Ginkgo Biloba

G ModelPHYMED-51597; No. of Pages 7 ARTICLE IN PRESS Phytomedicine xxx (2014) xxx–xxx Contents lists available at ScienceDirect Phytomedicine journal homepage: www.elsevier.de/phymed Adulteration of Ginkgo biloba products and a simple method to improve its detection Hans Wohlmuth a,b,∗ , Kate Savage c , Ashley Dowell c , Peter Mouatt a,c a b c Medicinal Plant Herbarium, Southern Cross University, PO Box 157, Lismore, NSW 2480, Australia Integria Healthcare, Gallans Road, Ballina, NSW 2478, Australia Southern Cross Plant Science, Southern Cross University, PO Box 157, Lismore, NSW 2480, Australia a r t i c l e i n f o Article history: Received 14 November 2013 Received in revised form 27 November 2013 Accepted 26 January 2014 Keywords: Ginkgo biloba Ginkgo extract Adulteration Quality control Ginkgo flavonol glycosides Genistein a b s t r a c t 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. © 2014 Elsevier GmbH. All rights reserved. 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$30 million (Lindstrom et al., 2013). Ginkgo is also one of the most intensely studied medicinal plants, with more than 3000 scientific papers published on the topic between 2001 and 2009 alone (van Beek and Montoro, 2009). Ginkgo leaf extracts are recommended for a range of conditions, including cerebral insufficiency, vertigo and tinnitus of vascular ∗ Corresponding author at: Integria Healthcare, Gallans Road, Ballina, NSW 2478, Australia. Tel.: +61 2 6620 5180; fax: +61 2 6622 3459. E-mail addresses: [email protected], [email protected] (H. Wohlmuth). origin, and peripheral arterial disease (Blumenthal, 2003; Bone and Mills, 2013). The pharmacologically active compounds in ginkgo leaf are considered to be flavonol glycosides (quercetin, kaempferol and isorhamnetin being the principal aglycones) and terpene lactones (bilobalide and ginkgolides). Most ginkgo leaf extracts on the market are produced by selective, multi-step extraction processes involving organic solvents and carry quantitative claims concerning their content of flavonol glycosides and terpene lactones. Accordingly, most ginkgo leaf extracts are more high-tech and high-cost than typical botanical extracts. Botanical raw materials including extracts present special challenges in terms of quality control and assurance due to their chemical complexity and inherent natural variability. The most fundamental aspects of quality assurance for such materials are to ensure the correct morphological part(s) from the right botanical taxon is used, and that the material is not adulterated with other botanical or extraneous material. Adulteration, either accidental or intentional and economically motivated, is a well-known issue for botanicals, and one that potentially can jeopardise not only the quality but also the safety of the finished product (Khan, 2006; Walker and Applequist, 2012). The potential safety issues associated with adulterated or sub-standard ginkgo extracts have been 0944-7113/$ – see front matter © 2014 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2014.01.010 Please cite this article in press as: Wohlmuth, H., et al., Adulteration of Ginkgo biloba products and a simple method to improve its detection. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.01.010 respectively.1016/j. Sample preparation Ginkgo leaf samples were ground to a fine powder in a Retsch MM301 Mixer Mill (Haan.G Model PHYMED-51597.5 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.07 ␮g/ml.000625–0. 0. time filter enabled. System control and data evaluation were achieved using ChemStation for HPLC software. They were authenticated by an experienced pharmacognosist (H. USA). MA. which is not native to ginkgo. USA) 1100 HPLC system fitted with a Phenomenex (Torrance. peak width 0. The USP-NF also provides monographs for Ginkgo Tablets and Ginkgo Capsules.0025–0. Australia)..) column.5 mg/ml for kaempferol and 0.005% trifluoroacetic acid added. corona 4. 