Fast Equivalency Estimation of Unknown Enzyme Inhibitors in

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Fast Equivalency Estimation of Unknown Enzyme Inhibitors in situ the Effect-Directed Fingerprint, shown for Bacillus Lipopeptide Extracts Maryam Jamshidi-Aidji, and Gertrud Elisabeth Morlock Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03407 • Publication Date (Web): 11 Nov 2018 Downloaded from http://pubs.acs.org on November 13, 2018

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Analytical Chemistry

Fast Equivalency Estimation of Unknown Enzyme Inhibitors in situ the Effect-Directed Fingerprint, shown for Bacillus Lipopeptide Extracts Maryam Jamshidi-Aidjia and Gertrud E. Morlocka,* aChair

of Food Science, Institute of Nutritional Science, and Interdisciplinary Research Center, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany *Corresponding Author: fax +49-641-99-39149; [email protected] ABSTRACT: The hyphenation of high-performance thin-layer chromatography (HPTLC) with enzyme inhibition assays followed by high-resolution mass spectrometry (HRMS) represents a targeted profiling required in the development of new natural pharmaceuticals, functional food and cosmetics. This direct combination of a chromatogram with an enzymatic assay substantially extents the understanding of inhibitor properties in vitro. For the first time, a straightforward workflow was established for estimating the equivalency of unknown inhibitors directly in the autogram. Exemplarily, lipopeptides produced as secondary metabolites by five different Bacillus strains were analyzed by HPTLC hyphenated with the tyrosinase and acetylcholinesterase (AChE) assays. Lipopeptides that showed an inhibition were characterized by HPTLC-HRMS. Among the many reports about the biological properties of lipopeptides, their enzyme inhibitory properties are new. The most intense inhibitors were identified as surfactin and iturin A according to reference substances and exact masses. Three further inhibitors were supposedly assigned as fengycin, iturin C, and surfactin methyl ester according to their exact masses. The inhibitory activities of surfactin and iturin A were quantitatively compared with kojic acid and piperine, as references for common natural inhibitors. Their equivalently calculated tyrosinase inhibition showed that 1 µg kojic acid was equal to 1.8 µg and 3.2 µg of iturin A and surfactin, respectively; regarding to AChE inhibition, 1 µg piperine was equal to 1.7 µg and 0.6 µg of iturin A and surfactin, respectively. Further unknown enzyme inhibitors found in the sample were exemplarily calculated as surfactin, iturin A, kojic acid and piperine equivalents to estimate their importance.

Developing a new natural product, starting from the original idea up to the market product, is a multidisciplinary pipeline, which takes many years for in vitro and in vivo investigation of the product properties.1 The basic research starts with the screening and identification of bioactive molecules from natural sources. This research ideally leads to find potent molecules in vitro, termed drug development candidates for investigation in vivo. While secondary metabolites are promising agents for natural product development, new analytical tools are greatly required, which expedite the discovery of bioactive metabolites within the chemical diversity of natural samples.2,3 In the conventional strategy, compounds separated from a complex sample in a chromatographic column were split according to their retention times and spotted as fractions onto a microtiter plate for effect-directed analysis (EDA).4–6 In contrast, high-performance thin-layer chromatography (HPTLC) hyphenated in situ to any EDA and structure elucidation technique is capable to provide a fast profiling of different complex samples in parallel with regard to single bioactive compounds. 7–9 Especially, modern quantitative direct bioautography (DB) together with the immense flexibility of HPTLC to obtain comprehensive information on unknown compounds proved to be highly efficient and straigthforward.9–14

For further characterization, the detected bioactive zones were eluted directly from the adsorbent bed to high-resolution mass spectrometry (HRMS) by an elution head-based interface or were collected into vials for nuclear magnetic resonance (NMR) or infrared (IR) spectroscopy.15–17 A direct desorption of bioactive compounds directly from the bioautogram via scanning Direct Analysis in Real Time (DART)-MS was recently reported as a straightforward workflow (DB-DARTMS).18-19 In contrast to column-derived techniques, HPTLC hyphenation is capable to evaluate the activity of active components detected in different samples directly in the chromatogram.20–22 Such quantitative chromatographic and spectroscopic/-metric information were obtained in complex sample matrices, analyzed in parallel in a single chromatographic run. Recently, hyphenations in HPTLC were shown as reliable tool for quantification of biologically active substances directly in the bioautogram based on their biological responses.23-25 The HPTLC hyphenation is capable to estimate the importance of the discovered active components detected in different samples via activity equivalency calculation referred to common drugs. So far, estimating the equivalency of enzyme inhibition for unknown bioactive compound zones has not been reported. The equivalency estimating of tyrosinase and acetylcholinesterase (AChE) inhibitors were chosen as case

