consequentially on the particular manufacturing process.

For the quantified Ginkgo extract, the active markers specified in the monographs are the flavonol glycosides (specified at 22.0 to 27.0%) and terpene trilactones (specified at 2.6 to 3.2% for bilobalide and 2.8 to 3.4% for ginkgolides A, B, and C). However, both groups of compounds add up to only approximately 30% of the extract mass balance, whereas the remaining 70% of the compounds are either unspecified or even unknown. According to the concept of the monograph “Herbal Drug Extracts” of the Ph. Eur. in case of quantified extracts, it is implied that additional compounds are relevant for clinical efficacy.

Recently, we published a new method with the objective to enable quantification of an additional group of compounds called proanthocyanidins (PACs), with a remarkable phytochemical complexity in Ginkgo extract [10]. PACs belong to the flavonoid family and are composed of polymerized flavan-3-ol units. These units, also known as catechins, are linked either by carbon-carbon bonds in B-type PACs or with an additional ether bond in A-type PACs. The degree of polymerization of proanthocyanidins can vary, resulting in different molecular weights and chain lengths. The predominant PACs found in G. biloba include B-type proanthocyanidins, which are primarily composed of (epi-)gallocatechin and (epi-)catechin units linked through carbon-carbon bonds at positions 4β → 6 and 4β → 8 [10], [11] (see [Fig. 1]).

Fig. 1 Chemical basic structures of catechin monomers and proanthocyanidins.

Phytochemical properties of PACs were reported in detail [10] for the proprietary Ginkgo leaf extract EGb 761 [12], which is one of the herbal active ingredients most intensely investigated in clinical studies and can thus be regarded as a benchmark for evidence-based herbal medicinal products. A consistent quantity of approximately 7% PACs was found in Ginkgo extract EGb 761 [10]. This portion can be considered as relevant in the mass balance of the extract since it is in the same percentage range compared to, e.g., the specified terpene trilactones. Preclinical studies conducted on PACs from G. biloba [13], [14], [15], [16] reported pharmacological activities such as neuroprotection or antiamnesic activity, with potential relevance for the treatment of neurological disorders, suggesting that PACs might contribute to the overall efficacy profile.

In contrast to the well-controlled quantified markers flavonol glycosides and terpene trilactones, which are part of Ginkgo leaf extract specifications in herbal medicinal products, there is scarce knowledge concerning the contents of PACs in different G. biloba leaf extracts and no systematic comparisons have been reported so far. Given the potential contribution of PACs to the pharmacological activity of Ginkgo extracts, we were interested in assessing the levels of PACs in different herbal medicinal products containing G. biloba leaf extracts in the market. For this reason, a HPLC method for PACs in G. biloba leaf extract was applied after suitable sample preparation to several herbal medicinal products containing G. biloba leaf extracts. Choosing Germany as a benchmark market was due to the high level of regulation for Ginkgo extracts and a number of Ginkgo herbal medicinal products available from a range of marketing authorization holders. All 14 chosen Ginkgo products were purchased in Germany. All these products received marketing authorization as herbal medicinal products by the German authority BfArM [17]. The Ginkgo extracts contained in these products must comply with the monograph “Ginkgo dry extract, refined and quantified” [7], with mandatory specifications on flavonol glycosides and terpene trilactones. Hence, these parameters were not considered in our assessment, and the focus was solely on the PACs.

Our interest was to get an idea to what extent a so far little noticed group of compounds like PACs are present in the products, and to assess batch-to-batch consistency. Therefore, for each product, three batches were analyzed for PACs, covering a period of several years during which these herbal medicinal products were marketed.

Finally, we transferred the analytical method to a third-party laboratory to show suitability of the method for industrial analytical purposes.

Results and Discussion

The results obtained by HPLC for PAC contents in the 14 herbal medicinal products are summarized in [Table 1]. Results obtained by us, and results obtained by the third-party laboratory are indicated.

Table 1 Results for PACs expressed as % values calculated in respect to the declared amount of Ginkgo extract in a single dose of each product; 14 herbal medicinal products with Ginkgo extract and 3 individual batches each. Laboratory 1 is Dr. Willmar Schwabe GmbH & Co. KG and laboratory 2 is the Central Laboratory of German Pharmacists e. V. (Zentrallaboratorium Deutscher Apotheker e. V.).