2013).. No. Krenn et al.. Methanol (HPLC grade) was obtained from Merck (Kilsyth. Four of these were tablets and four were capsules. VIC.6 mm i. We demonstrate that current pharmacopoeial methods are inadequate for the detection of this type of adulteration.5%) was obtained from Chromadex (Irvine. including the United States Pharmacopoeia-National Formulary (USP-NF) (United States Pharmacopeial Convention. To achieve hydrolysis of flavonol glycosides. v/v) to A/B (5:95. phosphoric acid from Ajax Finechem (Sydney. of Pages 7 ARTICLE IN PRESS H. B and C.5% aqueous phosphoric acid. Limits of detection for quercetin. 2009).6 mm i. 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%. nebuliser 60 psig. In addition. Here we report on ginkgo retail products found to be adulterated with free flavonol aglycones and also containing the isoflavone genistein. trifluoroacetic acid.org/10. 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. CA. USA) Synergi C18 4 ␮M (250 mm × 4. Materials and methods Plant materials and botanicals Five samples of dried Ginkgo biloba leaf were obtained from commercial suppliers.2 ml/min. but the ranges for these differ between the USP-NF and the BP/EP. Germany). Depending on the label claim for flavonol glycoside content. kaempferol and isorhamnetin) were dissolved in DMSO at a concentration of 2. gas temperature 350 ◦ C. dimethyl sulfoxide (DMSO). CA. and the injection volume was 10 ␮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. kaempferol and isorhamnetin were 0.d.750 ml/min. Wohlmuth et al.. MS parameters in the positive ionisation mode were: Vcap 3000 V. The gradient eluting mobile phase was A/B (90:10.010 . scan range 100–1500 m/z. the column temperature 40 ◦ C. 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. LC–MS analysis was performed on an Agilent (Palo Alto. except for the addition of hydrochloric acid. CA. The gradient eluting mobile phase was A/B (60:40. Quercetin dihydrate (98. LC–MS Chemicals and reagents Water was obtained from an in-house Milli-Q system (Millipore.14 mg/ml for isorhamnetin. 0.00028–0. Mobile phase A consisted of water and mobile phase B of acetonitrile.8%) and genistein (>98%) from Sigma-Aldrich (Sydney. drying gas flow rate 5. using an in-house validated method based on the USP34-NF29. Australia). Eight retail products containing Ginkgo biloba as the sole active ingredient were purchased in Australia (6) and Denmark (2). USA) 1100 LC/MSD system equipped with an atmospheric pressure chemical ionisation (APCI) source and fitted with a Phenomenex (Torrance. H. v/v) to A/B (50:50.doi. 2012).5 mg/ml for quercetin. These leaf samples came from ginkgo cultivated in China (3). HPLC Reverse-phase HPLC analysis was performed on an Agilent (Palo Alto.W. Once cooled. The range for precision. 2013. and hydrochloric acid (AR grade).d. step size 0.). vaporiser 350 ◦ C.. between 300 and 1000 mg of the powder was extracted by sonication. / Phytomedicine xxx (2014) xxx–xxx 2 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. Pharmacopoeial monographs play an important role in the quality assurance of botanicals and herbal medicinal products (Vlietinck et al. v/v) over 40 min. Australia). Sichuan. CA. Adulteration of Ginkgo biloba products and a simple method to improve its detection. the European Pharmacopoeia (EP) and the British Pharmacopoeia (BP) (British Pharmacopoeia Commission.2014.01. v/v) over 18 min followed by a 3 min column wash with A/B (5:95). and we propose a simple modification of the USP-NF method that addresses this problem. http://dx. Spain). Calculation