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study. For showing the specificity of the discovered inhibitors, the opposite assay could be taken as negative control. Tyrosinase inhibitors were applied in the treatment of some dermatological issues, e.g. hyperpigmentation and malignant melanoma,26 as well as the preventing of undesirable browning of fresh-cut fruits and vegetables.27 Kojic acid, a common natural tyrosinase inhibitor, is a fungal secondary metabolite produced by Aspergillus, Penicillium, and Acetobacter.28 AChE inactivation is used as treatment in Alzheimer's disease and by insecticides in agriculture.29 Piperine, the main alkaloid of Piper nigrum, is a famous natural AChE inhibitor.30 Bacteria are of commercial interest for natural products development due to the possibility to yield the target metabolite through an industrial bioprocess.31,32 Bacterial cyclic lipopeptides (LPs) are the most common secondary metabolites produced by Bacillus species.33 These amphiphilic compounds are constituted by a hydrophobic fatty acid chain linked to a hydrophilic cyclic peptide moiety. Several isoforms can be produced by the same strain, which differs by the length of the fatty acid chain, the amino acid composition of the peptide moiety and the type of link between the two parts. According to strain types and environmental conditions, Bacillus cultures may involve different types and amounts of LPs. For bacterial extract analysis, hyphenated HPTLC allows a qualitative and quantitative tracking of single bioactive metabolites in varying cultivated strains analyzed in parallel. Thereby, HPTLC-EDA-(HR)MS/NMR is able to present a discriminating benefit compared to column-derived techniques, since the prioritization34 of microbial strains of high potential for producing the target metabolites as well as the characterization of single bioactive metabolites in various bacterial extracts can be simultaneously performed in the same bioanalytical run. However, literature showing the potential of hyphenated HPTLC for bacterial metabolite analysis is limited. In this study, a bioanalytical tool was demonstrated for bacterial strain screening, targeting specific secondary metabolites. The LPs found in five different Bacillus strains showing inhibitory activity were detected via HPTLCtyrosinase/AChE assays and HPTLC-HRMS. For the first time, a quantitative planar chromatographic enzyme assay was employed for estimating the equivalency of tyrosinase and AChE inhibiting LPs extracted from Bacillus cultures.

Experimental Section Chemicals and Materials. Aceton, chloroform, ethyl acetate, ethanol, isopropyl acetate, and methanol (all HPLC quality) as well as dipotassium phosphate, disodium phosphate, 3-[(3-cholamidopropyl) dimethylammonio]-1propanesulfonate (CHAPS), polyethylene glycol (PEG) 8000 and kojic acid (KA, 5-hydroxy-2-hydroxymethyl-4H-pyran-4one, 99%) were purchased from Carl Roth, Karlsruhe, Germany. Bidistilled water was prepared with Destamat Bi 18E (Heraeus, Hanau, Germany). Surfactin (98%) and iturin A (95%) from Bacillus subtillis, tyrosinase from mushroom, AChE from Electrophorus electricus, piperine (PIP, 1piperoylpiperidine) (97%), fast blue salt B (95%), primuline (≥50% as sodium salt), tris(hydroxy-methyl) aminomethane (TRIS, 99.8%), methanol (MS quality) and bovine serum albumin (BSA) were obtained from Sigma-Aldrich, Steinheim, Germany. HPTLC plates silica gel 60 (20 cm×10 cm) were delivered by Merck, Darmstadt, Germany. Levodopa ((2S)-2amino-3-(3,4-dihydroxyphenyl) propanoic acid, 97%) and α-

