Printer Friendly

Adulteration of Ginkgo biloba products and a simple method to improve its detection.

ABSTRACT

Extracts of ginkgo (Ginkgo biloba) leaf are widely available worldwide in herbal medicinal products, dietary supplements, botanicals and complementary medicines, and several pharmacopoeias contain monographs for ginkgo leaf, leaf extract and finished products. Being a high-value botanical commodity, ginkgo extracts may be the subject of economically motivated adulteration. We analysed eight ginkgo leaf retail products purchased in Australia and Denmark and found compelling evidence of adulteration with flavonol aglycones in three of these. The same three products also contained genistein, an isoflavone that does not occur in ginkgo leaf.

Although the United States Pharmacopeia--National Formulary (USP-NF) and the British and European Pharmacopoeias stipulate a required range for flavonol glycosides in ginkgo extract, the prescribed assays quantify flavonol aglycones. This means that these pharmacopoeial methods are not capable of detecting adulteration of ginkgo extract with free flavonol aglycones.

We propose a simple modification of the USP-NF method that addresses this problem: by assaying for flavonol aglycones pre and post hydrolysis the content of flavonol glycosides can be accurately estimated via a simple calculation. We also recommend a maximum limit be set for free flavonol aglycones in ginkgo extract.

Keywords:

Ginkgo biloba

Ginkgo extract

Adulteration

Quality control

Ginkgo flavonol glycosides

Genistein

Introduction

Extracts of ginkgo (Ginkgo biloba L.) leaf are sold worldwide as the active ingredient of numerous dietary supplements, botanicals, herbal medicinal products and complementary medicines. Indeed, ginkgo is currently one of the most widely sold medicinal plants, and the global market for ginkgo has been estimated at more than US$700 million (Euromonitor International Ltd., 2009). In the United States, the most recent (2012) data show the retail market for ginkgo products to be worth US$30 million (Lindstrom et al., 2013).

Ginkgo is also one of the most intensely studied medicinal plants, with more than 3000 scientific papers published on the topic between 2001 and 2009 alone (van Beek and Montoro, 2009). Ginkgo leaf extracts are recommended for a range of conditions, including cerebral insufficiency, vertigo and tinnitus of vascular origin, and peripheral arterial disease (Blumenthal, 2003; Bone and Mills. 2013).

The pharmacologically active compounds in ginkgo leaf are considered to be flavonol glycosides (quercetin, kaempferol and isorhamnetin being the principal aglycones) and terpene lactones (bilobalide and ginkgolides). Most ginkgo leaf extracts on the market are produced by selective, multi-step extraction processes involving organic solvents and carry quantitative claims concerning their content of flavonol glycosides and terpene lactones. Accordingly, most ginkgo leaf extracts are more high-tech and high-cost than typical botanical extracts.

Botanical raw materials including extracts present special challenges in terms of quality control and assurance due to their chemical complexity and inherent natural variability. The most fundamental aspects of quality assurance for such materials are to ensure the correct morphological part(s) from the right botanical taxon is used, and that the material is not adulterated with other botanical or extraneous material. Adulteration, either accidental or intentional and economically motivated, is a well-known issue for botanicals, and one that potentially can jeopardise not only the quality but also the safety of the finished product (Khan, 2006; Walker and Applequist, 2012). The potential safety issues associated with adulterated or sub-standard ginkgo extracts have been highlighted by recent toxicology and carcinogenicity studies using such inferior materials, while other studies using pharmaceutical quality ginkgo extract have found it to be safe (Koch et al., 2013; Krenn et al., 2013).

Pharmacopoeial monographs play an important role in the quality assurance of botanicals and herbal medicinal products (Vlietinck et al., 2009). Monographs for ginkgo raw materials (leaf and extract) can be found in various pharmacopoeias, including the United States Pharmacopoeia-National Formulary (USP-NF) (United States Pharmacopeial Convention, 2013), the European Pharmacopoeia (EP) and the British Pharmacopoeia (BP) (British Pharmacopoeia Commission, 2012). The USP-NF monograph for Powdered Ginkgo Extract and the BP/EP monograph for Refined and Quantified Ginkgo Dry Extract specify a flavonoid content of 22-27%, calculated as flavonol/flavone glycosides and a maximum content of 5 ppm for ginkgolic acids (putative allergens). In addition, specifications are provided for the terpene lactones, bilobalide and ginkgolide A, B and C, but the ranges for these differ between the USP-NF and the BP/EP. The USP-NF also provides monographs for Ginkgo Tablets and Ginkgo Capsules.

Here we report on ginkgo retail products found to be adulterated with free flavonol aglycones and also containing the isoflavone genistein, which is not native to ginkgo. We demonstrate that current pharmacopoeial methods are inadequate for the detection of this type of adulteration, and we propose a simple modification of the USP-NF method that addresses this problem.