No	Product	Company*	Batch	Lab. 1	Lab. 2	PACs [%] single	PACs [%] mean
* The term company refers to the entity responsible for placing the respective product on the market according to the valid marketing authorization
1	Gingonin	TAD Pharma GmbH, Cuxhaven	SC5122	x		0.86	0.66
			SD5445	x		0.80
			SH9656		x	0.31
2	Ginkgo-Maren	HERMES Arzneimittel GmbH, Großhessenlohe	92 102C	x		0.34	0.42
			00 901A	x		0.60
			10 702A		x	0.32
3	Ginkgovital Heumann	Heumann Pharma GmbH & Co. KG, Nürnberg	1 183 020 700	x		0.83	0.58
			1 362 720 500		x	0.27
			1 425 210 500	x		0.65
4	Kaveri	KSK-Pharma Vertriebs AG, Berghausen	173 084	x		0.63	0.49
			181 120	x		0.44
			192 057	x		0.40
5	Gingobeta	betapharm Arzneimittel GmbH, Augsburg	1 324 390 100	x		1.00	0.69
			1 324 390 300		x	0.37
			1 394 210 200	x		0.70
6	Doppelherz Ginkgo	Queisser Pharma GmbH & Co. KG, Flensburg	3 001 079	x		0.59	0.60
			50 129		x	0.55
			010 031	x		0.65
7	Binko	Klinge Pharma GmbH, Bad Ems	1 290 980 700		x	0.43	0.47
			1 290 980 600	x		0.48
			1 290 980 500	x		0.50
8	Ginkgo AL	Aliud Pharma GmbH, Laichingen	91 232	x		0.62	0.61
			93 440		x	0.40
			145 223E	x		0.81
9	Ginkgo-ADGC	KSK-Pharma Vertriebs AG, Berghausen	181 122	x		0.32	0.30
			192 081	x		0.36
			132 105		x	0.21
10	Ginkgo AbZ	AbZ Pharma GmbH, Ulm	138 415A		x	0.31	0.66
			143 481B	x		0.55
			144 046A	x		1.13
11	Ginkgo 1 A Pharma	1 A Pharma GmbH, Holzkirchen	KF8018		x	0.38	0.70
			LP5482-AA	x		0.98
			LW8733	x		0.74
12	Ginkobil	Ratiopharm GmbH, Ulm	2 440 220	x		4.80	4.50
			2 740 622		x	4.21
			2 750 622		x	4.48
13	Gingium	Hexal AG, Holzkirchen	LP6994		x	0.95	1.29
			LP7481		x	1.09
			LV9288	x		1.84
14	Tebonin	Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe	0 300 421		x	6.17	5.86
			0 480 122		x	5.77
			0 490 322		x	5.64

Twelve of the fourteen products consistently showed very low PAC contents of approximately 1%, calculated on the basis of the labelled extract quantity, whereas the product with EGb 761 contributed approximately 6% PACs. One product was found to have PAC contents at a level in-between.

Three batches of a product were analyzed, and the mean value is reported. Except for product number 4, which is no longer available on the German market, at least one batch of each product was tested in the third-party laboratory as indicated in the table. For product number 14, only results of this laboratory are reported.

The results from [Table 1] are visualized in [Fig. 2], indicating the origin of each individual data point.

Fig. 2 Results for products numbers 1 to 14 as summarized in [Table 1]. Mean values are indicated by the bars, while the corresponding individual results of single batches are indicated by dots (laboratory 1) or squares (laboratory 2), respectively. Laboratory 1 is Dr. Willmar Schwabe GmbH & Co. KG and laboratory 2 is the Central Laboratory of German Pharmacists e. V. (Zentrallaboratorium Deutscher Apotheker e. V.).

Differences are also obvious in exemplary chromatograms of the 14 products as shown in [Fig. 3]. In all samples, peaks for delphinidin and cyanidin are detectable. Intensities of the peaks correlate to the calculated quantities of PACs. Only product number 14 shows relevant areas, as indicated by the dark-labelled peaks on the top of the figure.

Fig. 3 Overlay of exemplary chromatograms received by reversed-phase C18 HPLC at 530 nm for the 14 herbal medicinal products. After acidic hydrolysis of the PACs in the samples, the anthocyanidins delphinidin and cyanidin were detectable and could be used for quantification. The assignment of products numbers 1 to 14 corresponds to [Table 1].

