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From Bioprofiling and Characterization to Bioquantification of Natural Antibiotics by Direct Bioautography Linked to High Resolution Mass Spectrometry, Exemplarily Shown for Salvia miltiorrhiza Root Maryam Jamshidi-Aidji, and Gertrud Elisabeth Morlock Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02648 • Publication Date (Web): 21 Oct 2016 Downloaded from http://pubs.acs.org on October 22, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

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From Bioprofiling and Characterization to Bioquantification of Natural

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Antibiotics by Direct Bioautography Linked to High Resolution Mass

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Spectrometry, Exemplarily Shown for Salvia miltiorrhiza Root

7 8 Maryam Jamshidi-Aidjia and Gertrud E. Morlocka, *

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a

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Science, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany

Interdisciplinary Research Center (IFZ) and Institute of Nutritional Science, Department of Food

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*Corresponding author. Tel.: +49-641-99-39141; fax: +49-641-99-39149.

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E-mail addresses: [email protected] (G. E. Morlock), Maryam.Jamshidi-Aidji@uni-

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giessen.de (M. Jamshidi)

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ACS Paragon Plus Environment

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ABSTRACT: Phytochemicals are promising agents in the development of new antibiotics. A

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streamlined strategy for rapid screening and reliable characterization of antibiotics in botanicals was

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demonstrated in contrast to the commonly applied chromatographic column fractionation followed by

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microtiter plate assay. Modern direct bioautography hyphenated to structure elucidation techniques is a

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straightforward bioanalytical tool, especially if microbiological assays were taken into account. At one

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go, lipophilic antimicrobials in Salvia miltiorrhiza root samples were analyzed using high-performance

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thin-layer chromatography (HPTLC) in direct combination with Aliivibrio fischeri and Bacillus subtilis

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bioassays. The most intense antimicrobials were characterized via HPTLC-high resolution mass

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spectrometry. As proof of this streamlined strategy, dihydrotanshinone, cryptotanshinone and

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tanshinone IIA were identified and also compared with a reference. Two further antimicrobial zones in

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the bioautograms were tentatively assigned to be methylenetanshinquinone and tetrahydrotanshinone (or

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its structural isomer methylenedihydrotanshinquinone). In another run, a validation study was

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performed for the bioquantification of ciprofloxacin and marbofloxacin via HPTLC-Bacillus subtilis.

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This pointed out the improved quality of the performance that was reached. Cryptotanshinone was

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biologically quantified in two S. m. root samples.Antimicrobials without an available reference standard

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were calculated as cryptotanshinone-bioequivalents. The results were of relevance, as 1 ng

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cryptotanshinone was calculated to be bioequivalent to 0.6 ng and 2 ng of the synthetic antibiotics,

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ciprofloxacin and marbofloxacin, respectively. For the first time, quantitative direct bioautography via

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HPTLC-Bacillus subtilis was shown as reliable tool for streamlined bioprofiling of complex samples.

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Introduction

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Scientists looking for new evidence-based natural medicine need a time-saving streamlined method

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covering screening to structure elucidation and quantification of bioactive compounds. Three major

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strategies for screening, discovery and characterization of unknown bioactive substances in complex

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mixtures are evident in literature. In the conventional strategy, compounds separated in a

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chromatographic column, were split according to their retention times and transferred or spotted onto a

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microtiter plate for the biological/biochemical/chemical assay. Such an offline coupling between

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column chromatography and biochemical detection1 was demonstrated as a high resolution fractionation

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after the high-performance liquid chromatography (HPLC)2 and gas chromatography (GC)3 column

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using a respective spotter technology.4 A split after the column transferred the eluent to mass

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spectrometry (MS) and biochemical detection (BCD).5-7

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The second strategy was an online coupling between HPLC and biochemical/chemical detection. In

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such a workflow, the eluent was split after passing the column. One stream was oriented to the

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ultraviolet (UV) detector, while the other one was mixed with a biochemical solution (in one step or two

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steps depending on the assay) and pumped to the reaction coil. After a respective incubation time, the

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eluent was directed to the visible (Vis) detector.7,8

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The third strategy was hyphenated planar chromatography.9-11 The most efficient combination of high-

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performance thin-layer chromatography (HPTLC) with any effect-directed assay (EDA) was performed

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by immersion. In such a workflow, complex sample mixtures were applied on a HPTLC plate and

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separated in parallel. All chromatograms obtained on one plate were subjected to the

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biological/biochemical/chemical assays via immersion and the single bioactive compounds were

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directly detected in their adsorbent bed. The discovered bioactive compounds were eluted and

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transferred to mass spectrometry or collected into vials for nuclear magnetic resonance (NMR) or

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infrared (IR) spectroscopy by an elution head-based interface.10-13 Such hyphenations were ideally