Materials and methods

Plant materials and botanicals

Five samples of dried Ginkgo biloba leaf were obtained from commercial suppliers. These leaf samples came from ginkgo cultivated in China (3), New Zealand (1) and Australia (1). They were authenticated by an experienced pharmacognosist (FEW.), and voucher materials were deposited in the Medicinal Plant Herbarium (PHARM) at Southern Cross University.

Eight retail products containing Ginkgo biloba as the sole active ingredient were purchased in Australia (6) and Denmark (2). Four of these were tablets and four were capsules.

Chemicals and reagents

Water was obtained from an in-house Milli-Q system (Millipore, Billerica, MA, USA). Methanol (HPLC grade) was obtained from Merck (Kilsyth, VIC, Australia), ethanol (AR grade) from Chem-Supply (Gillman, SA, Australia), acetonitrile from Scharlau (Sentmenat, Spain), phosphoric acid from Ajax Finechem (Sydney, Australia), and hydrochloric acid (AR grade), trifluoroacetic acid, dimethyl sulfoxide (DMSO), kaempferol (97.8%) and genistein (>98%) from Sigma-Aldrich (Sydney, Australia). Quercetin dihydrate (98.5%) was obtained from Chromadex (Irvine, CA, USA) and isorhamnetin (>98%) from Chengdu Biopurify Phytochemicals (Chengdu, Sichuan, China).

Standard preparation

Reference standards (quercetin, kaempferol and isorhamnetin) were dissolved in DMSO at a concentration of 2.0 mg/mL, then serially diluted in methanol using a Hamilton Microlab 500 Diluter (Reno, NV, USA). Genistein was dissolved in methanol.

Sample preparation

Ginkgo leaf samples were ground to a fine powder in a Retsch MM301 Mixer Mill (Haan, Germany). Approximately 5.5 g of powdered leaf material was placed in a 250-ml round bottom flask with 50 ml ethanol and 20 ml Milli-Q water and sonicated for 15 min (Soniclean Ultrasonic bath, Thebarton, SA, Australia). To achieve hydrolysis of flavonol glycosides, 8 ml of 37% hydrochloric acid was added (with boiling chips) and the mixture refluxed at moderate temperature in a fume-hood for 2 h 15 min. Once cooled, the solution was transferred quantitatively to a 100-ml volumetric flask and diluted to volume with Milli-Q water. Samples not hydrolysed were treated identically, except for the addition of hydrochloric acid.

Tablets were extracted by combining a quantity of 20 and grinding them to a fine powder in a Retsch MM301 Mixer Mill. Depending on the label claim for flavonol glycoside content, between 300 and 1000 mg of the powder was extracted by sonication, as detailed for the leaf samples. Capsules were opened and their content extracted in the same way as the tablets. All samples were extracted in triplicate.

HPLC

Reverse-phase HPLC analysis was performed on an Agilent (Palo Alto, CA, USA) 1100 HPLC system fitted with a Phenomenex (Torrance, CA, USA) Synergi C18 4 [micro]M (250 mm x 4.6 mm i.d.) column, using an in-house validated method based on the USP34-NF29. Mobile phase A consisted of 0.5% aqueous phosphoric acid; mobile phase B consisted of methanol. The gradient eluting mobile phase was A/B (60:40, v/v) to A/B (50:50, v/v) over 40 min. This was followed by a 5 min column wash with A/B (5:95) and a 5 min equilibration period with A/B (60:40) prior to the next injection. Mobile phase was pumped at 1.2m!/min, the column temperature 40[degrees]C, and the injection volume was 10 [micro]l. Data were collected using a UV/visible light diode array detector collecting absorption spectra from 200 to 400 nm with quantification performed at 270 nm. System control and data evaluation were achieved using ChemStation for HPLC software. Limits of detection for quercetin, kaempferol and isorhamnetin were 0.625[micro]g/ml, 0.25 [micro]g/ml and 0.07 [micro]g/ml, respectively. The range for precision, accuracy and linearity was 0.0025-0.5 mg/ml for quercetin, 0.000625-0.5 mg/ml for kaempferol and 0.00028-0.14 mg/ml for isorhamnetin.

LC-MS

LC-MS analysis was performed on an Agilent (Palo Alto, CA, USA) 1100 LC/MSD system equipped with an atmospheric pressure chemical ionisation (APC1) source and fitted with a Phenomenex (Torrance, CA, USA) Luna C18 3 [micro]M (100 mm x 4.6 mm i.d.) column. Mobile phase A consisted of water and mobile phase B of acetonitrile, both with 0.005% trifluoroacetic acid added. The gradient eluting mobile phase was A/B (90:10, v/v) to A/B (5:95, v/v) over 18 min followed by a 3 min column wash with A/B (5:95), to A/B (90:10) over 3 min and a 5 min equilibration period with A/B (90:10). Rate of mobile phase flow was 0.750 ml/min, the column temperature was 40[degrees]C, and the injection volume was 5 [micro]l

MS parameters in the positive ionisation mode were: Vcap 3000 V, nebuliser 60psig, drying gas flow rate 5.01/min, gas temperature 350[degrees]C, corona 4.0[micro]A, vaporiser 350[degrees]C, scan range 100-1500m/z, step size 0.15m/z, peak width 0.1 min, time filter enabled, fragmenter 150 V. System control and data evaluation were performed with ChemStation for LC/MS software.

