Molecular characterization of Perilla frutescens extracts & chromatographic

In order to estimate the quantity of elutable PF compounds within the online coupled continuous flow mixing system, molecular composition of extracts was characterized beforehand using a HILIC-RPLC coupling with UV and MS detection [195, 249]. By means of this method non-polar as well as polar compounds can be captured in a single run, which allows a comprehensive overview of the entirety of contained molecules. Knowledge about the polarity distribution of extract compounds is eminently important for the establishment of the chromatographic separation implemented into the online coupled continuous flow mixing system.

ESI-MS

I Column ESI-MS

Detection of enzymatic activity

Continuous flow assay

Online coupled continuous flow mixing assay

Adjustments

- further adjustment of substrate and enzyme concentrations

- addition of an internal standard

- implementation of a chromatographic separation may necessitate the use of organic solvents - finding of organic solvents compatible to enzymatic

assays

- assay characterization, e.g. determination of K_mand V_max

- decrease of assay component concentrations - substitute non-volatile assay additives and buffers - adjustment of pH with regard to efficient ionization - use of physiological substrates rather than artifical

chromophoric ones Photometric

assay

Enzyme

Extract

Substrate

59 Figure 14 Exemplarily displayed chromatographic separation of PF water (A) and 100% EtOH extract (B) using the HILIC-RPLC coupling. Mass spectrometrically detected m/z of contained molecules are plotted against their RTs.

The molecular characterization of PF extracts revealed clear differences between the extracts, which can be ascribed to the use of different solvents for the extraction (Figure 14).

The determination of the water extract composition showed distinctly more molecules to elute within the first 17 minutes of the chromatographic run, in which mainly compounds with logD values below 0, i.e. polar ones, can be found [195]. In contrast the separation of 100% EtOH extract was detected with an increase of eluting non-polar compounds after 25 minutes and a distinctly lower quantit ithi the pola ti e a ge up to minute 17, compared to PF water extract. Nevertheless both extracts contain polar compounds, which are potentially separable with the chromatographic method to be adapted for the utilization within the online coupled continuous flow mixing system. Due to the need of avoiding (high) organic solvent concentrations, which may negatively affect the enzyme, the chromatographic separation of PF extracts was first tested with an entirely aqueous isocratic elution. Chromatographic columns, which are stable to run with a 100% aqueous mobile phase, are available (Appendix IV, Supplementary material). However the quantity of eluting compounds as well as the MS intensities were found to be low (Figure 15, ). An efficient method to shorten the experimental time and to enhance the solubility and thus the elution of extract components is the application of a temperature gradient to the column [245]. The gradual heating of the mobile phase causes a decrease of its static permittivity, i.e. polarity,

0 200 400 600 800 1000

0 5 10 15 20 25 30 35

m/z

Retention time [min]

0 200 400 600 800 1000

0 5 10 15 20 25 30 35

m/z

Retention time [min]

Increasing hydrophilicity (logD < 0)

Increasing hydrophobicity

(logD > 0) Increasing hydrophilicity (logD < 0)

Increasing hydrophobicity (logD > 0)

A B

60 which results in an increase of elution strength [250]. In accordance with the column specifications a moderate temperature gradient up to 70°C was applied (Figure 15 &

Appendix IV, Figure 2). Due to the enhanced solution in the heated mobile phase an increasing quantity of eluting compounds (Figure 15, gray-shaded) and a distinct shift of retention times (Figure 15, arrows) was detected. Nevertheless also late-eluting compounds, like e.g. 623.1 or 639.1 (Figure 15), were still highly polar with logD values distinctly below 0 (Appendix IV, Table 3).

Figure 15 Chromatographic separation of PF water extract using a 100% aqueous mobile phase without () and with () the application of a moderate temperature gradient (TG) up to 70°C. Shaded compounds only elute after the application of a TG. Arrows mark the shifts in RT of a selection of eluting compounds, which are due to the increasing temperature applied to the column.

Consequently a low amount of eluting compounds is expected after the injection of semi- to non-polar extracts with a 100% aqueous mobile phase. To extent the polarity spectrum and to further accelerate the separation low organic solvent proportions were added to the mobile phase and tested for their suitability within the online coupled continuous flow mixing system (Appendix IV, Figure 2). Besides being a necessary stability requirement with

333.9 623.1

639.1

191.3 209.2

639.1

268.1

427.0

0 10 20 30 40 50 60 70 80

0 100 200 300 400 500 600 700

0 20 40 60 80 100

Temperature [°C]

m/z

Retention time [min]

TG no TG

61 most chromatographic columns, the use of organic solvents may also contribute to the improvement of MS intensities and signal stability (Appendix IV, Supplementary material, Figure S1) [14, 251]. Since organic solvents are likely to interfere with the enzyme´s catalytic activity (Appendix IV, Table 2, Figure 2 and Supplementary material) [5], their proportion has to be kept constant. This can either be achieved by performing an entirely isocratic separation (Appendix IV) or by the application of a counter gradient. By means of antagonizing the organic solvent proportion of the chromatographic gradient a constant solvent exposure to the enzyme can be preserved [26, 28, 29, 252, 253]. The additional flow will however cause the dilution of assay components and analytes, which potentially reduces the MS signal, wherefore a counter gradient was not applied here. Consequently a balance has to be found between the improvement of the separation by addition of an organic solvent and sufficient remaining enzymatic activity, wherefore the assessment of the enzymes compatibility with diverse organic solvents is a prerequisite for further measurements (Figure 9 & Appendix IV, Supplementary material). Moreover the injection of different PF extract polarities allows the comparison of chromatographic elution efficiency in terms of the contained polar, semi- and non-polar compounds (Appendix IV, Table). Due to the mostly polar properties of the employed mobile phases and the high content of polar compounds, most elutable features were found after the injection of PF water extract, followed by 50% EtOH extract and 90% MeOH, 0.5% FAc extract (Appendix IV). Injection of PF water extract revealed the best results with the applied most polar mobile phase containing 5% EtOH, whereas most elutable compounds of 50% EtOH and 90% MeOH, 0.5%

FAc extract were observed with 5% IPA. Due to a higher static permittivity of the 10% EtOH and 5% IPA mobile phase [250], the finding of an improved elution efficiency for less polar extracts is reasonable. Consequently the lowest amount of compounds was found to elute with the non-polar 90% MeOH, 0.5% FAc PF extract and the most polar mobile phase of 5%

EtOH (Appendix IV). Overall the applied chromatographic method was found to result in a good separation of PF extracts (Appendix IV, Figure 2). The plotting of known PF compound RTs vs their logDs revealed a logarithmic increase (Appendix IV, Figure 3). This correlation could then be utilized to tentatively assign unknown compounds e.g. like luteolin-6,8-di-C-glucoside (Appendix IV, Figure 3, B). Based on the observed logD values, the range of eluting molecule polarities can be determined. This is of special interest with regard to the detection

62 of enzymatic regulation by means of the online coupled continuous flow mixing system. The classification of inhibitors in terms of their chemical properties may be insightful for the finding and identification of further promising regulatory compounds.