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performed using one chromatographic run with parallel separations of the same sample on the same ACS Paragon Plus Environment

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plate, and after plate cut, subjection of the plate sections to the different hyphenations. The hyphenation

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HPTLC-UV/Vis/fluorescence detection (FLD)-EDA-MS/NMR/IR is in some aspects superior to

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column-derived techniques combined with EDA. Especially, the simple combination of (HP)TLC with

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bioassays performed by immersion, namely direct bioautography (DB), is advantageous for screening of

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the bioactive compounds directly in the sample chromatogram, as described by Morlock et al.10,11,15,

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Choma et al.13,16, Grzelak et al.17 and Klingelhöfer et al.18,19

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Apart from the superior separation number of HPLC and GC, the HPLC-BCD or GC-BCD were

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technically complex, labor-intensive and expensive2-8. Especially with regard to microbiological assays,

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there is still no report about the rational combination of HPLC or GC with these, as such a hyphenation

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seems impossible for incubation times of several hours. There was shown only one academic attempt for

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coupling of HPLC with the instantly luminescing Aliivibrio fischeri (A. f.) bacteria20, whose practical

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usability is limited due to the huge effort required.11 Also, correlating chromatographic results with

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microtiter plate readouts and MS spectra recorded, is challenging when broad bioactivity peaks match to

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several sharp peaks in the chromatogram21. In contrast, DB hyphenated to structure elucidation

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techniques, such as NMR, IR and high-resolution (HR)MS, gives the impression of being instrumentally

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simple,

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biological/biochemical/chemical assays and capable of high sample throughput and screening in

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parallel.9-12,22 The planar chromatogram is directly subjected to biological/biochemical/chemical assays,

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and thus the correlation of chromatogram and bioautogram, which are two images taken of the same

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plate, is much easier if compared to HPLC-BCD or GC-BCD. Via HPTLC-UV/Vis/FLD-EDA-

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(HR)MS/NMR/IR,

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bioquantitative information23 of known and unknown bioactive compounds is obtainable in varying,

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complex sample matrices, analyzed in parallel on the same plate in a single run.9-19 In a recent study24,

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hyphenated HPTLC substantially contributed to structure elucidation of bioactive compounds in

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Solidago virgaurea. A latest study developed a streamlined structure elucidation workflow based on

time-saving,

powerful

comprehensive

in

visual

interpretation,

chromatographic,

applicable

spectroscopic/-metric,


for

diverse

bioprofiling

and

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planar chromatography22. However, literature showing the potential of bioquantification of antimicrobial compounds using this bioanalytical approach is lacking. The discovery of antimicrobial compounds in natural sources was chosen as case study, as the challenge of antibiotic resistance was resembled as “ticking time bomb”.25 Botanicals are highly regarded as a natural source to discover new antibiotics.26 Furthermore, plant secondary metabolites have the potential to decrease the effective therapeutic dose of synthetic antibiotics through synergistic effects.27 Tanshinones, a subgroup of diterpenes as a large group of plant secondary metabolites, are important lipophilic bioactive compounds in Salvia miltiorrhiza (S. m.).28,29 Tanshinone constituents of this traditional medicinal plant, such as tanshinone IIA, tanshinone I, and cryptotanshinone, were already reported to treat cardiovascular diseases30 and to have a broad range of activity, including antitumor and antioxidant properties.31–33 In this study, a streamlined strategy from bioprofiling and characterization to bioquantification of natural antibiotics was demonstrated exemplarily for three S. m. root samples. HPTLC was linked to the Bacillus subtilis (B. s.) and A. f. bioassays. HPTLC-HRMS was employed to characterize the discovered antibiotics in the complex samples. For the first time, the feasibility for bioquantification of the natural and synthetic antibiotics was demonstrated with regard to the HPTLC-B. s. bioassay.

EXPERIMENTAL SECTION Chemicals and Materials. Methanol, petroleum ether (40-60 °C), ethyl acetate, n-hexane, thiazol blue tetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, MTT) and all mineral salts (NaCl, KCl, NaH2PO4, Na2HPO4, K2HPO4) required for producing of phosphate-buffered saline (PBS) were purchased from Carl Roth, Karlsruhe, Germany. Müller-Hinton broth, cryptotanshinone (CT, 98%), tanshinone I (TAI, 98%), tanshinone IIA (TAII, 97%), dihydrotanshinone I (DHT, 98%), ciprofloxacin (CIP, 98%) were obtained from Sigma-Aldrich, Steinheim, Germany. Marbofloxacin (MAR, 98%) was purchased from Tokyo Chemical Industry, Tokyo, Japan. HPTLC ACS Paragon Plus Environment