The majority of randomized placebo-controlled clinical trials on G. biloba leaf extracts have been conducted with EGb 761 [18], [19]. The results of these studies have considerably contributed to the HMPC assessment report and monograph, which is a reference for marketing authorizations in the European Union and other countries. The HMPC monograph includes the same quality parameters as the monograph in the Ph. Eur., in which, however, only approximately one-third of the Ginkgo extract composition is described and specified. Accordingly, the vast majority of the extract components are defined neither in quality nor in quantity. However, it is to be expected that constituents other than the flavonol glycosides and terpene trilactones may also be of importance for the efficacy and safety of G. biloba leaf extracts.

The manufacturing process of the Ginkgo extract EGb 761 has been published in detail [20]. In the course of the last few years, more and more companies have applied for a certificate of suitability (CEP) for their G. biloba leaf extracts to confirm compliance with the monographs of the Ph. Eur. and many certificates have been granted [21]. The parameters relating to the requirements of the Ph. Eur. can be considered compliant in this case. However, the knowledge about other quality aspects is unknown to the public. Therefore, we started a product screening for the quantification of PACs. This was the first time the previously published HPLC method for Ginkgo PACs was used to analyze herbal medicinal products containing Ginkgo leaf extract at the finished product level.

The results for PACs reported in this study show very low amounts in some products and quantitatively relevant amounts on the other side of the spectrum ([Table 1]). In the case of the products with low amounts of PACs, it can be assumed that additional compounds are present to account for the unknown portion in the extract composition.

Most of the products show PAC contents below 1%, whereas product number 14 has a content of approximately 6% and product number 12 is between 1 and 6%. The individual results are consistent for each product. The results obtained by laboratory 1 and laboratory 2 are comparable, which confirms suitability of this methodology and transferability between different laboratories.

The reasons for these differences in the composition of the extracts may be due to differences concerning the drug material used or due to details of the extraction process of the different manufacturers, which are, however, not disclosed to the public in all cases.

The slightly lower content of 6% of product number 14 in comparison to the quantity published before for the corresponding Ginkgo extract EGb 761 [10] is believed to be correlated to the complex sample preparation caused by matrix effects in the herbal medicinal product consisting of a variety of pharmaceutical excipients.

Our analytical comparison demonstrates that in some of the herbal medicinal products containing G. biloba leaf extracts, the contents of PACs is quite significant, up to 6%, and thus in the same quantitative range as the Ph. Eur. specified amount of Ginkgo terpene trilactones (5.4 to 6.6%). At the same time, PACs have been reported to exert various pharmacological effects, such as antioxidant or anti-inflammatory activities. Although it has not yet been established whether and to what extent PACs contribute to the overall pharmacological or clinical efficacy of Ginkgo extracts, a potential impact of different contents in PACs cannot be ruled out [22].

Our findings lead us to the conclusion that the phytochemical composition of Ginkgo herbal medicinal products available on the German market differs substantially. Such differences should also be considered when the clinical evidence generated with one product is transferred to products with an obviously different phytochemical composition, which can be distinguished by a closer look at their phytochemistry. Therefore, we suggest to further characterize the composition of G. biloba leaf dry extracts in general in order to support a serious discussion on whether extracts or their effects can be compared or not. It is in the realm of possibility that many extracts considered “bioequivalent” based on todayʼs requirements of the Ph. Eur. are actually quite different in their composition, and thus may differ in their clinical efficacy and safety.

Materials and Methods

Test samples

Fourteen products containing Ginkgo extracts available in German pharmacies were selected. Thirteen were film-coated tablets that contained either 120 or 240 mg Ginkgo extract as single-dose active pharmaceutical ingredient as indicated on the label and in the package leaflet. One product was a hard gelatin capsule with 120 mg Ginkgo extract. All of them were authorized as herbal medicinal products in Germany by the Federal Institute for Drug and Medical Devices. From each product, three individual batches were purchased in German pharmacies. Among the products, only product number 14 (Tebonin) contained Ginkgo extract EGb 761 [12], the material used in the recently reported development of a new HPLC method for quantification of PACs in Ginkgo extract [10]. Voucher specimens of all products are deposited in the archive of Dr. Willmar Schwabe GmbH & Co. KG.