Calculation offlavonol glycoside content

Flavonol glycoside content was calculated according to the method provided in the Ginkgo Tablet and Ginkgo Capsule monographs in USP36-NF31. Briefly, this method involves the acid hydrolysis of an extract of the sample, quantification of quercetin, kaempferol and isorhamnetin by HPLC against reference standards, and calculation of the quantity (in mg) of each flavonol glycoside in the sample using the formula:

[Glycoside.sub.quantity] = ([r.sub.u/r.sub.s] x [C.sub.s] x F x 50

where [r.sub.u] is the peak area of the relevant aglycone in the sample solution, [r.sub.s] is the peak area of the aglycone in the corresponding standard solution, [C.sub.s] is the concentration (mg/mL) of the aglycone in the standard solution, and F is the factor used to convert each aglycone into a flavonol glycoside with a mean molecular mass of 756.7 (2.504 for quercetin, 2.588 for kaempferol, and 2.437 for isorhamnetin). The total quantity of flavonol glycosides in the sample is calculated by summing the values for quercetin, kaempferol and isorhamnetin glycosides.

Results

The content of quercetin, kaempferol and isorhamnetin in unhydrolysed and hydrolysed extracts of five ginkgo leaf samples, four tablet samples and four capsule samples is shown in Table 1. None of the unhydrolysed leaf samples contained detectable levels of any of these flavonol aglycones. The quercetin content in the hydrolysed leaf samples varied almost five-fold (range 0.87-4.30 [per thousand] w/w), the kaempferol content more than three-fold (range 1.35-4.29 [per thousand]) and the isorhamnetin content 1.6-fold (range 0.36-0.59 [per thousand]). The hydrolysed extract of the Australian grown leaf sample LI-AU had the highest content of all three flavonols (total 9.17 [per thousand]), followed by the New Zealand sample (6.97 [per thousand]) and the three leaf samples from China (2.59-2.76 [per thousand]).

Three of the tablets and two of the capsules contained no or very low levels of free flavonols when not subjected to acid hydrolysis. However, unhydrolysed samples of one tablet (T3-AU) and two capsules (C2-AU and C3-AU) contained high levels of quercetin (range 2.48-7.42 [per thousand]) and kaempferol (range 1.84-7.50 [per thousand]), whereas the same samples contained only very low concentrations (0.01 [per thousand]) of isorhamnetin. As shown in Table 2, the concentration of free flavonol aglycones in these three samples comprised up to 51% (quercetin) and 41% (kaempferol) of the concentration measured in the same products post hydrolysis. Whether or not high levels of quercetin and kaempferol were present pre hydrolysis was obvious by visual inspection of HPLC chromatograms (Fig. 1).

The flavonol glycoside content of the retail products, calculated both pre and post hydrolysis using the formula provided for Content of Flavonol Glycosides in the USP-NF monographs, is shown in Table 3. As is evident from this table, the presence of significant amounts of flavonol aglycones in three of the products resulted in the flavonol glycoside content being overestimated between 29.0 and 40.9% when applying the pharmacopoeial method.

As is also shown in Table 3, 75% of the products analysed contained within [+ or -] 10% of their label claim for flavonol glycosides, when the pharmacopoeial method was used. One product contained 14% less and another 34% more than the label claim by this method.

Fig. 2 shows the ratios between kaempferol and quercetin and between isorhamnetin and quercetin in the hydrolysed samples of retail product. The minimum ratios stipulated in the monographs are shown as horizontal bars (kaempferol to quercetin NLT 0.7; isorhamnetin to quercetin NLT 0.1). All products conformed to the relevant USP-NF monographs with respect to flavonol ratios after acid hydrolysis.

All samples were examined for the presence of genistein by LC-DAD and LC-MS using positive selective ion monitoring (Fig. 3). Genistein was identified only in the three samples (T3-AU, C2-AU and C3-AU) shown to contain high levels of free quercetin and kaempferol (Table 2). The presence of genistein was confirmed by re-analysis by LC-DAD and LC-MS after spiking with a genistein reference standard. Genistein was also readily observable in the HPLC chromatogram as a peak located between the peaks for quercetin and kaempferol (Fig. 1).