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plates silica gel 60, also with F254 (20 cm x 10 cm) and B. s. spore suspension (BGA) were delivered by Merck, Darmstadt, Germany. Sample Preparation. Three S. m. root samples S1, S2 and S3 were obtained from meine-teemischung, Augsburg, Germany, HerbaSinica Hilsdorf, Rednitzhembach, Germany, and an experimental field study34, respectively. Samples S1 and S2 (both meine-teemischung) were separately ground, 100 mg each was suspended with 20 mL ethanol-water (1:1, V/V) and extracted using a magnetic stirrer for 15 min, followed by 30 min ultrasonification and centrifugation at 756 x g for 10 min. The obtained extracts were filtered through a 0.2 µm membrane filter in a brown vial and stored at -20°C until use. Sample S3 was a methanolic extract of ground S. m. roots obtained from a field study33. Its concentration was not exact, as the approximately 1-mL solvent volume of the extract was evaporated, which we redissolved in 1 mL methanol. Hence, this sample was used only quantitatively. Standard and Positive Control Solutions. Methanolic stock solutions (80 µg/mL each) were prepared for CT, DHT, TAI and TAII. For a standard mixture (diluted 1:4), 400 µL of each standard solutions were pipetted into a vial (20 µg/mL each). Stock solutions of MAR and CIP were prepared in methanol and 0.1 M hydrochloric acid (1 mg/mL), respectively, as CIP was insoluble at a neutral pH. Each stock solution was diluted with methanol to obtain a standard solution (0.5 µg/mL) that was used as a positive control solution and stored at -20°C until use. HPTLC-UV/Vis/FLD. The sample extracts (10 µL) were applied as 8-mm band on the HPTLC plate using the Automatic TLC Sampler 4, developed with 10 mL petroleum ether – cyclohexane – ethyl acetate 5:2.8:2.2 (V/V/V)34 in a twin trough chamber (20 x 10 cm or 10 x 10 cm), dried for 2 min using a stream of warm air, documented under white light illumination, at UV 254 nm and UV 366 nm using the TLC Visualizer and scanned at 264 nm (absorbance measurement) using the TLC Scanner 4 (all CAMAG, Muttenz, Switzerland). HPTLC-B. s. bioassay. Müller-Hinton broth was obtained ready to use. The bacterial suspension was prepared according to a recently published, optimized workflow.23 The cell number was monitored ACS Paragon Plus Environment

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during cultivation using a hemocytometer and the optical density (OD) at 600 nm was measured using a spectrophotometer (M501, Spectronic Camspec, Leeds, UK). The cell number and the OD600 of the medium were adjusted to be 5.5 × 106 cells in 1 mL growth broth and 0.8 ≤ OD600 ≤ 0.9, respectively, for immersion. The chromatogram was automatically dipped into the bacterial suspension using the TLC Immersion Device (CAMAG; immersion time 6 s, immersion speed 3.5 cm/s). The plate seeded with bacteria was horizontally incubated at 37°C for 2 h, in a moistened plastic box (KIS 26.5 x 16 x 10 cm, ABM, Wolframs-Eschenbach, Germany), which was covered with wet filter papers (wetted with bidistilled water) at the bottom and sides and, for temperature equilibration, already placed in an incubator before. After the incubation time, the plate was immersed into a 0.2% PBS-buffered MTT solution using the TLC Immersion Device (CAMAG; immersion time 1 s, immersion speed 3.5 cm/s). Biological reduction of MTT to a colored formazan was performed during further incubation at 37°C for 30 min. Then, the plate was completely dried on the TLC Plate Heater (CAMAG) at 50°C for 5 min. The outcome was documented with the TLC Visualizer (CAMAG) under white light illumination in the reflectance mode. The biodensitogram was recorded using inverse scanning at 546 nm23 with the TLC Scanner 4 and winCATS software v1.4.7 (both CAMAG). HPTLC-A. f. bioassay. The chromatogram was dipped (TLC Immersion Device, CAMAG; immersion time 2 s and immersion speed 3.5 cm/s) into a A. f. suspension prepared according to DIN EN ISO 11348-1.35 As A. f. is an instant assay, the bioluminescence of the seeded plate was directly documented using the BioLuminizer (CAMAG). The exposure time was 50 s for each image and ten images were taken in time intervals of 3 min to monitor a period of 30 min. HPTLC-HRMS. The bioactive tanshinone-like zones of interest on the HPTLC chromatogram were marked with a soft pencil according to their natively visible color. The marked zones were eluted with methanol (flow rate 0.1 mL/min) using the TLC-MS Interface 2 (CAMAG) or Plate Express (Advion, Ithaca, NY, USA) coupled to the QExactive Plus mass spectrometer (Thermo Fisher Scientific, Dreieich, Germany). Full scan mass spectra (m/z 100 − 800) were recorded in the positive ionization ACS Paragon Plus Environment

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mode with the following settings: ESI voltage 3.3 kV, capillary temperature 320°C and collision energy 35 eV. Nitrogen was produced by a SF2 compressor (Atlas Copco Kompressoren und Drucklufttechnik, Essen, Germany). Data evaluation and background subtraction were performed by Xcalibur 3.0.63 software (Thermo Fisher Scientific).