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naphthyl acetate (97%) were obtained from Santa Cruz Biotechnology, Dallas, USA, and Panreac, Barcelona, Spain, respectively. Standard and Positive Control Solutions. Stock solutions of 1 mg/mL were prepared for surfactin and iturin A with methanol. Each stock solution was diluted with methanol to obtain a standard mixture that was used for quantification (250 ng/µL) or limit of detection (LOD) and quantification (LOQ) studies (100 ng/µL). For positive control solutions, stock solutions of 1 mg/mL were prepared with methanol for PIP and KA. Each stock was 1:10 diluted with methanol to obtain standard solutions of 100 ng/µL. All solutions were stored at -20 C until use. Description of Samples. Five strains of the genus Bacillus were selected from the large microbiological collection belonging to Professor Dr. Slaviša Stanković, Faculty of Biology, University of Belgrade. The selected strains of different locations in Serbia named in the collection SS-10.7, SS-12.6, SS-13.1 and SS-38.4 were isolated from soil samples, and the strain SS-27.2 was from manure. Each Bacillus culture supernatant was extracted by Dr. Ivica Dimkić et al. as published.35,36 Briefly, the dried ethyl acetate extract obtained from 1 L cell-free bacteria broth (ca. 25 mg) was either dissolved in 1 mL methanol (Ex-1)35,36 or the cell-free broth supernatant was acidified with hydrochloric acid and the resulting precipitated pellet was dissolved in 1 mL methanol (Ex-2)37. Both extracts had a concentration of about 25 mg/mL. HPTLC Method. The sample (1 or 2 µL) and standard solutions (1 µL, for validation 0.1-20 µL) were applied as 8mm band on the HPTLC plate using the Automatic TLC Sampler 4, developed with 5 mL or 10 mL ethyl acetate – methanol - water 75:14:11 (V/V/V) in a twin trough chamber (10 cm×10 cm or 20 cm×10 cm). The chromatogram was dried for 2 min using a stream of cold air, scanned at 190 nm using the TLC Scanner 4, immersed into the primuline reagent (0.5 g primuline in 100 mL acetone - water 4:138), dried for 2 min using a stream of cold air and documented at UV 366 nm using the TLC Visualizer. Data were evaluated using winCATS software (all CAMAG, Muttenz, Switzerland). HPTLC-Tyrosinase Assay. Tyrosinase inhibitors were detected using the Derivatizer (CAMAG) based on Taibon et al.39,40, however, the workflow was improved41. The substrate solution was prepared by dissolving 45 mg levodopa, 25 mg CHAPS and 75 mg PEG 8000 in 10 mL phosphate buffer (0.02 M, pH 6.8) and stored at -20 C until use. The chromatogram was placed in the Derivatizer on a sheet of filter paper, sprayed with 2 mL of substrate solution, and subsequently dried for 2 min using a stream of cold air. Then, it was sprayed with 2 mL enzyme solution (800 units in 2 mL phosphate buffer) and horizontally incubated in a moistened plastic box (KIS 26.5 cm × 16 cm × 10 cm, ABM, WolframsEschenbach, Germany), which was covered with wet filter papers at room temperature for 15 min. The humid plate was dried using the drying mode of the Automatic Developing Chamber 2 (ADC 2) for 8 min. The autogram was documented under white light illumination (reflectance mode) using the TLC Visualizer. The maximum wavelength of the assay background color (530 nm) on the HPTLC plate was measured by recording of UV/vis spectra. To avoid the peak conversion, the inverse measurement of the absorbance (selecting fluorescent mode with no filter) was performed at this