Solvents, reagents, and chemicals

Methanol (p. a.), o-phosphoric acid, and hydrochloric acid were purchased from Merck. Deionized water was obtained by a water purification system (Evoqua, Water Technologies). For the preparation of the hydrolysis solution, 1 part methanol and 1 part 1.5 M hydrochloric acid were mixed (v/v). The hydrolysis solution must be prepared fresh for each analysis.

Reference standard

Two sources for the reference standard were used: procyanidin B2 with a purity ≥ 98% was purchased from Cayman Chemical Company. Additionally, procyanidin B2 was purchased with a purity of 93% from Phytolab. The reference standard procyanidin B2 was hydrolyzed in the reference solution to form the reference peak cyanidin in situ (see below).

Preparation of standard solutions

The reference standard (procyanidin B2) was weighed exactly into appropriate volumetric flasks and dissolved in the hydrolysis solution to obtain a standard solution with 100 µg procyanidin B2/mL. For an example chromatogram, see Fig. 1S, Supporting Information. This standard solution was diluted with the hydrolysis solution to prepare an additional standard solution with 10 µg procyanidin B2/mL. For an example chromatogram, see Fig. 2S, Supporting Information. The standard solutions were hydrolyzed in parallel and in the same way as described for the sample solutions (see below). The standard solutions appeared clear after hydrolysis and were used directly for HPLC analysis without prior filtration.

Preparation of sample solutions

All products that presented as film-coated tablets were milled without removing the film coating at room temperature in a mixer mill (Retsch MM 400, 30 Hz, 1 min) resulting in a fine powder. For one product that presented as a hard gelatin capsule, the capsule was opened manually, and the filling powder was used directly. The weighed-in quantity for the obtained powder was calculated with reference to the labelled quantity of Ginkgo extract in the individual product. In all cases, quantities of the powder corresponding to 100 mg Ginkgo extract were weighed exactly into 25 mL volumetric flasks, filled up to volume with hydrolysis solution, and dissolved in an ultrasonic bath (SONOREX Super RK510, 160/320 W, 35 kHz, Company Bandelin) for 10 min at room temperature. The solution was stirred for additional 20 min followed by centrifugation (2900 g, 10 min). The obtained test solutions were hydrolyzed in a boiling water bath in tightly closed 10 mL hydrolysis tubes with screw caps remaining above the surface. The time of 45 min was determined to be the optimal duration to yield a complete hydrolysis. After hydrolysis, the closed tubes were cooled in an ice bath and stored at ambient conditions until they reached room temperature (23 °C). The sample solutions obtained were completely clear, and no additional filtration or centrifugation was applied. The sample solutions were transferred to HPLC vials and used directly for HPLC analysis. For chromatograms of product numbers 8 and 14, see Figs. 3S and 4S, Supporting Information.

High-performance liquid chromatograpy analysis

The HPLC analysis was performed as previously described [10] on a Thermo UltiMate 3000 system with autosampler WPS-3000 TRS, pump LPG-3400 RS, detector MWD-3000 RS, and column oven TCC-3000 SD (Thermo) using a Kromasil C18 (5 µm, 4 × 125 mm) column (MZ-Analysentechnik) without a pre-column. The mobile phase consisted of water adjusted to pH 2.0 with o-phosphoric acid 85% solution (phase A) and methanol (phase B). The following gradient was applied at a flow rate of 1.0 mL/min: isocratic from 0.00 – 1.00 min at 60% eluent A, from 1.00 – 8.00 min linear from 60% eluent A to 54.5% eluent A following 8.00 – 9.00 min linear 0% eluent A, 4 min column wash with 0% eluent A, from 13.00 – 13.50 min to 60% eluent A and 6.5 min equilibration period with 60% eluent A, resulting in a total run time of 20.00 min. A visual light detection wavelength of 530 nm, a column temperature of 25 °C, and an injection volume of 10 µL were applied.

The retention time for delphinidin was approximately 2.9 min, for cyanidin 4.6 min, and for pelargonidin 6.3 min. Quantitation was done for the peaks of delphinidin and cyanidin individually using the standard solutions of hydrolyzed procyanidin B2 and both results were added for further calculation. Delphinidin was calculated as cyanidin. Smaller peaks like pelargonidin were not considered in the sample solutions for quantitation due to being off the dynamic detection range.