RESULTS AND DISCUSSION Bioprofiling of the S. m. root extracts using two bioassays. The three S. m. root sample extracts S1S3 were investigated by HPTLC. The plate was developed with a mobile phase mixture consisting of petroleum ether – cyclohexane – ethyl acetate 5:2.8:2.2 (V/V/V), which was slightly adjusted from our previous study.34 The chromatograms were documented at UV 254 nm, UV 366 nm and under white light illumination (Figure S1). All three samples S1–S3 showed the same chromatographic fingerprint. Thus, the subsequent bioprofiling was demonstrated for the S. m. root sample extract S3. The S3 solution was applied 3-fold on the HPTLC plate, separated and documented (Figure 1 A-C). The dried plate was cut into three sections (Figure S2). Each plate section containing a S3 sample chromatogram was subjected to a bioassay. Three different antibiotic zones were detected in the sample via the B. s. bioassay (Figure 1 D, zones a-c). These three B. s.-active zones were also active against A. f. bacteria; however, also further substances (zones d-f) showed an antimicrobial activity against the latter bacteria (Figure 1 E, zones a-f). As a benefit of HPTLC, attention is paid to the whole sample extract. Further, polar bioactive compounds were evident, which remained at the start region for the given chromatographic system (Figure 1 E) and would require a more polar mobile phase for separation. Thus, it is advantageous to have access to the full sample extract for bioprofiling and to get attracted to bioactive components remained at the adsorbent start. This would not be observable for column-derived techniques: what is remained at the adsorbent start will not reach the detector. Hence, such components are overlooked and not discovered as bioactive sample components. ACS Paragon Plus Environment

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The A. f. bioassay with its instant bioluminescence inhibition turned out to be a fast and simple microbiological assay, and at the same time, very informative with regard to the outcome. So, the complexity of these natural extract samples was reduced to five active zones via HPTLC-A. f .bioassay, which were further characterized in a highly targeted way. One plate section out of the three was still available for further analysis of the bioactive target compounds. Chromatographic and spectral characterization of the unknown antimicrobials. The five unknown antimicrobial compounds discovered in the S. m. root sample extract (S3) were further characterized. The comparison of the hRF values of zones in sample and standard solutions (Figure 2 A) showed that the bioactive zone a had an almost equal hRF value to DHT (hRF 30 versus 29), whereas the bioactive zones b and d had absolutely equal hRF values to CT (hRF 35) and TAII (hRF 72), respectively. Also, TAI was detected in the sample via comparison to the TAI standard zone of an equal hRF value of 48, whereas substance zone c was not assignable to one of the reference compounds (Figure 2 A). The absorbance measurement, exemplarily recorded at the maximal absorbance of the CT standard compound at 264 nm (Figure S3), showed three further absorbing and less intense substance peaks (*), but these unknowns turned out to be not bioactive at the given amounts on the plate (Figure 2 F versus G), and thus, were neglected. Also, TAI did not show any antimicrobial activity at the given sample volume applied (Figure 2 C, D versus G). Opposite to these non-actives, the antimicrobial activity of substances a-c as well as standards DHT and CT was confirmed via their biodensitograms recorded by inverse scanning at 546 nm (Figure 2 G). The overlaid standard/sample densitograms at 264 nm confirmed that substance a and DHT did not match perfectly (Figure 2 F), which needed further clarification. To exclude any matrix effect on the slight hRF value shift, the sample and standard solutions were applied on the HPTLC plate in an overlapped mode (Figure S4). This showed that the CT standard migrated to the same position of substance b in the sample. Hence, the overlapped application, and thus chromatography of the CT standard in matrix, confirmed the previous assignment. In the case of DHT, a ACS Paragon Plus Environment