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Analytical Chemistry maximum wavelength using the TLC Scanner 4 (all CAMAG devices, if not stated otherwise). HPTLC-AChE Assay. AChE inhibitors were detected according to a recently optimized workflow.42 Briefly, the chromatogram was immersed into an enzyme solution (666 units AChE and 100 mg BSA in 100 mL TRIS buffer, 0.05 M, pH 7.8) using the TLC Chromatogram Immersion Device (immersion time 5 s, immersion speed 2.5 cm/s), incubated in a horizontal position in a plastic box (ABM) under humidified atmosphere at 37 C for 25 min and immersed into the substrate solution (25 mg α-naphthyl acetate and 50 mg Fast Blue Salt B in 90 mL ethanol - water, 1:2, immersion time 1 s, immersion speed 2.5 cm/s). Documentation and densitometry were performed as for the tyrosinase assay (all CAMAG devices, if not stated otherwise). HPTLC-HRMS. LPs were marked with a soft pencil (LPs fluoresce bluely at UV 366 nm due to physisorption of primuline) and directly eluted with methanol (60 s, flow rate 0.1 mL/min) using an elution head-based interface (Plate Express, Advion, Ithaca, NY, USA) coupled to the QExactive Plus mass spectrometer (Thermo Fisher Scientific, Dreieich, Germany). All full scan mass spectra (m/z 200-3000) were recorded in the negative/positive ionization mode with a resolution of 140.000 using automatic maximum injection time 10/200 ms, spray voltage ±3.5 kV and capillary temperature 270 °C. The obtained data was processed with Xcalibur 3.0.63 software (Thermo Fisher Scientific). A representative plate background (showing the primuline mass signal in the positive mode) was subtracted from the analyte mass spectrum. Method Validation. The peak areas obtained from densitograms before and after enzymatic assays were used for quantification. The method was validated regarding to linearity and precision of the calibration slop. LOD and LOQ were calculated according to the guidance of the U.S. Food and Drug Administration43 by signal-to-noise ratios (S/N) 3 and 10 as well as calibration curve method based on DIN 32645.

chromatographic detection enabled a rapid, visual evaluation of the chromatograms obtained by each mobile phase. The mobile phase 10 consisting of ethyl acetate – methanol - water 75:14:11 (V/V/V) showed the best separation performance among the investigated alternative for the mobile phase used in previous study (Table S-1).50 Enzyme Inhibition Profiling of Bacillus Extracts. The LP samples extracted by two methods (Ex1 and Ex2) from five cultivated Bacillus strains (SS-10.7, SS-12.6, SS-13.1, SS-27.2 and SS-38.4) were investigated. The ten extracts were applied twice, i.e. on two HPTLC plates. The plates were developed by the mobile phase optimized in this study. The obtained chromatograms were subjected to the tyrosinase and AChE assays and afterwards documented under white light illumination. White or light inhibition zones were detected on a purple background for the AChE assay and on a greyish background for the tyrosinase assay (Figure 1). Regarding both assays, the SS-13.1 strain showed least inhibition activity, if compared with all other strains. The SS-12.6 strain had an activity profile like the SS-27.2 strain, as zones a-c were only detectable in these two strains. Zones a (hRF 35) and b (hRF 40) inhibited both AChE and tyrosinase, while zones c (hRF 42) and f (hRF 85) were selective for AChE versus tyrosinase inhibition. Zone e (hRF 61) showed a very intensive AChE inhibition in all samples (except SS-13.1), whereas it was slightly tyrosinase inhibiting. Zone d (hRF 59), migrating very close to zone e, showed a slight AChE inhibition in the most samples. These discovered inhibitors were further characterized. All in all, the samples showed a higher AChE inhibition compared with the tyrosinase inhibition.

Results and Discussion The combination of HPTLC with enzymatic assays reduced the whole complexity of a sample to comprehensive information about few inhibitors. This hyphenation was efficiently performed via automated immersion or automated spraying. Both techniques are crucial for a homogeneous background, and thus, for quantitative studies. For further characterization, the detected bioactive zones were eluted directly from the adsorbent bed to HRMS (Figure S-1) Mobile Phase Development. To help chemists in the selection of more healthy and environmentally friendly solvents, the American Chemical Society’s Green Chemistry Institute, Pharmaceutical Roundtable, published a green solvent selection guide (GSG).44,45 The mobile phase previously demonstrated for separation of LPs on a (HP)TLC silica gel layer included 70% of chloroform.36,46 In this study, the use of solvents banned in GSG was avoided by developing an alternative mobile phase. Several solvent mixtures were investigated, exemplarily for Ex2 of SS-27.2 (Table S-1). The chromatogram was detected at UV 366 nm after immersion in the primuline reagent (Figure S-2). Physisorption of primuline at the lipophilic fatty acid chain of the LPs makes these detectable as blue fluorescent zones at UV 366 nm. This post-

Figure 1. Profiling of AChE (A) and tyrosinase (B) inhibitors a–f: autograms of five Bacillus strains (1 µL/band) extracted according to Ex1 (1) and Ex2 (2) documented under white light illumination.

