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H NMR-based metabolomics combined with HPLC-PDA-MS-SPE-NMR for investigation of standardized Ginkgo biloba preparations.

Agnolet S, Jaroszewski JW, Verpoorte R, Staerk D - Metabolomics (2010)

Bottom Line: The standardized extracts originated from Denmark, Italy, Sweden, and United Kingdom, and the results show that (1)H NMR spectra allow simultaneous assessment of the content as well as identity of flavonoid glycosides and TTLs based on a very simple sample-preparation procedure consisting of extraction, evaporation and reconstitution in acetone-d(6).The present study shows that (1)H NMR-based metabolomics is an attractive method for non-selective and comprehensive analysis of Ginkgo extracts.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-009-0195-x) contains supplementary material, which is available to authorized users.

View Article: PubMed Central - PubMed

ABSTRACT
Commercial preparations of Ginkgo biloba are very complex mixtures prepared from raw leaf extracts by a series of extraction and prepurification steps. The pharmacological activity is attributed to a number of flavonoid glycosides and unique terpene trilactones (TTLs), with largely uncharacterized pharmacological profiles on targets involved in neurological disorders. It is therefore important to complement existing targeted analytical methods for analysis of Ginkgo biloba preparations with alternative technology platforms for their comprehensive and global characterization. In this work, (1)H NMR-based metabolomics and hyphenation of high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) were used for investigation of 16 commercially available preparations of Ginkgo biloba. The standardized extracts originated from Denmark, Italy, Sweden, and United Kingdom, and the results show that (1)H NMR spectra allow simultaneous assessment of the content as well as identity of flavonoid glycosides and TTLs based on a very simple sample-preparation procedure consisting of extraction, evaporation and reconstitution in acetone-d(6). Unexpected or unwanted extract constituents were also easily identified in the (1)H NMR spectra, which contrasts traditional methods that depend on UV absorption or MS ionizability and usually require availability of reference standards. Automated integration of (1)H NMR spectral segments (buckets or bins of 0.02 ppm width) provides relative distribution plots of TTLs based on their H-12 resonances. The present study shows that (1)H NMR-based metabolomics is an attractive method for non-selective and comprehensive analysis of Ginkgo extracts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-009-0195-x) contains supplementary material, which is available to authorized users.

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Top: Relative distribution plots of GA, GB, GC, GJ, and BB obtained from buckets, i.e., automatically integrated spectral segments of 0.02 ppm, of the H-12 singlet. The total sum of intensities of the H-12 singlets of GA, GB, GC, GJ, and BB are normalized and expressed as a fraction of 6 (standard composition of EGb761 is 6% TTLs). Bottom: Correlation plots comparing automated integration (AMIX) of bucketed data obtained as above with data from manual integration of H-12 resonances
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Fig5: Top: Relative distribution plots of GA, GB, GC, GJ, and BB obtained from buckets, i.e., automatically integrated spectral segments of 0.02 ppm, of the H-12 singlet. The total sum of intensities of the H-12 singlets of GA, GB, GC, GJ, and BB are normalized and expressed as a fraction of 6 (standard composition of EGb761 is 6% TTLs). Bottom: Correlation plots comparing automated integration (AMIX) of bucketed data obtained as above with data from manual integration of H-12 resonances

Mentions: A comparison between the relative amounts of TTLs based on integrals of 0.02-ppm buckets representing the H-12 resonances of GA, GB, GC, GJ, and BB is shown in Fig. 5. The total integral area representing all H-12 resonances in each preparation was normalized and expressed as a fraction of 6, which is the standard composition of the extract EGb 761 containing 6% of TTLs (3.1% of ginkgolides and 2.9% of bilobalide) (Drieu and Jaggy 2000), assumed to be the case for the commercial preparations studied here. The majority of the preparations follow this distribution between the ginkgolides and bilobalide, although preparation 11 and to a smaller degree preparations 9, 12, 13, and 14 differ from this by having relatively high values of GA, and to some degree of GB. It is worth emphasizing that Fig. 5 shows the distribution between the individual components, and not absolute amounts. However, because the exact pharmacological role of the individual constituents remains unknown and because the plot is easily prepared from information already available from the data reduction, this method is suggested to be implemented as a routine procedure for characterization of Ginkgo biloba extracts to be used in future pharmacological studies as a supplement to determination of absolute amount of total TTLs. To show that the relative distribution of TTLs obtained by automatic integration of buckets is, as expected, in agreement with the distributions obtained by manual integration of individual peaks as described elsewhere (Choi et al. 2003; Li et al. 2004), a correlation plot was prepared (Fig. 5). It demonstrates excellent correlation for GA (R2 = 0.9942), GB (R2 = 0.9955), GC (R2 = 0.9918), and BB (R2 = 0.9927). Only GJ showed a significantly lower correlation (R2 = 0.9038), which is presumably due to the low S/N-ratio for this signal.Fig. 5


H NMR-based metabolomics combined with HPLC-PDA-MS-SPE-NMR for investigation of standardized Ginkgo biloba preparations.