Since the PAC fraction purified from Ginkgo extract EGb 761 was set to be 100% by convention as described in a previous publication [10], a conversion factor was needed to determine the contents of PACs using procyanidin B2. The applied HPLC assay correlates to a response factor of 2.12 for the water-free fraction of PACs. Results obtained as procyanidin B2 in the products were multiplied by 2.12 and these results are shown in [Table 1] for direct comparison.

Data analysis

Data processing and analysis was carried out using Chromeleon 7.2 SR5 software (Thermo). [Fig. 2] and Fig. S5, Supporting Information, was produced with GraphPad Prism, version 9.3.4, for Windows (GraphPad Software).

External results (laboratory 2)

As already described in the Introduction, besides analysis in the laboratory of the authors, results from an external laboratory were also included in the study. Only in the case of product number 4 was this not possible, as this product was no longer available on the German market at the time the external analysis took place. For product number 14, only external results are reported. For analysis by a third-party laboratory, the method described above was transferred to the Central Laboratory of German Pharmacists e. V. (Zentrallaboratorium Deutscher Apotheker e. V., https://zentrallabor.com/). Comparability of the results of both laboratories was evaluated and the results were considered to be equivalent. The method was applied to challenge the validity of the method and exclude any result bias by independent external reproducibility. These external results are labelled in the table of the individual results ([Table 1]). Variability of results are the consequence of the variability of the batches tested.

Validation data

The method was comprehensively validated addressing the parameters precision, intermediate precision, linearity, accuracy, selectivity, and robustness. As we found significantly different contents of PACs, validation of precision and intermediate precision was done on the 1 and 6% levels, and we selected a representative product for the lower level (number 4) and for the higher level (number 14). The test for precision with n = 6 individual sample preparations showed a relative standard deviation of 8.48% for the lower level and 7.14% for the higher level. The test was repeated after 1 week for evaluation of intermediate precision with new sample preparation and new calibration and showed a relative standard deviation of 7.91% for the lower level and 6.30% for the higher level. Linearity of the method was already tested in a previous publication [10] by linear regression, with 12 concentrations between 0.602 µg procyanidin B2/mL to 120.400 µg procyanidin B2/mL. The correlation coefficient was 0.999 946 with a y-intercept of − 0.0181 area and a slope of 0.0353 area/µg/mL. As a real recovery rate of PACs for the evaluation of accuracy in the products cannot be tested directly due to the indirect principle of the method using the reference standard procyanidin B2, and because the scope of the study was a comparison of products, we also applied an indirect approach similar to the previous validation for the extract level [10]. As the reference substance cyanidin is formed in situ by the acidic hydrolysis of procyanidin B2, accuracy was addressed by the addition of several selected levels of procyanidin B2 to an accurately weighed quantity of the milled herbal medicinal products, which was done for all 14 herbal medicinal products individually. This was necessary, as except for product number 14, no samples of authentic excipient mixtures were available as well as no samples of the individual pure Ginkgo extracts without excipients. The quantity of the milled herbal medicinal products was half, as described in the test procedures, and was supplemented by the addition of procyanidin B2 at five concentration levels each. For the evaluation of accuracy, we focused on a graphical interpretation with a view on the linear relationship of the received peak areas and the x- and y-intercepts, respectively. Fig. 5S (Supporting Information) shows that the method is applicable for all 14 products, independent of the individual composition of pharmaceutical excipients, which offers direct comparability of the obtained results. Selectivity was shown with a view on the peak identification of delphinidin and cyanidin and their good chromatographic separation, as already described in a previous publication [10]. Robustness of the method was tested by variation of the method parameters described in the test procedures, such as column temperature and pH value of mobile phase A within relevant ranges, and was considered to be suitable. Also, the time required for an analysis series was suitable to guarantee stability of reference and sample solutions.

Supporting information

Chromatograms of standard solutions at different concentrations and different samples as well as a graphical figure of the standard addition results are available as Supporting Information.

Contributorsʼ Statement

Sample preparation and analysis: T. Ritter; Design of the study: M. Wurglics, S. Germer; Interpretation of the data: M. Wurglics, S. Germer; Drafting the manuscript: S. Germer

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