of flavonol glycoside content Flavonol glycoside content was calculated according to the method provided in the Ginkgo Tablet and Ginkgo Capsule Please cite this article in press as: Wohlmuth. Mobile phase A consisted of 0. New Zealand (1) and Australia (1). USA).phymed. ethanol (AR grade) from Chem-Supply (Gillman.25 ␮g/ml and 0.0 mg/mL. 2013). to A/B (90:10) over 3 min and a 5 min equilibration period with A/B (90:10). fragmentor 150 V. Genistein was dissolved in methanol. then serially diluted in methanol using a Hamilton Microlab 500 Diluter (Reno. specifications are provided for the terpene lactones. acetonitrile from Scharlau (Sentmenat. as detailed for the leaf samples. Rate of mobile phase flow was 0.15 m/z. kaempferol (97. USA) Luna C18 3 ␮M (100 mm × 4. and voucher materials were deposited in the Medicinal Plant Herbarium (PHARM) at Southern Cross University. calculated as flavonol/flavone glycosides and a maximum content of 5 ppm for ginkgolic acids (putative allergens).) column. Billerica. SA.0 ␮A. Capsules were opened and their content extracted in the same way as the tablets.0 l/min. NV. Phytomedicine (2014). and the injection volume was 5 ␮l. et al. System control and data evaluation were performed with ChemStation for LC/MS software. SA. the solution was transferred quantitatively to a 100-ml volumetric flask and diluted to volume with Milli-Q water. Australia). USA) and isorhamnetin (>98%) from Chengdu Biopurify Phytochemicals (Chengdu. Standard preparation Reference standards (quercetin. China). All samples were extracted in triplicate. Thebarton. bilobalide and ginkgolide A. CA. mobile phase B consisted of methanol.1 min.625 ␮g/ml. Australia). Monographs for ginkgo raw materials (leaf and extract) can be found in various pharmacopoeias. accuracy and linearity was 0. Approximately 5. both with 0. Samples not hydrolysed were treated identically. Tablets were extracted by combining a quantity of 20 and grinding them to a fine powder in a Retsch MM301 Mixer Mill. the column temperature was 40 ◦ C. capsule.291 2.000 0.715 0. T.000 ± ± ± ± ± ± ± ± ± ± ± ± ± 0.122 0. Results The content of quercetin.026 0.000 0.000 0.094 26. 2003.000 0. of Pages 7 ARTICLE IN PRESS H. The minimum ratios stipulated in the monographs are shown as horizontal bars (kaempferol to quercetin NLT 0.132 0. Being a high-value botanical commodity.42‰) and kaempferol (range 1. whereas the same samples contained only very low concentrations (0.59‰). China.961 0..000 0.362 0. He and Roller. kaempferol and isorhamnetin in unhydrolysed and hydrolysed extracts of five ginkgo leaf samples.500 2.84–7. and F is the factor used to convert each aglycone into a flavonol glycoside with a mean molecular mass of 756. leaf.000 0. All samples were examined for the presence of genistein by LC–DAD and LC–MS using positive selective ion monitoring (Fig.000 0.000 0.020 0. rs is the peak area of the aglycone in the corresponding standard solution.000 Hydrolysed 4. is shown in Table 3. Whether or not high levels of quercetin and kaempferol were present pre hydrolysis was obvious by visual inspection of HPLC chromatograms (Fig. Three of the tablets and two of the capsules contained no or very low levels of free flavonols when not subjected to acid hydrolysis.1016/j. Cs is the concentration (mg/mL) of the aglycone in the standard solution.832 4..010 0.000 0. C.36–0. but in both cases the glycoside content is calculated from the content of free flavonol aglycones. b Quercetin Unhydrolysed L1-AU L2-NZ L3-CH L4-CH L5-CH T1-AU T2-AU T3-AU T4-DK C1-AU C2-AU C3-AU C4-DK a b 0.112 2. the flavonol glycoside rutin (quercetin 3-rutinoside) or with other plant extracts containing flavonols has been reported (Chandra et al. when the pharmacopoeial