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matrix shift of DHT towards zone a was not evident. Instead of migrating to a common compound zone, the two compound zones were still visible in the overlapped region (Figure S4) and substance a was found to be different to DHT. Although UV/Vis spectra do not offer the same degree of confidence as mass spectra, it can be an additional helpful tool for characterization of compounds with very similar or even identical hRF values. The in situ absorbance spectra of the unknown substances and standard compounds were recorded between 200 and 700 nm using the TLC Scanner 4 with an automated switch of the deuterium (working range 190 to 450 nm) and tungsten lamp (working range 370 to 800 nm). The spectra comparison showed a good correlation between the standard CT and substance b (Figure S3), whereas substance a did not correlate with the standard DHT between 200 and 500 nm, despite of their almost identical maximal absorption wavelengths (Figure 2 E). This confirmed the outcome of the overlapped application that substance a was different to the standard DHT. Characterization of the antimicrobials by HPTLC-HRMS. The hyphenation of HPTLC and HRMS allowed a targeted characterization of bioactive substances at the analytical scale. Although the unknown antimicrobial substance b was confirmed to be CT based on the chromatographic and spectral properties, HRMS was employed as proof. Out of question was the need for recording of HRMS spectra for substance a and standard DHT as well as substance c. However, structure elucidation of these substances demand hyphenation of HPTLC to NMR. As substances a and c migrate very close to other compounds, the isolation of these substances is focus of another study. After UV/Vis spectral analysis, the third chromatogram (third plate section, Figure S2) was still useable for HRMS. The bioactive zones a-d detected in the bioautograms were extrapolated and marked in the respective chromatogram for MS. These zones were eluted to the HRMS via an elution head-based interface. The unknown zone a showed two pronounced mass signals at m/z 301.08352 and m/z 303.09993 (Figure 3 A and Table 1). In this study, all four tanshinone standards showed the respective sodium adduct ACS Paragon Plus Environment

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signals as base peak. Regarding to this, the high mass signal at m/z 301.08352 in zone a was identified as DHT [M1+Na]+ (Table 1), as it was proven by MS2 to be an independent mass signal and not a fragment of the mass signal at m/z 303.09993. The still unknown mass signal at m/z 303.09993 was tentatively assigned to be a sodium adduct [M2+Na]+ (Table 1). It was supposedly assigned36,37 to be one

of

the

two

structural

isomers

of

methylenedihydrotanshinquinone

(MDHT)

and

tetrahydrotanshinone (THT) The identification of compound b, previously assigned as CT based on the spectral and chromatographic properties, was verified by comparing the mass spectra of the zones b and CT. In the mass spectra recorded in the positive ionization mode, the respective base peaks equaled to m/z 319.13058 [M+Na]+ and the unknown bioactive zone b was identified as CT based on its exact mass (Figure 3 B, Table 1). For zone c (Figure 3 C and Table 1), the mass signal at m/z 301.08355 was tentatively assigned36,37as methylenetanshinquinone (MTQ). Similarity of its structure to TAII supported this hypothesis, as substance c and TAII were located closely to each other on the plate at hRF values 66 and 72, respectively (Figure 3, track 1). In the mass spectrum of zone c, another smaller mass signal at m/z 303.06274 was assigned to be the oxidized MTQ (Figure 3C), which is assumedly transferred during analysis. Zone d already assigned as TAII by its chromatographic and spectral properties (Figure 2) was confirmed by HRMS (Figure 3 D and Table 1) via its mass signal at m/z 317.11477 [M+Na]+. Tanshinone IIA itself is stable, but via exposure to daylight or UV light, it might be changed to oxidation-induced substances. The mechanism of photo-oxidation of tanshinone II A was already reported.37 Hence, the other mass signal at m/z 333.10937 was tentatively assigned as the oxidized molecule of TAII, which might be produced during sample preparation or HPTLC analysis. The exact mass signal at m/z 333.10937 also fits to the hydroxylated secondary metabolite tanshinone IIB (TAIIB). However, an additional hydroxyl group makes the molecule more polar and would lead to a


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lower hRF value in contrast of TAII. Hence, we consider this mass signal as artefact. The mass signal at m/z 611.24265 was supposedly the dimer of TAII (Table 1). Implementation and validation of bioquantification via DB. According to the microorganisms` response, DB allows a quantification of bioactive substances directly in the bioautogram (bioquantification). The amount of bioactive substances can be calculated as equivalents to a reference, based on their biological activity (bioequivalence). In this regard, DB presents an outstanding capability in comparison with traditional bioassay techniques, e. g. cuvette and microtiter plate assays. Traditionally, a planimeter was applied to manually measure the inhibition zones in bioautography.38 But for improved bioautograms in modern DB (narrow zones on a homogeneous background), biodensitometry can be applied for a digital quantification of the bioautogram.23 As proof-of-principle, the mixture of two antibiotics, CIP and MAR, was applied on the HPTLC plate in the range of 2.5- 45.0 ng/band each, separated39 at hRF values 55 and 74, respectively, and documented at UV 366 nm. Three chromatograms of CIP and MAR (three different plates) were subjected to the B. s. bioassay on three different days and documented under white light illumination (Figure 4 A). The bioautograms were scanned inversely.22 In the biodensitograms (Figure 4 B), a mean intermediate precision (%RSD, n = 9) of 7.4% and 6.3% was calculated for the nine mean precisions of the CIP and MAR peak areas (n = 3, over three different days), respectively. The 9 calibration points were chosen for validation, but can be reduced to 5 calibration points for routine quantification. The logarithmic amounts per zone of both antibiotics correlated well to the corresponding signal intensities (Figure 4 C). For the three different days, the precision (%RSD) of the mean slop of the three different functions was calculated to be 6.7% for CIP; for MAR, it was 4.6% (Table S1). This meant that the calibration curve was highly reproducible and thus the bioassay detection worked reliably. The biocalibration (Figure 4 C) of CIP and MAR revealed that B. s. was more sensitive to CIP than MAR, as the slop for CIP was higher than that for MAR. Exemplarily, the antimicrobial equivalence determined for these two antibiotics showed that 40 ng/zone MAR was equal to the antimicrobial activity of 12 ng/zone CIP. ACS Paragon Plus Environment