Discussion

Ginkgo extract is used in numerous dietary supplements, botanicals, herbal medicinal products and complementary medicines around the world. Being a high-value botanical commodity, ginkgo extract is a target for economically motivated adulteration, and suspected adulteration with the flavonols quercetin and kaempferol, the flavonol glycoside rutin (quercetin 3-rutinoside) or with other plant extracts containing flavonols has been reported (Chandra et al., 2011; Harnly et al., 2012; He and Roller, 2011; Liu et al., 2005; Sloley et al., 2003; Tawab, 2010; van Beek and Montoro, 2009).

The pharmacopoeial methods for determining the content of flavonol glycosides in ginkgo extract and products differ somewhat between the USP-NF and BP/EP monographs, but in both cases the glycoside content is calculated from the content of free flavonol aglycones, quantified by HPLC following acid hydrolysis. The fact that flavonol glycosides are not quantified directly, but instead calculated based on the aglycone content after hydrolysis, means that adulteration with flavonol aglycones may not be detectable using the current pharmacopoeial methods. In fact, the inadequacy of calculating the flavonol glycoside content in ginkgo solely on the basis of the aglycone content determined post hydrolysis has previously been identified by other workers (Liu et al., 2005; Sloley et al., 2003).

As an aid to detecting adulteration of ginkgo extracts, the USP-NF monographs for Powdered Ginkgo Extract (but not the equivalent EP/BP monograph), Ginkgo Tablets and Ginkgo Capsules include specifications for the relative abundance of quercetin, kaempferol and isorhamnetin after acid hydrolysis. These criteria are provided in Identification test B in the monograph for Powdered Ginkgo Extract but form part of Identification test A in the monographs for Ginkgo Tablets and Ginkgo Capsules. Since November 2011, the USP-NF monographs have stipulated a kaempferol to quercetin HPLC peak ratio of not less than 0.7 and an isorhamnetin to quercetin peak ratio of not less than 0.1 (United States Pharmacopeial Convention, 2013). However, while this test may assist in the detection of some types of adulteration, it cannot effectively detect adulteration with free flavonol aglycones.

Adulteration of ginkgo products

In the present study, we have shown that five ginkgo leaf samples, sourced from three different countries, contained levels of quercetin-, kaempferol- and isorhamnetin glycosides that varied up to five-fold between samples. However, none of these leaf samples contained detectable levels of free quercetin, kaempferol or isorhamnetin aglycones. Consistent with the virtual absence of free flavonols in the crude raw material, five retail products with ginkgo extract as the sole active ingredient contained no or negligible amounts of free flavonols. One of these products (Tl-AU) was Tebonin[R] (Schwabe Pharmaceuticals), which as its active ingredient contains EGb761[R], arguably the classic gold standard ginkgo leaf extract (for which Schwabe holds a worldwide patent). These results indicate that neither crude ginkgo leaf nor quality ginkgo leaf extracts would be expected to contain free flavonols, except in very low concentrations in some cases.

In contrast, three other retail products (T3-AU, C2-AU and C3-AU) contained significant levels of free quercetin (2.5-7.4[per thousand] w/w) and kaempferol (1.8-7.5 [per thousand]). The levels of isorhamnetin in these products were considerably lower, ranging from 0.01 to 0.11[per thousand]. The same three products were shown to contain the isoflavone genistein (4',5,7-trihydroxyisoflavone). Isoflavones occur predominantly in leguminous plants (family Fabaceae), but have been reported from 60 other, mostly angiosperm families (Lapcik, 2007). In addition to the Fabaceae, genistein has been reported from the monocotyledonous family Iridaceae and the dicotyledonous families Moraceae, Rosaceae, Myristicaceae and Rutaceae (Reynaud et al., 2005). Isoflavones including genistein have never been reported from Ginkgo biloba plant material, but there is one published report of genistein isolated from a Chinese ginkgo leaf extract (Wang et al., 2007). The chemistry of ginkgo leaf and leaf extracts is exceedingly well known (van Beek, 2002; van Beek and Montoro, 2009), and we believe there can be little doubt that this report concerns an adulterated ginkgo extract.

The presence of genistein combined with significant levels of free quercetin and kaempferol in three of the retail products analysed provides compelling evidence of adulteration of these products (all of which listed Ginkgo biloba leaf extract as the sole active ingredient). While we have not been able to identify the adulterant, an extract containing both quercetin and kaempferol (but not isorhamnetin) as well as genistein (or their glycosides) would appear most likely. One such candidate is Styphnolobium japonicum (L.) Schott, (syn. Sophora japonica L), a well-known Chinese medicinal plant known as Japanese pagoda or Chinese scholar tree. Two botanical drugs derived from this plant are listed in the Pharmacopoeia of the People's Republic of China: huaihua, comprising the dried flower and flower bud, and huaijiao, which consists of the dried fruit (Chinese Pharmacopoeia Commission, 2005). The pericarp of this species contains a number of genistein, quercetin and kaempferol glycosides (Qi et al., 2007), and it has previously been identified as a likely adulterant of ginkgo extracts (Chandra et al., 2011; He and Roller, 2011; Tawab, 2010). The adulterated ginkgo products analysed by us contained genistein, quercetin and kaempferol in free aglycone form, so if Styphnolobium japonicum were indeed the adulterant, the extract would likely have undergone processing leading to the hydrolysis of the flavonoid glycosides.