method was used. 3).229 2.449 1.000 0. monographs in USP36-NF31.000 0.833 1.116 0.000 Kaempferol Hydrolysed 4. tablet.000 0.002 0.048 0. Samplea . ginkgo extract is a target for economically motivated adulteration. four tablet samples and four capsule samples is shown in Table 1. and calculation of the quantity (in mg) of each flavonol glycoside in the sample using the formula: Glycosidequantity = r  u rs × Cs × F × 50 where ru is the peak area of the relevant aglycone in the sample solution.000 0. http://dx. No. Wohlmuth et al.000 0.060 0.230 0. Liu et al.000 0.504 for quercetin.76‰).011 0. et al.000 0. Discussion Ginkgo extract is used in numerous dietary supplements.140 Unhydrolysed 0.015 0.422 0.01‰) of isorhamnetin.000 0.000 0. The quercetin content in the hydrolysed leaf samples varied almost five-fold (range 0. None of the unhydrolysed leaf samples contained detectable levels of any of these flavonol aglycones.000 0.. 2010.17‰).022 0.425 17.311 4. quantification of quercetin.000 7.835 15. 2011..000 0.825 1.7 (2.000 0.113 0.01.008 0. C2-AU and C3-AU) shown to contain high levels of free quercetin and kaempferol (Table 2).060 0.006 0.007 0.000 0.0 and 40.027 ± ± ± ± ± ± ± ± ± ± ± ± ± 0. 2012.317 ± ± ± ± ± ± ± ± ± ± ± ± ± 0.G Model PHYMED-51597.010 0.7.416 14.835 0.052 0. but instead calculated based on the aglycone content after hydrolysis. 2009).35–4. 75% of the products analysed contained within ±10% of their label claim for flavonol glycosides.000 0. and 2. H.014 0. and suspected adulteration with the flavonols quercetin and kaempferol.111 0..032 0. However.870 4. unhydrolysed samples of one tablet (T3-AU) and two capsules (C2-AU and C3-AU) contained high levels of quercetin (range 2.624 5. Country of origin (leaf) or purchase (retail products): AU. DK. As is also shown in Table 3.000 0. herbal medicinal products and complementary medicines around the world.000 1.org/10.718 5. van Beek and Montoro. Phytomedicine (2014). 1).036 2. New Zealand.1). Denmark. quantified by HPLC following acid hydrolysis.000 0.9% when applying the pharmacopoeial method.272 7.000 0. 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.051 0. The total quantity of flavonol glycosides in the sample is calculated by summing the values for quercetin.120 17.869 13.000 Hydrolysed 0.292 3.349 15.001 0.287 1. this method involves the acid hydrolysis of an extract of the sample.019 0. Harnly et al.036 0.062 12.000 0. kaempferol and isorhamnetin by HPLC against reference standards.416 0.000 0.000 ± ± ± ± ± ± ± ± ± ± ± ± ± 0.022 0.006 0.000 0.000 0.006 0.470 1.000 0.000 0.000 0.phymed.643 3.034 0.000 0. kaempferol and isorhamnetin glycosides.522 ± ± ± ± ± ± ± ± ± ± ± ± ± 0.102 Isorhamnetin Unhydrolysed 0.025 0. means that Please cite this article in press as: Wohlmuth.29‰) and the isorhamnetin content 1.000 0.030 0.375 3.083 0.97‰) and the three leaf samples from China (2.383 1.59–2.765 ± ± ± ± ± ± ± ± ± ± ± ± ± 0.000 0.30‰ w/w). As is evident from this table. kaempferol and isorhamnetin in unhydrolysed and hydrolysed samples of ginkgo leaf and retail products (calculated as quercetin. 2 shows the ratios between kaempferol and quercetin and between isorhamnetin and quercetin in the hydrolysed samples of retail product. botanicals.064 0. the presence of significant amounts of flavonol aglycones in three of the products resulted in the flavonol glycoside content being overestimated between 29.48–7. CH.000 0.6-fold (range 0. followed by the New Zealand sample (6.2014.046 0.635 15. The flavonol glycoside content of the retail products.010 .005 0.909 0.000 0.099 0.421 0.031 0.593 13. Briefly.000 0. isorhamnetin to quercetin NLT 0. The fact that flavonol glycosides are not quantified directly.000 0.004 0.000 0. 2011.069 L. 2005. 