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Bioquantification of CT in two different S. m. samples. The CT standard solution (20-200 ng/zone) and two sample extracts of different S. m. roots (S1 and S2; each in four application volumes of 1-4 µL/zone) were applied and analyzed on three different HPTLC plates and days. Ten calibration points were chosen for validation, but can be reduced to five for routine quantification. After separation, a positive control pattern each of CIP and MAR (three concentrations of 20, 40 and 60 ng/zone) was applied on the same plate (edge track). The plate was documented under white light illumination and at UV 366 nm. The densitograms were recorded at 264 nm (the maximal absorption wavelength of CT) and evaluated via the peak area (Table 2). For bioquantification of CT, this plate was subjected to the standardized workflow of the B. s. bioassay and documented under white light illumination (Figure 5 A). The biodensitograms (Figure 5 B) were recorded at 546 nm using inverse scanning. The limit of detection (LOD) and quantification (LOQ) of the substance CT were calculated as 20 ng/zone and 35 ng/zone according to the signal to noise ratio (S/N) 3 and 10 in the biodensitogram, respectively. The biocalibration curve of CT was performed by correlation of the peak areas and the logarithm of the substance amount per zone. The precision value (%RSD) of the slop for the CT biocalibration curve on three different days was 12.2%. The IC50 value of CT was determined to be 65 ng/zone, which was equal to 365 µg S1 root powder and 203 µg S2 root powder. For bioquantification, the concentration of CT in samples was calculated according to the intensity of its antimicrobial activity against the B. s. bacteria (Table 2). Intermediate precisions (%RSD, n = 3) of the CT content in the samples S1 and S2 were 16.3% and 15.2 %, respectively. Since such a detailed HPTLC-B. s. bioquantification study was reported for the first time, a comparison with literature data was not possible. To improve the reproducibility of DB results, all steps of DB, i.e. physicochemical condition of cultivation, analysis time and MTT source, were standardized in our laboratory as discussed in23. Though it is a challenge for a bioanalytical method to obtain an as good intermediate precision as for a densitometric quantification, a mean intermediate precision (%RSD, n = 3, three different days) of 15.7% was yielded for the CT content in two samples using this standardized workflow. The mean intermediate precision (%RSD) of ACS Paragon Plus Environment

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the inhibition areas of CIP and MAR (obtained by evaluation of the positive control patterns) was 13.2% for three different days. Antimicrobial equivalency in vitro. The bioquantification of known bioactive substances allowed the parallel investigation of the biological equivalent content of unknown bioactive substances in a sample. Thus, the inhibition areas of the unknown substances a and c were calculated via the CT biocalibration curve and their impact was expressed as CT bioequivalents (Table 2). In sample S1, substance c showed the most intense antimicrobial activity, whereas its biological activity in sample S2 (even when 4 µL applied) was less than the activity of the minimal CT concentration (LOD 20 ng/zone) and a calculation was not possible. The substance a showed a higher antimicrobial activity in sample S2, compared to sample S1 (Table 2). The mean precision (%RSD) of all CT bioequivalent contents determined was 10.8% for three different days. For comparison of CT as natural antibiotic with CIP and MAR as synthetic antibiotics, the peak areas of three different concentrations of CIP and MAR were calculated in the biocalibration curve of CT. The peak area of 20 ng/zone MAR was less than that of the minimal CT concentration (20 ng/zone). These values were considered as semi-bioequivalency values, as CIP and MAR were not developed on the plate (only applied as positive control pattern). It was calculated that 1 ng/zone CT showed an equivalent antimicrobial activity to 0.6 ng/zone CIP and 2.0 ng/zone MAR. The mean intermediate precision (%RSD, n = 3) of this semi-bioequivalency calculation was 17.6%.