Characterization of Discovered Inhibitors by hRF Values. All samples were analyzed along with surfactin and iturin A (each 1 µg/zone) and the densitogram was recorded at 190 nm. The comparison of the hRF values of the zones a–f in the samples with the standards showed that the zone b and e had equal hRF values to iturin A (hRF 40) and surfactin (hRF 61),



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Figure 2. HPTLC-ESI--HRMS spectra of inhibition zones a (A), b (B), c (C), d (D), e (E), and f (F) in Ex2 sample of SS-27.2 (2 µL/band) recorded after detection with the primuline reagent at UV 366 nm on HPTLC plate silica gel 60 as well as HPTLC images after primuline detection at UV 366 nm (tracks 1 and 2: overlapped application of sample with standard mixture of 1-µg/band surfactin and 250-ng/band iturin A on track 2), tyrosinase (track 3) and AChE (track 4) assays, both latter under white light illumination.

respectively (Figure S-3). An additional substance was evident in the surfactin standard, which migrated close to the main compound and showed an equal hRF value of 59 to zone d. To exclude any matrix effect on the hRF value shift, the sample and standard solutions were applied on the HPTLC plate in an overlapped mode, separated and detected with the primuline reagent, exemplarily shown for Ex2 of SS-27.2 stain (Figure 2, tracks 1 and 2). It showed that the reference substances completely migrated in the equal position of zone b, e, and d in the sample. This preliminary assignment was proved by HRMS in the following. Characterization of Discovered Inhibitors by HRMS. The hyphenation of HPTLC and HRMS allowed further characterization of the discovered AChE/tyrosinase-inhibiting LPs. As primuline is a lipophilic dye that is only physisorbed at the LPs, the chromatogram visualized by the primuline reagent was used for HRMS analysis. The blue fluorescent LP bands were marked at UV 366 nm with a soft pencil and eluted into HRMS via an elution head-based interface.

For zone a, the mass spectrum recorded in the negative ionization mode showed a mass signal cluster ranged from m/z 1461.7825 to m/z 1517.8540. The positive ionization mode provided their respective sodium adducts (m/z 1485.7827 to m/z 1541.8487, Figure 2A and Table 1). According to previous studies,47,48 these m/z series were tentatively assigned as fengycin homologues. The mass signals differed in 14 Da, confirming the difference of a methylene (–CH2) group in the fatty acid chain or amino acid substitution of valin (Val) by alanine (Ala) in the peptide moiety, which occurred biologically in the bacterial LP metabolism.49 The high mass signals in the negative and positive ionization modes at m/z 1503.8375 ± 1.3 ppm [M4-H]- and m/z 1527.8330 ± 0.6 ppm [M4+Na]+ were tentatively assigned to be C17/Ala fengycin or C16/Val fengycin according to their exact masses (Figure 2A). The zone b was preliminary assigned as iturin A based on its chromatographic and spectral properties, if compared to the iturin A standard. This assignment was verified and further specified to be C15-iturin A by HRMS due to pronounced


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Analytical Chemistry mass signals in the negative and positive ionization mode at m/z 1055.5529 [M2-H]- and m/z 1057.5681 [M2+H]+, followed by its sodium adduct at m/z 1079.5499 [M2+Na]+ (Table 1 and Figure 2B). Another small mass signal at m/z 1041.5363 [M1-H]- as well as at m/z 1043.5523 [M1+H]+ was identified as C14-iturin A. Pronounced mass signals recorded for zone c in the negative ionization mode were at m/z 1042.5245 [M1-H]- and m/z 1056.5373 [M2-H]-, differing by a methylene substitute. In the positive ionization mode, the protonated molecules were obtained at m/z 1044.5372 [M1+H]+ and m/z 1058.5527 [M2+H]+, followed by the respective sodium adducts at m/z 1066.5188 [M1+Na]+ and m/z 1080.534 [M2+Na]+. These mass signals were supposedly assigned as iturin C homologues according to their exact masses as well as previous literature,47 in which iturin C contained a peptide moiety with aspartic acid instead of asparagine in iturin A. Table 1. Discovered enzyme inhibitors a–f in Ex1 sample of Bacillus strain SS-27.2 detected by HPTLC-AChE/tyrosinase assays and assigned by HPTLC-HRMS Measured m/z