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. Tawab. as per USP36-NF31). Sloley et al.50‰).000 0.013 7. / Phytomedicine xxx (2014) xxx–xxx 3 Table 1 Content (‰ w/w) of flavonol aglycones quercetin.000 0.368 0.061 0. 1). Fig.014 0.013 0.437 for isorhamnetin).006 0. the kaempferol content more than three-fold (range 1.477 0.124 0.87–4.000 0.000 0. Genistein was also readily observable in the HPLC chromatogram as a peak located between the peaks for quercetin and kaempferol (Fig.000 0.000 0.002 0.361 18. The presence of genistein was confirmed by re-analysis by LC–DAD and LC–MS after spiking with a genistein reference standard.226 0. All products conformed to the relevant USP-NF monographs with respect to flavonol ratios after acid hydrolysis.008 0.991 13. Adulteration of Ginkgo biloba products and a simple method to improve its detection.000 0. Australia. The hydrolysed extract of the Australian grown leaf sample L1-AU had the highest content of all three flavonols (total 9.011 0.588 for kaempferol.000 0. NZ. As shown in Table 2.592 0.doi.. One product contained 14% less and another 34% more than the label claim by this method. calculated both pre and post hydrolysis using the formula provided for Content of Flavonol Glycosides in the USP-NF monographs. 2. Genistein was identified only in the three samples (T3-AU. Adulteration of Ginkgo biloba products and a simple method to improve its detection.org/10. HPLC chromatograms of adulterated (A) and unadulterated (B) ginkgo products pre and post hydrolysis. / Phytomedicine xxx (2014) xxx–xxx 4 mA U A-pre 50 40 Q 30 K 20 G 10 0 5 10 15 mA U 20 25 Q 35 m in K A-post 50 30 40 30 20 0 I G 10 5 10 15 20 25 30 35 m in 10 15 20 25 30 35 m in mA U B-pre 50 40 30 20 10 0 5 mA U 50 Q B-post K 40 30 20 I 10 0 5 10 15 20 25 30 35 m in Fig. (G) genistein.01. http://dx..010 . (K) kaempferol. Phytomedicine (2014).doi. Please cite this article in press as: Wohlmuth. et al.1016/j. (Q) quercetin. 1. of Pages 7 H. No.2014. Wohlmuth et al.. (I) isorhamnetin.G Model ARTICLE IN PRESS PHYMED-51597.phymed. H. These results indicate that neither crude ginkgo leaf nor quality ginkgo leaf extracts would be expected to contain free flavonols. kaempferol or isorhamnetin aglycones..and isorhamnetin glycosides that varied up to five-fold between samples. However.3 0.5‰). As an aid to detecting adulteration of ginkgo extracts. Sloley et al.0 0. 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.71 31.0 − − − − − − − + − − + + − Table 3 Flavonol glycoside content and percentage of label claims for ginkgo retail products.9 29. ranging from 0. Presence (+) or absence (−) of genistein in unhydrolysed samples is shown.. except in very low concentrations in some cases.5–7. kaempferol and isorhamnetin after acid hydrolysis. Overestimation of flavonol glycoside content using USP-NF post hydrolysis method (%) 0. In fact.0 30.40 20.00 0.0 0. One of these products (T1-AU) was Tebonin® (Schwabe Pharmaceuticals). Isoflavones occur predominantly in leguminous plants (family Fabaceae). / Phytomedicine xxx (2014) xxx–xxx 5 Table 2 Free flavonol aglycones and genistein in unhydrolysed ginkgo products. calculated pre and post acid hydrolysis. it cannot effectively detect adulteration with free flavonol aglycones.5 109.24 37.0 0. The same three products were shown to contain the isoflavone genistein (4 .0 0. Consistent with the virtual absence of free flavonols in the crude raw material. 2005. the USP-NF monographs have stipulated a kaempferol to quercetin HPLC peak ratio of not less than 0.0 0.0 0.7-trihydroxyisoflavone).01. which as its active ingredient contains EGb761® . five retail products with ginkgo extract as the sole active ingredient contained no or negligible amounts of free flavonols.0 4.91 27.0 44.4 34.0 0.2 0.3 94.47 0. C2-AU and C3AU) contained significant levels of free quercetin (2.4‰ w/w) and kaempferol (1.0 0.doi. sourced from three different countries.0 0.0 0. 2013).0 0. based on USP-NF formula for Content of Flavonol Glycosides.0 0. Ginkgo Tablets and Ginkgo Capsules include specifications for the relative abundance of quercetin.0 0.0 96. Product T1-AU T2-AU T3-AU T4-DK C1-AU C2-AU C3-AU C4-DK Calculated flavonol glycoside content using USP-NF formula (mg/dose unit) Calculated flavonol glycoside content as percentage of label claim (%) Pre hydrolysis Post hydrolysis USP-NF method (post hydrolysis) Due to free aglycones (pre hydrolysis) 0.65 101. H.6 0.7 and an isorhamnetin to quercetin peak ratio of not less than 0.0 0.6 0. http://dx.9 0. 2.4 13.0 0.04 9.85 9..5. but have been Please cite this article in press as: Wohlmuth.0 0.7 102.4 109.phymed. The levels of isorhamnetin in these products were considerably lower.0 0.0 0.0 0.1016/j. In contrast.0 0. Adulteration of ginkgo products In the present study.0 31.80 26. Sample Type Quercetin Kaempferol Isorhamnetin Genistein L1-AU L2-NZ L3-CH L4-CH L5-CH T1-AU T2-AU T3-AU T4-DK C1-AU C2-AU C3-AU C4-DK Leaf Leaf Leaf Leaf Leaf Tablet Tablet Tablet Tablet Capsule Capsule Capsule Capsule 0. while this test may assist in the detection of some types of adulteration.6 134.0 0.1 Ginkgo Extract but form part of Identification test A in the monographs for Ginkgo Tablets and Ginkgo Capsules.2 51.2014..1 47. However.01 29.4 32.0 0.0 1.8–7.4 0.0 0. of Pages 7 H.010 .org/10. contained levels of quercetin-. Since November 2011. These criteria are provided in Identification test B in the monograph for Powdered Fig. et al.0 0. kaempferol.1 (United States Pharmacopeial Convention.09 0. Concentration of free flavonols in unhydrolysed samples is shown as percentage of the concentration in hydrolysed samples. three other retail products (T3-AU.0 55.00 0. Adulteration of Ginkgo biloba products and a simple method to improve its detection. Wohlmuth et al. the USP-NF monographs for Powdered Ginkgo Extract (but not the equivalent EP/BP monograph).0 0.0 0.0 0.0 0. arguably the classic gold standard ginkgo leaf extract (for which Schwabe holds a worldwide patent).02 15. none of these leaf samples contained detectable levels of free quercetin.1 adulteration with flavonol aglycones may not be detectable using the current pharmacopoeial methods.95 38.8 0. Ratios between kaempferol and quercetin (solid bars) and between isorhamnetin and quercetin (patterned bars) peak areas in hydrolysed samples of tablets and capsules (see text for details).11‰.0 41.0 0. No. Phytomedicine (2014).0 0.0 86.G Model ARTICLE IN PRESS PHYMED-51597.9 0.01 to 0.0 0.0 40.29 10. we have shown that five ginkgo leaf samples. 2003). Ginkgo Tablets and Ginkgo Tablets. of Pages 7 ARTICLE IN PRESS H. 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). Tawab. In other words. 2004).. where A(glycosides) is the amount of flavonol aglycones derived from glycosides in the extract or product (and the value the pharmacopoeial test aims to determine). In unadulterated material. 2011. and to equivalent monographs in other pharmacopoeias. (syn. 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). comprising the dried flower and flower bud. 2010. / Phytomedicine xxx (2014) xxx–xxx 6 reported from 60 other. 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. e. (B) mass spectrum showing M + 1 = 271. The presence of the isoflavone genistein. and it has previously been identified as a likely adulterant of ginkgo extracts (Chandra et al. Isoflavones including genistein have never been reported from Ginkgo biloba plant material.010 . 2000). 