CONCLUSIONS HPTLC-UV/Vis/FLD-EDA-HRMS proved to be well-suited as a high-throughput bioanalytical tool. For the first time, the applied B. s. bioassay demonstrated the streamlined strategy from screening, characterization and identification to bioquantification of natural antibiotics in S. m. root extracts. Sample preparation and instrumental effort were kept simple. The bioassay was directly applied in the chromatogram, which made the direct correlation of chromatogram and bioautogram easy. The ACS Paragon Plus Environment

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antimicrobial activity in a S. m. root extract was characterized via chromatographic, spectroscopic and HRMS data. For bioquantification an inverse densitometric measurement was employed, and the impact of two unknown antibiotics was specified via calculation of their CT bioequivalency. The importance of the antimicrobial results was underlined via a semi-bioequivalency calculation referred to two synthetic antibiotics, CIP and MAR. All these calculations were performed in a single run on the same plate. This strategy can be installed in every analytical laboratory without much microbiological effort. The type of bacteria can be selected as required for the intended antibiotic development. HPTLC linked to microbiological bioassays with pathogenic bacteria will be of high relevance, in combination with HRMS/NMR/IR and bioquantification. The potential of this streamlined hyphenation can contribute to discovery of new antibiotic structures from natural sources, since antibiotic resistance is a current challenge of public health and pharmaceutical industry.

ASSOCIATED CONTENT

Supporting Information Supporting Information Available: Table S1 shows the validation of the HPTLC-B. s. bioquantification, exemplarily for the two synthetic antibiotics CIP and MAR, Figure S1: HPTLC chromatograms of three different S. m. root samples, Figure S2: HPTLC chromatograms showing the three-fold application and parallel development of sample S3 on a HPTLC plate, Figure S3: Overlaid absorption spectra of unknown substance b and CT standard recorded between 200-800 nm, Figure S4: HPTLC chromatograms via overlapped application and chromatography on HPTLC plates silica gel 60 of standard mixture and sample S3. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author


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*E-mail: [email protected]. Tel. +49-(0)641-99-39141. Fax +49-(0)641-99-39149.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

Thank is owed to Merck Millipore, Darmstadt, Germany, and CAMAG, Muttenz, Switzerland for support with regard to plates and instrumentation, respectively.


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(19) Klingelhöfer, I.; Morlock, G. E. Anal. Chem. 2015, 87 (21), 11098–11104. (20) Eberz, G.; Rast, H.-G.; Burger, K.; Kreiss, W.; Weisemann C. Chromatographia 1996, 43 (1−2), 294–301 (21) Falck, D.; de Vlieger, J. S. B.; Giera, M.; Honing, M.; Irth, H.; Niessen, W.M.A.; Kool, J. Anal. Bioanal. Chem. 2012, 403 (2), 367–375. (22) Yüce, I.; Morlock G.E. Streamlined structure elucidation of an unknown compound in a pigment formulation, J. Chromatogr. A, in print. (23) Jamshidi-Aidji, M.; Morlock, G. E. J. Chromatogr. A 2015, 1420, 110–118. (24) Móricz, Á. M.; Ott, P. G.; Häbe, T. T.; Darcsi, A.; Böszörményi, A.; Alberti, Á.; Krüzselyi, D.; Csontos, P.; Béni, S.; Morlock, G. E. Anal. Chem. 2016, 88 (16), 8202–8209 (25) Gaudêncio, S. P.; Pereira, F. Nat. Prod. Rep. 2015, 32 (6), 779–810. (26) Cowan, M. M. Clin. Microbiol. Rev. 1999, 12 (4), 564–582 (27) Shahverdi, A. R.; Abdolpour, F.; Monsef-Esfahani, H. R.; Farsam, H. J. Chromatogr. A, 2007, 850 (1-2), 528–530. (28) HU, P.; LUO, G.-A.; ZHAO, Z.; JIANG, Z.-H. Chem. Pharm. Bull. 2005, 53(5) 481—486 (29) Dong, Y.; Morris-Natschke, S. L.; Lee, K.-H. Nat. Prod. Rep. 2011, 28 (3), 529–542. (30) Chang, J.-Y.; Chang, C.-Y.; Kuo, C.-C.; Chen, L.-T.; Wein, Y.-S. Mol. Pharmacol 2004, 65 (1), 77–87 (31) Shang, Q.; Xu, H.; Huang, L. J Evid Based Complementary Altern Med., 2012, DOI:10.1155/2012/716459 (32) Wang, B.-Q. J. Med. Plants Res. 2010, 4 (25), 2813–2820. (33) Zhao, J.; Lou, J.; Mou, Y.; Li, P.; Wu, J.; Zhou, L. Molecules 2011, 16 (3), 2259–2267. (34) Morlock, G.E.; Scholl, I.; Sung,Y.H.; Yan, F.; Honermeier, B., Justus Liebig University Giessen, unpublished work of 2013, sample S3 was cultivated in the experimental field of Institute of Agronomy and Plant Breeding by YHS, FY and BH. A mobile phase was developed for separation of sample extracts and analysis was performed by IS and GEM, personal communication in May 2015. (35) DIN EN ISO 11348-1, Part 1, Section 5; Beuth-Verlag: Berlin, 2009. (36) Chang, H. M.; Cheng, K. P.; Choang, T. F.; Chow, H. F.; Chui, K. Y.; Hon, P. M.; Tan, F. W. L.; Yang, Y., Zhong, Z.P. J. Org. Chem. 1990, 55(11), 3537-3543 (37) Luo, H. In Dan Shen (Salvia miltiorrhiza) in Medicine: Biology and Chemistry, Yan, X., Ed.; Springer: Dordrecht, 2015; pp 143-189