Thus, we have identified three adulterated ginkgo retail products, all of which conformed to the relevant USP-NF monographs (Ginkgo Tablets or Ginkgo Capsules) in terms of ratios of HLPC peak areas between kaempferol and quercetin and between isorhamnetin and quercetin in hydrolysed samples (Fig. 2).

Improved quality control for ginkgo

According to the respective USP-NF monographs, ginkgo tablets and capsules must be prepared from Powdered Ginkgo Extract that contains between 22.0 and 27.0% flavonol glycosides. The adulterated products analysed by us contained significant levels of free quercetin and kaempferol. Because the pharmacopoeial method for calculating flavonol glycoside content is based on the quantity of flavonol aglycones measured after acid hydrolysis, the actual flavonol glycoside content in these products was overestimated by between 29% and 41% (Table 3). In other words, these products did not meet the pharmacopoeial standard for flavonol glycoside content, but this could not be detected by the prescribed pharmacopoeial method. Whether flavonoids are present in aglycone or glycoside form can have profound impact on their pharmacokinetic and pharmacodynamic characteristics (Murato and Terao, 2003; Walle, 2004), with obvious potential implications for the safety and efficacy of the product.

In order to address this problem, we propose a simple modification of the pharmacopoeial test for flavonol glycosides. This involves assaying for flavonol aglycone content (as per the pharmacopoeial monograph) before and after acid hydrolysis and basing the calculation of flavonol glycoside content on the difference between pre and post hydrolysis aglycone content:

[A.sub.(glycosides)] = [A.sub.(post-hydrolysis)] - [A.sub.(pre-hydrolysis)]

where [A.sub.(glycosides)] is the amount of flavonol aglycones derived from glycosides in the extract or product (and the value the pharmacopoeial test aims to determine), [A.sub.(post-hydroiysis)] is the total amount of flavonol aglycones after acid hydrolysis (representing the sum of free aglycones plus aglycones from glycosides) and [A.sub.(pre-hydrolysis]) is the amount of free flavonol aglycones in the unhydrolysed extract or product.

Applying this modified test ensures that only the amount of flavonol aglycones that exist in glycoside form in the extract or product is used in the calculation of flavonol glycoside content. Any free aglycones present in the extract or product are not included in this calculation. This approach prevents the overestimation of flavonol glycoside content that will result by applying the current pharmacopoeial method to gingko extracts or products that have been adulterated with free flavonol aglycones (illustrated by three of the products we analysed). The proposed change to the pharmacopoeial method, while adding an additional step, is both cheap and simple and provides for an accurate determination of the flavonol glycoside content. Accordingly, we believe this change to the method would represent a significant improvement to the USP-NF monographs for Powdered Ginkgo Extract, Ginkgo Tablets and Ginkgo Tablets, and to equivalent monographs in other pharmacopoeias, including the EP and the BP.

Our analysis of five commercial samples of ginkgo leaf grown in three different countries found no evidence (within the limits of detection) of quercetin, kaempferol or isorhamnetin in aglycone form. Free aglycones have been reported from unhydrolysed ginkgo leaf, but only in low concentrations (Song et al., 2010; van Beek and Montoro, 2009). These aglycones were absent from the five unadulterated products tested with the exception of quercetin, which was detected in three of the products at very low levels (0.013-0.036[per thousand] w/w). In unadulterated material, an increase in aglycone levels (with accompanying decrease in flavonol glycoside levels) is indicative of degradation during extraction, formulation or storage and thus undesirable (Sticher et al., 2000). On this basis we also suggest that a suitable maximum level for free flavonol aglycones be set for ginkgo leaf extract, e.g. 0.5% w/w. Such a limit would further contribute to the quality assurance of ginkgo extracts, including limiting adulteration with flavonol aglycones from extraneous sources.

The presence of the isoflavone genistein, a compound not occurring naturally in Ginkgo biloba, in the three products that were adulterated with flavonol aglycones suggests that the adulterant was a plant extract that contains flavonols as well as genistein. We therefore also recommend that manufacturers further improve the rigour of their quality control processes for ginkgo raw materials (especially extracts) by assaying for the presence of genistein and rejecting as adulterated ginkgo materials found to contain this extraneous compound.

Conclusions

The need for ongoing vigilance in relation to the quality of herbal medicinal products and botanicals has been highlighted by our finding that three out of eight ginkgo retail products tested showed clear evidence of adulteration. Pharmacopoeial monographs for botanical raw materials and dose forms, such as those in the USPNF, EP and BP, play an essential role in assuring the quality of such products. Here we have demonstrated that the tests currently prescribed in these monographs do not provide for the detection of adulteration of ginkgo extracts and products with free flavonol aglycones.

As a solution to this specific problem, we have proposed a simple modification of the USP-NF test for Content of Flavonol Glycosides that allows for the accurate estimation of these compounds, thus avoiding the overestimation that will result from the presence of free flavonol aglycones (likely of extraneous origin) when applying the current test.

We believe that the proposed test, along with the setting of a maximum acceptable level for free flavonol aglycones and testing for the presence of genistein (an extraneous compound) would make a significant contribution to the quality assurance of gingko leaf extracts and products.

ARTICLE INFO

Article history:

Received 14 November 2013

Received in revised form

27 November 2013

Accepted 26 January 2014

References

Blumenthal, M., 2003. The ABC Clinical Guide to Herbs. American Botanical Council, Austin, TX.

Bone, K., Mills, S., 2013. Principles and Practice of Phytotherapy: Modern Herbal Medicine, second ed. Churchill Livingstone, Edinburgh.

British Pharmacopoeia Commission, 2012. British Pharmacopoeia. The Stationary Office, London.

Chandra, A., Li, Y., Rana, J., Persons, K., Hyun, C, Shen, S., Mulder, T., 2011. Qualitative categorization of supplement grade Ginkgo biloba leaf extracts for authenticity. Journal of Functional Foods 3, 107-114.

Chinese Pharmacopoeia Commission, 2005. Pharmacopoeia of the People's Republic of China, English ed. People's Medical Publishing House, Beijing.

Euromonitor International Ltd., 2009. Country Sector Briefing, Vitamins and Dietary Supplements. Euromonitor International Ltd., London.

Harnly, J.M., Luthria, D., Chen, P., 2012. Detection of adulterated Ginkgo biloba supplements using chromatographic and spectral fingerprints. J AOAC Int 95, 1579-1587.

He, K., Roller, M., 2011. Identification of adulterated compounds in G. biloba extract. In: Dayan, N., Kromidas, L. (Eds.), Formulating, Packaging and Marketing of Natural Cosmetic Products. Wiley, Hoboken, NJ, pp. 378-385.

Khan, I.A., 2006. Issues related to botanicals. Life Sciences 78, 2033-2038.

Koch, E., Noldner, M., Leuschner.J., 2013. Reproductive and developmental toxicity of the Ginkgo biloba special extract EGb 761 in mice. Phytomedicine 21, 90-97.

Krenn, L, Bilia, A.R., do Ceu Costa, M., Hook, L, Steinhoff, B., Wegener, T., 2013. Now Ginkgo--10 years after Cimicifuga? Phytomedicine 21, 98-99.

Lapcik, O., 2007. Isoflavonoids in non-leguminous taxa: a rarity or a rule? Phytochemistry 68, 2909-2916.

Lindstrom, A., Ooyen, C, Lynch, M.E., Blumenthal, M., 2013. Herb supplement sales increase 5.5% in 2012: herbal supplement sales rise for 9th consecutive year: turmeric sales jump 40% in natural channel. Herbalgram 99.

Liu, C, Mandai, R., Li, X.F., 2005. Detection of fortification of ginkgo products using nanoelectrospray ionization mass spectrometry. Analyst 130, 325-329.

Murato, K., Terao, J., 2003. Antioxidative flavonoid quercet implication of its intestinal absorption metabolism. Archives of Biochemistry Biophysics 417, 12-17.

Qi, Y., Sun, A., Liu, R., Meng, Z., Xie, H., 2007. Isolation and purification of flavonoid and isoflavonoid compounds from the pericarp of Sophora japonica L. by adsorption chromatography on 12% cross-linked agarose gel media. Journal of Chromatography A 1140, 219-224.

Reynaud, J., Guilet, D., Terreux, R., Lussignol. M., Walchshofer, N., 2005. Isoflavonoids in non-leguminous families: an update. Natural Products Reports 22, 504-515.

Sloley, B.D.. Tawfik, S.R., Scherban, K.A., Tam, Y.K., 2003. Quality control analyses for ginkgo extracts require analysis of intact flavonol glycosides. Journal of Food and Drug Analysis 11, 102-107.

Song, J., Fang, G., Zhang, Y., Deng, Q., Wang, S., 2010. Fingerprint analysis of Ginkgo biloba leaves and related health foods by high-performance liquid chromatography/electrospray ionization-mass spectrometry. Journal of AOAC International 93, 1798-1805.

Sticher, O., Meier, B., van Beek, T.A., 2000. The analysis of ginkgo flavonoids. In: van Beek, T.A. (Ed.), Ginkgo biloba. Harwood Academic Publishers, Amsterdam, pp. 179-202.

Tawab, M., 2010. Nahrungserganzungsmittel mit Ginkgo unter der Lupe, Pharmazeutische Zeitung Online. Govi-Verlag.

United States Pharmacopeial Convention, 2013. United States Pharmacopoeia and National Formulary (USP36-NF31). United States Pharmacopeial Convention, Rockville, MD.

van Beek, T.A., 2002. Chemical analysis of Ginkgo biloba leaves and extracts. Journal of Chromatography A 967, 21-55.

van Beek, T.A., Montoro, P., 2009. Chemical analysis and quality control of Ginkgo biloba leaves, extracts, and phytopharmaceuticals. Journal of Chromatography A 1216, 2002-2032.

Vlietinck, A., Pieters, L, Apers, S., 2009. Legal requirements for the quality of herbal substances and herbal preparations for the manufacturing of herbal medicinal products in the European Union. Planta Medica 75, 683-688.

Walker, K.M., Applequist, W.L., 2012. Adulteration of selected unprocessed botanicals in the U.S. retail herbal trade. Economic Botany 66, 321-327.

Walle, T., 2004. Absorption and metabolism of flavonoids. Free Radical Biology and Medicine 36, 829-837.

Wang. F., Jiang, K., Li, Z., 2007. [Purification and identification of genistein in Ginkgo biloba leaf extract]. Chinese Journal of Chromatography 25, 509-513.

Hans Wohlmuth (a, b), *, Kate Savage (c), Ashley Dowell (c), Peter Mouatt (a, c)

(a) Medicinal Plant Herbarium, Southern Cross University, PO Box 157, Lismore, NSW 2480, Australia

(b) Integria Healthcare, Callans Road, Ballina, NSW 2478, Australia

(c) Southern Cross Plant Science, Southern Cross University, PO Box 157, Lismore, NSW 2480, Australia

* Corresponding author at: Integria Healthcare, Gallans Road, Ballina. NSW 2478, Australia. Tel.: +61 2 6620 5180; fax: +61 2 6622 3459.

E-mail addresses: hans.wohlmuth@scu.edu.au, hans.wohlmuth@integria.com (H. Wohlmuth).

http://dx.doi.org/10.1016/j.phymed.2014.01.010

Table 1
Content ([per thousand] w-w) of flavonol aglycones quercetin,
kaempferol and isorhamnetin in unhydrolysed and hydrolysed samples of
ginkgo leaf and retail products (calculated as quercetin, as per
USP36-NF31).

Sample (a,b)    Quercetin

                Unhydrolysed             Hydrolysed

L1-AU           0.000 [+ or -] 0.000      4.292 [+ or -] 0.046
L2-NZ           0.000 [+ or -] 0.000      3.715 [+ or -] 0.026
L3-CH           0.000 [+ or -] 0.000      0.961 [+ or -] 0.010
L4-CH           0.000 [+ or -] 0.000      0.909 [+ or -] 0.013
L5-CH           0.000 [+ or -] 0.000      0.869 [+ or -] 0.031
T1-AU           0.000 [+ or -] 0.000     13.643 [+ or -] 0.099
T2-AU           0.036 [+ or -] 0.006      3.718 [+ or -] 0.014
T3-AU           2.477 [+ or -] 0.006      5.593 [+ or -] 0.019
T4-DK           0.000 [+ or -] 0.000     13.062 [+ or -] 0.132
C1-AU           0.013 [+ or -] 0.000     12.635 [+ or -] 0.064
C2-AU           7.272 [+ or -] 0.034     15.416 [+ or -] 0.060
C3-AU           7.421 [+ or -] 0.124     14.425 [+ or -] 0.226
C4-DK           0.027 [+ or -] 0.000     17.317 [+ or -] 0.102

Sample (a,b)    Kaempferol

                Unhydrolysed             Hydrolysed

L1-AU            0.000 [+ or -] 0.000      4.291 [+ or -] 0.061
L2-NZ           0.000 [+ or -] 0.000      2.833 [+ or -] 0.022
L3-CH           0.000 [+ or -] 0.000      1.383 [+ or -] 0.020
L4-CH           0.000 [+ or -] 0.000      1.470 [+ or -] 0.032
L5-CH           0.000 [+ or -] 0.000      1.349 [+ or -] 0.048
T1-AU           0.000 [+ or -] 0.000     15.311 [+ or -] 0.122
T2-AU           0.000 [+ or -] 0.000      4.624 [+ or -] 0.005
T3-AU           1.835 [+ or -] 0.015      5.991 [+ or -] 0.001
T4-DK           0.000 [+ or -] 0.000     13.835 [+ or -] 0.116
C1-AU           0.000 [+ or -] 0.000     15.361 [+ or -] 0.060
C2-AU           7.500 [+ or -] 0.030     18.120 [+ or -] 0.083
C3-AU           2.230 [+ or -] 0.111     17.094 [+ or -] 0.368
C4-DK           0.000 [+ or -] 0.000     26.522 [+ or -] 0.140

Sample (a,b)    Isorhamnetin

                Unhydrolysed             Hydrolysed

L1-AU            0.000 [+ or -] 0.000     0.592 [+ or -] 0.008
L2-NZ           0.000 [+ or -] 0.000     0.422 [+ or -] 0.025
L3-CH           0.000 [+ or -] 0.000     0.416 [+ or -] 0.006
L4-CH           0.000 [+ or -] 0.000     0.362 [+ or -] 0.004
L5-CH           0.000 [+ or -] 0.000     0.375 [+ or -] 0.011
T1-AU           0.000 [+ or -] 0.000     3.449 [+ or -] 0.036
T2-AU           0.000 [+ or -] 0.000     1.287 [+ or -] 0.006
T3-AU           0.010 [+ or -] 0.002     1.112 [+ or -] 0.007
T4-DK           0.000 [+ or -] 0.000     2.832 [+ or -] 0.022
C1-AU           0.000 [+ or -] 0.000     4.229 [+ or -] 0.051
C2-AU           0.113 [+ or -] 0.008     2.825 [+ or -] 0.011
C3-AU           0.014 [+ or -] 0.002     1.870 [+ or -] 0.052
C4-DK           0.000 [+ or -] 0.000     4.765 [+ or -] 0.069

(a) L, leaf; T, tablet; C, capsule.

(b) Country of origin (leaf) or purchase (retail products): AU,
Australia; NZ, New Zealand; CH, China; DK, Denmark.

Table 2
Free flavonol aglycones and genistein in unhydrolysed ginkgo products.
Concentration of free flavonols in unhydrolysed samples is shown as
percentage of the concentration in hydrolysed samples. Presence (+) or
absence (-) of genistein in unhydrolysed samples is shown.

Sample   Type       Quercetin   Kaempferol   Isorhamnetin   Genistein

L1-AU    Leaf        0.0         0.0         0.0            -
L2-NZ    Leaf        0.0         0.0         0.0            -
L3-CH    Leaf        0.0         0.0         0.0            -
L4-CH    Leaf        0.0         0.0         0.0            -
L5-CH    Leaf        0.0         0.0         0.0            -
T1-AU    Tablet      0.0         0.0         0.0            -
T2-AU    Tablet      1.0         0.0         0.0            -
T3-AU    Tablet     44.3        30.6         0.9            +
T4-DK    Tablet      0.0         0.0         0.0            -
C1-AU    Capsule     0.1         0.0         0.0            -
C2-AU    Capsule    47.2        41.4         4.0            +
C3-AU    Capsule    51.4        13.0         0.8            +
C4-DK    Capsule     0.2         0.0         0.0            -

Table 3
Flavonol glycoside content and percentage of label claims for ginkgo
retail products, based on USP-NF formula for Content of Flavonol
Glycosides, calculated pre and post acid hydrolysis.

Product    Calculated flavonol glycoside
           content using USP-NF formula
           (mg/dose unit)

           Pre hydrolysis    Post hydrolysis

T1-AU       0.00             29.29
T2-AU       0.04             10.91
T3-AU       9.47             27.80
T4-DK       0.00             26.24
C1-AU       0.02             37.95
C2-AU      15.85             38.71
C3-AU       9.09             31.40
C4-DK       0.01             20.65

Product    Calculated flavonol glycoside          Overestimation of
           content as percentage of label         flavonol glycoside
           claim (%)                              content using USP-NF
                                                  post hydrolysis
           USP-NF method       Due to free        method (%)
           (post hydrolysis)   aglycones
                               (pre hydrolysis)

T1-AU      101.7                0.0                0.0
T2-AU      102.0                0.4                0.4
T3-AU       96.5               32.9               34.0
T4-DK      109.3                0.0                0.0
C1-AU       94.6                0.0                0.0
C2-AU      134.4               55.0               40.9
C3-AU      109.0               31.6               29.0
C4-DK       86.0                0.1                0.1
COPYRIGHT 2014 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2014 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Wohlmuth, Hans; Savage, Kate; Dowell, Ashley; Mouatt, Peter
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUDE
Date:May 15, 2014
Words:5668
Previous Article:Conversion of salvianolic acid B into salvianolic acid A in tissues of Radix Salviae Miltiorrhizae using high temperature, high pressure and high...
Next Article:Essential oil composition of Senecio graciliflorus DC: comparative analysis of different parts and evaluation of antioxidant and cytotoxic activities.
Topics:

Terms of use | Privacy policy | Copyright © 2020 Farlex, Inc. | Feedback | For webmasters