Theoretical Error Ionization m/z [ppm] mode

Molecular formula

Assignmen t

Bioactive zone a 1485.7827

1485.7852

1.7

1461.7825

1461.7887

4.2

1499.8027

1499.8008

1.3

1475.8069

1475.8043

1.8

1513.8179

1513.8165

0.9

1489.8159

1489.8199

2.7

1527.8330

1527.8321

0.6

1503.8375

1503.8356

1.3

1541.8487

1541.8478

0.6

1517.8540

1517.8513

1.8

[M1+Na]+ C72H110N12O2 0 [M1-H][M2+Na]+ C73H112N12O2 0 [M2-H][M3+Na]+ C74H114N12O2 0 [M3-H]-

Fengycin

[M4+Na]+ C75H116N12O2 0 [M4-H][M5+Na]+ C76H118N12O2 0 [M5-H]-

The zones d and e had equal hRF values to the two substance zones of the surfactin standard, and were preliminary assigned as such (Figure 2, tracks 1 and 2). The mass signals obtained for zones d and e differed in a methylene group in the fatty acid chain or substitution of Val and Ala amino acids in the peptide moiety, indicating surfactin homologues. For zone d, the base peak at m/z 1020.6619 [M1-H]- and its sodium adduct at m/z 1044.6573 [M1+Na]+ were obtained, while the zone e provided two pronounced deprotonated molecules at m/z 1034.6776 [M2-H]- and m/z 1048.6931 [M3-H]- as well as the corresponding potassium and sodium adducts in the positive ionization mode at m/z 1074.6459 [M2+K]+, 1058.6732 [M2+Na]+ and 1088.6611 [M3+K]+ (Table 1, Figure 2D and E). The respective zones of the surfactin standard provided the same base peaks (data not shown). Thus, the identification of surfactin in the sample was verified. Zone f provided a major deprotonated molecule at m/z 1062.7087 [M-H]-. The respective sodium adduct was obtained at m/z 1086.7044 [M+Na]+. This zone was tentatively assigned as dimethyl surfactin based on its exact mass. This substance was formed by a methyl esterification of glutamic acid and aspartic acid in the peptide moiety, as reported previously.50 This reaction might be caused during the acidic culture extraction or biologically by strains, as reported for the biologically produced methylated surfactin.51 Quantification of Surfactin and Iturin in Samples. Surfactin and iturin A was applied from 50 ng/zone to 2 µg/zone together with all samples (each 1 µL) on the same HPTLC plate. After separation, the densitogram was recorded by absorbance measurement at 190 nm (Figure 3B). This was reproduced 4 times, and thus, analyzed on five different days (n = 5). The calibration curves (peak areas) of surfactin and iturin A showed a good linear regression (R2>0.999). For visualization of LPs, the chromatogram was immersed in the primuline reagent and documented at UV 366 nm (Figure 3A).

Bioactive zone b 1043.5523

1043.5520

0.3

[M1+H]+

1041.5363

1041.5369

0.5

[M1-H]-

1057.5681

1057.5677

0.4

[M2+H]+

1079.5499

1079.5496

0.3

1055.5529

1055.5526

0.3

[M2-H][M1+H]+

C48H74N12O14 Iturin A

[M2+Na]+ C49H76N12O14

Bioactive zone c 1044.5372

1044.5360

1.1

1066.5188

1066.5180

0.8

1042.5245

1042.5215

2.9

[M1-H]-

1058.5527

1058.5517

0.9

[M2+H]+

1080.5343

1080.5336

0.6

1056.5373

1056.5371

0.2

[M2-H]-

1044.6567

0.6

[M1+Na]+

1020.6619 1020.6602 1074.6459 1074.6463 1058.6732 1058.6724 1034.6776 1034.6759 1088.6611 1088.6620 1048.6931 1048.6915 Bioactive zone f 1086.7044 1086.7036 1062.7087 1062.7072