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(38) Grzelak, E. M.; Majer-Dziedzic, B.; Choma, I. M.; Pilorz, K. M. J AOAC Int. 2013, 96 (2), 386– 391. (39) Cardak, A. D.; Morlock, G.E. Poster at 43. Deutscher Lebensmittelchemikertag, Giessen, 2014.


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Table 1. The antimicrobial substances a-d in the S. m. extract S3 detected by HPTLC-B. s. bioassay and assigned via HPTLC-HRMS

substance hRF zone

observed m/z

theoretical m/z

formula

error (ppm)

assignment

[M1+Na]+

301.08352

301.08406

C18H14NaO3+

-1.8

DHT**

[M2+Na]+

303.09993

303.09971

C18H16NaO3+

0.7

MDHT/THT*

a

30

b

35

[M+Na]+

319.13058

319.13047

C19H20NaO3+

0.3

CT**

c

66

[M+Na]+

301.08355

301.08352

C18H14NaO3+

0.1

MTQ*

[M+Na]+

317.11477

317.11482

C19H18NaO3+

-0.2

TAII**

[2M+Na]+

611.24265

611.24041

C38H36NaO6+

2.8

dimer of TAII*

d *

measured m/z

72

were tentatively assigned according to exact mass were assigned according to exact mass and confirmed with reference substances

**


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Table 2 Bioquantification results of substance CT obtained by biodensitometry at 546 nm versus results of densitometry at 264 nm and mean CT bioequivalency calculation of substances a and c in S1 (for 3 and 4 µL applied) and S2. S. m. root extract sample

450 451 biodensitometry versus densitometry S1

S2

CT content in S. m. (mg/g)

1.8 versus 1.6

3.2 versus 3.9

repeatability (%RSD, n=2)

8.7 versus 0.4

7.0 versus 0.9

intermediate precision (%RSD, n=3)

16.3 versus 1.4

15.2 versus 1.6

a

1.1 ± 0.09

2.2 ± 0.02

c

2.7 ± 0.03

< LOD

mean CT bioequivalent content in S. m. ± SD (mg/g, n=2)


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Figure legends Figure 1. HPTLC chromatograms of the S. m. root sample extract S3 (10 µL) recorded at UV 254 nm (A), 366 nm (B), under white light illumination (C); bioautograms after B. s. (D) and A. f. (E) bioassays. Figure 2. HPTLC chromatograms of a standard mixture consisting of DHT, CT, TIA and TIIA (track 1, 40 ng/band each) and S. m. root sample extract S3 (track 2, 5 µL) recorded under white light illumination (A), at UV 366 nm (B), after A. f. (C) and B. s. bioassays (D). HPTLC spectra (200-700 nm) of substance a in sample S3 as well as DHT (E). Overlaid densitograms of absorbance measurement of tracks 1 and 2 recorded at 264 nm (F); Overlaid biodensitograms of tracks 1 and 2 recorded after B. s. bioassay by inverse scanning at 546 nm (G); chromatogram tracks were cut in the upper part. Figure 3. Mass spectra (ESI+) recorded for the antimicrobial zones a (A), b (B), c (C) and d (D) in sample S3 (5 µL) eluted into the HRMS system via an elution-head based interface as well as HPTLC chromatogram (10 µL, track 1), after B. s. (track 2) and A. f. bioassays (track 3), recorded under white light illumination and with the BioLuminizer, respectively;, chromatogram tracks were cut in the upper part. Figure 4. B. s. bioautograms of a mixture of CIP and MAR at nine concentrations between 2.5 ng/zone (track 1) and 45.0 ng/zone (track 9) each with an interval of 5.0 ng/zone (A); the biodensitograms were extracted from the chromatogram by inverse scanning at 546 nm (B); biocalibration curves were shown for CIP and MAR (C). Figure 5. HPTLC-B. s. bioautograms documented under white light illumination (A) for the bioprofiling of substances a-c in the samples S1, S2 (1, 2, 3 and 4 µL applied), CT standard (20-200 ng/zone with intervals of 20 ng/zone) and a positive control pattern of CIP and MAR (20, 40 and 60 ng/zone each) applied on the edge track (P); respective biodensitograms recorded by inverse scanning at 546 nm (B). ACS Paragon Plus Environment

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Figure 1


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Figure 2


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Figure 3


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Figure 4


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Figure 5


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For TOC only


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