Agnolet S, Jaroszewski JW, Verpoorte R, Staerk D - Metabolomics (2010)

Top: Relative distribution plots of GA, GB, GC, GJ, and BB obtained from buckets, i.e., automatically integrated spectral segments of 0.02 ppm, of the H-12 singlet. The total sum of intensities of the H-12 singlets of GA, GB, GC, GJ, and BB are normalized and expressed as a fraction of 6 (standard composition of EGb761 is 6% TTLs). Bottom: Correlation plots comparing automated integration (AMIX) of bucketed data obtained as above with data from manual integration of H-12 resonances
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2874492&req=5

Fig5: Top: Relative distribution plots of GA, GB, GC, GJ, and BB obtained from buckets, i.e., automatically integrated spectral segments of 0.02 ppm, of the H-12 singlet. The total sum of intensities of the H-12 singlets of GA, GB, GC, GJ, and BB are normalized and expressed as a fraction of 6 (standard composition of EGb761 is 6% TTLs). Bottom: Correlation plots comparing automated integration (AMIX) of bucketed data obtained as above with data from manual integration of H-12 resonances
Mentions: A comparison between the relative amounts of TTLs based on integrals of 0.02-ppm buckets representing the H-12 resonances of GA, GB, GC, GJ, and BB is shown in Fig. 5. The total integral area representing all H-12 resonances in each preparation was normalized and expressed as a fraction of 6, which is the standard composition of the extract EGb 761 containing 6% of TTLs (3.1% of ginkgolides and 2.9% of bilobalide) (Drieu and Jaggy 2000), assumed to be the case for the commercial preparations studied here. The majority of the preparations follow this distribution between the ginkgolides and bilobalide, although preparation 11 and to a smaller degree preparations 9, 12, 13, and 14 differ from this by having relatively high values of GA, and to some degree of GB. It is worth emphasizing that Fig. 5 shows the distribution between the individual components, and not absolute amounts. However, because the exact pharmacological role of the individual constituents remains unknown and because the plot is easily prepared from information already available from the data reduction, this method is suggested to be implemented as a routine procedure for characterization of Ginkgo biloba extracts to be used in future pharmacological studies as a supplement to determination of absolute amount of total TTLs. To show that the relative distribution of TTLs obtained by automatic integration of buckets is, as expected, in agreement with the distributions obtained by manual integration of individual peaks as described elsewhere (Choi et al. 2003; Li et al. 2004), a correlation plot was prepared (Fig. 5). It demonstrates excellent correlation for GA (R2 = 0.9942), GB (R2 = 0.9955), GC (R2 = 0.9918), and BB (R2 = 0.9927). Only GJ showed a significantly lower correlation (R2 = 0.9038), which is presumably due to the low S/N-ratio for this signal.Fig. 5

Bottom Line: The standardized extracts originated from Denmark, Italy, Sweden, and United Kingdom, and the results show that (1)H NMR spectra allow simultaneous assessment of the content as well as identity of flavonoid glycosides and TTLs based on a very simple sample-preparation procedure consisting of extraction, evaporation and reconstitution in acetone-d(6).The present study shows that (1)H NMR-based metabolomics is an attractive method for non-selective and comprehensive analysis of Ginkgo extracts.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-009-0195-x) contains supplementary material, which is available to authorized users.

View Article: PubMed Central - PubMed

ABSTRACT
Commercial preparations of Ginkgo biloba are very complex mixtures prepared from raw leaf extracts by a series of extraction and prepurification steps. The pharmacological activity is attributed to a number of flavonoid glycosides and unique terpene trilactones (TTLs), with largely uncharacterized pharmacological profiles on targets involved in neurological disorders. It is therefore important to complement existing targeted analytical methods for analysis of Ginkgo biloba preparations with alternative technology platforms for their comprehensive and global characterization. In this work, (1)H NMR-based metabolomics and hyphenation of high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) were used for investigation of 16 commercially available preparations of Ginkgo biloba. The standardized extracts originated from Denmark, Italy, Sweden, and United Kingdom, and the results show that (1)H NMR spectra allow simultaneous assessment of the content as well as identity of flavonoid glycosides and TTLs based on a very simple sample-preparation procedure consisting of extraction, evaporation and reconstitution in acetone-d(6). Unexpected or unwanted extract constituents were also easily identified in the (1)H NMR spectra, which contrasts traditional methods that depend on UV absorption or MS ionizability and usually require availability of reference standards. Automated integration of (1)H NMR spectral segments (buckets or bins of 0.02 ppm width) provides relative distribution plots of TTLs based on their H-12 resonances. The present study shows that (1)H NMR-based metabolomics is an attractive method for non-selective and comprehensive analysis of Ginkgo extracts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-009-0195-x) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus