Screening of Natural Products for Cathepsin B Activity

We analyzed various natural extracts that were previously tested in a fluorescence-based cathepsin B assay in order to demonstrate the applicability of the current method for screening real-life samples. The screening result of a nonspiked fungi extract is represented in Fig. 5.6. A methanol gradient was used for elution of the sample components. The increasing amount of methanol resulted in an improved ESI-MS sensitivity for AMC and Z-FR, but simultaneously decreased enzymatic activity. The corresponding baseline instability can clearly be seen in the mass chromatograms of AMC and Z-FR. Nevertheless, data interpretation was still possible, even at the highest methanol concentration level applied. The AMC and Z-FR mass chromatograms (see Fig. 5.6b, c) show several synchronous negative peaks, of which the first peak at 3.0 min is the injection peak. In a procedure similar to the one described above, mass spectra were constructed from the negative peaks at tR 43.3, 51.8, and 55.6 min. As an example, the EICs of the possible bioactive compounds for peak 43.3 min are shown in Fig. 5.6f-j. The retention times of the peaks in Fig. 5.6g, i, j match with the negative peak at 43.3 min. Considering the peak shape of m/z 230, it is unlikely that this compound has caused the negative peak at tR 43.3 min, because the peak shape is rather different than the peak shape of the bioactive peak. It is more reliable that either

Lcms Esi Shimadzu

Fig. 5.6 On-line HPLC bioactivity screeningof a fungi extract using acetylcholinesterase as biological target. MS instrument: Shimadzu LCMS-2010 single-stage quadrupole mass spectrometer. (a) TIC chromatogram of the mixture, scan range m/z 50-1000; (b) mass chromatogram ofAMC (m/z 176.0); (c) mass chromatogram ofZ-FR (m/z 456.0); (d) mass chromatogram of SMC1 (m/z 245.0); (e)

Fig. 5.6 On-line HPLC bioactivity screeningof a fungi extract using acetylcholinesterase as biological target. MS instrument: Shimadzu LCMS-2010 single-stage quadrupole mass spectrometer. (a) TIC chromatogram of the mixture, scan range m/z 50-1000; (b) mass chromatogram ofAMC (m/z 176.0); (c) mass chromatogram ofZ-FR (m/z 456.0); (d) mass chromatogram of SMC1 (m/z 245.0); (e)

mass chromatogram of SMC2 (m/z 330.0); (f-j) mass chromatogram of various m/z values, which were present as an abundant peak in the mass spectrum recorded at tR = 43.3 min: (f) mass chromatogram of m/z 211.0; (g) mass chromatogram of m/z 230.0; (h) mass chromatogram of m/z 292.9; (i) mass chromatogram of m/z 677.3; (j) mass chromatogram of m/z 693.2.

m/z 677.3 or m/z 693.2 or both compounds were bioactive, because the peak shapes are identical. Most likely is that m/z 677.3 and m/z 693.2 are [M+Na] + and [M+K]+ of a molecule with a molecular mass of 654 Da. Regarding the other negative peaks, possible bioactive compounds showed an m/z 199.1 for the peak at 51.8 min, and an m/z 279.1 for the peak at 55.6 min (EICs not shown). The results of this screening measurement, that is, the number of active compounds in the extract, their retention times, and molecular masses serve as starting points for further structure elucidation experiments (data not shown).

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