2007). a compound not occurring naturally in Ginkgo biloba. an increase in aglycone levels (with accompanying decrease in flavonol glycoside levels) is indicative of degradation during extraction. 2).doi.. 2010). In addition to the Fabaceae. No. Improved quality control for ginkgo According to the respective USP-NF monographs. and we believe there can be little doubt that this report concerns an adulterated ginkgo extract. The pericarp of this species contains a number of genistein.. The proposed change to the pharmacopoeial method..). van Beek and Montoro. 2007). but only in low concentrations (Song et al. we believe this change to the method would represent a significant improvement to the USP-NF monographs for Powdered Ginkgo Extract... The adulterated ginkgo products analysed by us contained genistein. 2003. 2009). Myristicaceae and Rutaceae (Reynaud et al. We therefore also recommend that manufacturers further improve the rigour of their quality control processes for ginkgo raw materials Please cite this article in press as: Wohlmuth.g. 2011. which consists of the dried fruit (Chinese Pharmacopoeia Commission.) Schott. Wohlmuth et al. Adulteration of Ginkgo biloba products and a simple method to improve its detection. Because the pharmacopoeial method for calculating flavonol glycoside content is based on the quantity of flavonol aglycones measured after acid hydrolysis. 2009). the extract would likely have undergone processing leading to the hydrolysis of the flavonoid glycosides. including the EP and the BP. the actual flavonol glycoside content in these products was overestimated by between 29% and 41% (Table 3). In order to address this problem. Any free aglycones present in the extract or product are not included in this calculation.0% flavonol glycosides. kaempferol or isorhamnetin in aglycone form. formulation or storage and thus undesirable (Sticher et al. Accordingly. Free aglycones have been reported from unhydrolysed ginkgo leaf.01. is both cheap and simple and provides for an accurate determination of the flavonol glycoside content. Thus. http://dx. 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. so if Styphnolobium japonicum were indeed the adulterant. The adulterated products analysed by us contained significant levels of free quercetin and kaempferol. These aglycones were absent from the five unadulterated products tested with the exception of quercetin. an extract containing both quercetin and kaempferol (but not isorhamnetin) as well as genistein (or their glycosides) would appear most likely. 2002. et al. One such candidate is Styphnolobium japonicum (L. Walle. which was detected in three of the products at very low levels (0. (A) UV spectrum showing max 261. but this could not be detected by the prescribed pharmacopoeial method. Sophora japonica L. The chemistry of ginkgo leaf and leaf extracts is exceedingly well known (van Beek. van Beek and Montoro. 3. H.phymed... we propose a simple modification of the pharmacopoeial test for flavonol glycosides.2014. 2005). mostly angiosperm families (Lapcik. quercetin and kaempferol in free aglycone form.G Model PHYMED-51597. 2007).1016/j.org/10. Such a limit would further contribute to the quality assurance of ginkgo extracts. and huaijiao.5% w/w. Our analysis of five commercial samples of ginkgo leaf grown in three different countries found no evidence (within the limits of detection) of quercetin. 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.036‰ w/w). 0. we have identified three adulterated ginkgo retail products. 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(glycosides) = A(post-hydrolysis) − A(pre-hydrolysis) Fig. Genistein in adulterated ginkgo product. these products did not meet the pharmacopoeial standard for flavonol glycoside content. Whether flavonoids are present in aglycone or glycoside form can have profound impact on their pharmacokinetic and pharmacodynamic characteristics (Murato and Terao. 2005). On this basis we also suggest that a suitable maximum level for free flavonol aglycones be set for ginkgo leaf extract. A(post-hydrolysis) is the total amount of flavonol aglycones after acid hydrolysis (representing the sum of free aglycones plus aglycones from glycosides) and A(pre-hydrolysis) is the amount of free flavonol aglycones in the unhydrolysed extract or product. Two botanical drugs derived from this plant are listed in the Pharmacopoeia of the People’s Republic of China: huaihua. with obvious potential implications for the safety and efficacy of the product. quercetin and kaempferol glycosides (Qi et al. ginkgo tablets and capsules must be prepared from Powdered Ginkgo Extract that contains between 22. Phytomedicine (2014).013–0. including limiting adulteration with flavonol aglycones from extraneous sources. While we have not been able to identify the adulterant. a well-known Chinese medicinal plant known as Japanese pagoda or Chinese scholar tree.0 and 27. He and Roller. Rosaceae. but there is one published report of genistein isolated from a Chinese ginkgo leaf extract (Wang et al. genistein has been reported from the monocotyledonous family Iridaceae and the dicotyledonous families Moraceae. while adding an additional step. J. 325–329. Luthria. Euromonitor International Ltd.. Isoflavonoids in non-leguminous families: an update. N. http://dx. London. Blumenthal. 321–327.. Isolation and purification of flavonoid and isoflavonoid compounds from the pericarp of Sophora japonica L.. 2013. United States Pharmacopoeia and National Formulary (USP36-NF31)..R. Song. Edinburgh.. Hyun. 102–107. United States Pharmacopeial Convention. 2013. The analysis of ginkgo flavonoids. Vitamins and Dietary Supplements. 2013.01... Walle. Bone. Noldner. The ABC Clinical Guide to Herbs. pp. Euromonitor International Ltd. 2909–2916.. Terreux. Austin. M... Mills. L. H. C.E.. Analyst 130. Walker. 2009... 504–515. 2010. Journal of Functional Foods 3....org/10. Wang.. Chemical analysis and quality control of Ginkgo biloba leaves. I. J AOAC Int 95.A. Roller. / Phytomedicine xxx (2014) xxx–xxx (especially extracts) by assaying for the presence of genistein and rejecting as adulterated ginkgo materials found to contain this extraneous compound.. M. K. 2009. such as those in the USPNF.5% in 2012: herbal supplement sales rise for 9th consecutive year. K.. 2005. B. G.. Z.. Reproductive and developmental toxicity of the Ginkgo biloba special extract EGb 761 in mice. S.M. 829–837. T.M. H. Life Sciences 78. Y. Amsterdam. Harwood Academic Publishers. 21–55. Fang.A.... Nahrungsergänzungsmittel mit Ginkgo unter der Lupe... A. Fingerprint analysis of Ginkgo biloba leaves and related health foods by high-performance liquid chromatography/electrospray ionization-mass spectrometry. Li. Mandal. Wegener. Antioxidative flavonoid quercet implication of its intestinal absorption metabolism. X. Formulating.A.D. K. Guilet. MD. P. A. Issues related to botanicals. by adsorption chromatography on 12% cross-linked agarose gel media. Pharmacopoeial monographs for botanical raw materials and dose forms. Meng. 1579–1587. Tawab. Chinese Pharmacopoeia Commission.. J. 2006. Now Ginkgo – 10 years after Cimicifuga? Phytomedicine 21.. Principles and Practice of Phytotherapy: Modern Herbal Medicine. 2000. 2007. S. T. T. 2013... Lapcik.010 .G Model PHYMED-51597. British Pharmacopoeia. Persons. Scherban. Sun. F. [Purification and identification of genistein in Ginkgo biloba leaf extract]. EP and BP.... of Pages 7 ARTICLE IN PRESS H. Adulteration of Ginkgo biloba products and a simple method to improve its detection.. 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