1.7 0.4 0.7 1.6 0.8 1.5

C52H91N7O13 [M1-H][M2+K]+ [M2+Na]+ C53H93N7O13 [M2-H][M3+K]+ C54H95N7O13 [M3-H]-

0.7 1.4

[M1+Na]+ Surfactin C55H97N7O13 methyl ester [M1-H]-

[M1+Na]+ C48H73N11O15 Iturin C

[M2+Na]+ C49H75N11O15

Bioactive zones d and e 1044.6573

Surfactin

Figure 3. HPTLC chromatogram at UV 366 nm by detection with primuline reagent (A) for profiling of LPs produced by five different Bacillus strains extracted with two different methods Ex1 and Ex2 (each 1 µL applied on tracks 1 and 2) along with standard mixture of surfactin and iturin A applied at five concentrations (S1-S5: 50, 100, 250, 1500 and 2000 ng/zone) as well as respective 3D densitogram at 190 nm (B) recorded before immersion into the primuline reagent.



The reproducibility (%RSD, n = 5) of the slop of five linear functions on different days (and HPTLC plates) was calculated to be 3.8% and 4.4% for surfactin and iturin A, respectively. For calculation of the LODs and LOQs, a standard mixture of surfactin and iturin A (each 100 ng/µL) was applied with increasing application volumes corresponding to nine concentrations between 10 ng/zone and 2 µg/zone on three different days and plates. LOD and LOQ values were calculated (calibration curve method) as 15 and 36 ng/zone for surfactin as well as 12 and 27 ng/zone for iturin A. In this case, the reproducibility (%RSD, n = 3) of the slop of three linear functions on different days (and HPTLC plates) was calculated to be 3.4% for surfactin and 2.8% for iturin A. The quantification performed after detection with the primuline reagent showed by factor of 9 and 11 higher LOD values for iturin A and surfactin, respectively (data not shown). The contents of surfactin and iturin A were calculated in five dried cell-free Bacillus cultures extracted with two different methods. A corresponding peak for surfactin was not detectable in the both extractions of strain SS-13.1, whereas its Ex1 showed a small iturin A peak at the given amounts on the plate. The results showed that the contents of both substances were higher in Ex1 than in Ex2 samples of all strains (Table 2). However, the substances showing a low hRF (10 to 40) in Ex2 samples of SS-12.6 and SS-27.2, were not evident in their respective Ex1 samples. The strains SS-38.4 and SS-27.2 showed the highest contents of surfactin and iturin A, respectively (Table 2). The results illustrated the variability with respect to surfactin and iturin A contents in different strains of Bacillus as well as extraction methods. Geissler et al.46 calculated surfactin contents in media of B. subtilis DSM 10, B. amyloliquefaciens DSM 7T and B. methylotrophicus DSM 23117 as 58, 2.1 and 3.1 µg/mL, respectively. They pointed out that the amounts of iturin A were lower than the calculated LOQ (39 ng/zone) in all three strains. In contrast, the strains investigated in our study (dried extracts of ca. 25 mg obtained from a 1-L broth) produced lower amount of surfactin (0.9-1.7 µg/mL, if calculated for the 1-L broth), whereas they showed strong potential to produce iturin A (0.52.0 µg/mL, if calculated for the 1-L broth).

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concentrations, ranged from 0.4 to 1.4 µg/zone. Then, the chromatograms were subjected to tyrosinase and AChE assays (Figure 4A and B). The maximum wavelength of the background color of each assay on the HPTLC plate was measured via recording of its UV/vis spectra (Figure S-4, 200700 nm). For the inverse densitometric measurement of the inhibition zones in both enzymatic assays, the maximum light wavelength absorbed by the purple background in the AChE assay (530 nm) was selected for both assays, as the greyish color of the background in tyrosinase assay absorbed all visible light wavelengths (Figure 4C and D). Based on peak areas, the LODs and LOQs of surfactin, iturin A and positive controls (PIP and KA) were calculated (Table 3) and respective dose-response curves were obtained for both assays (Figure 4E and F). In the AChE assay, surfactin showed the most intense inhibition (even more than PIP), whereas its activity in the tyrosinase assay was less than the activity of iturin A and KA (Figure 4E versus F). Equivalencies of surfactin and iturin A to positive controls were calculated. The peak area of 1 µg/zone of positive controls was calculated in the inhibitory calibration curves each of surfactin and iturin A. It was determined that KA showed by a factor of 2 and 3 higher tyrosinase inhibition than iturin A and surfactin, respectively (Table 3 and Figure 3E). For the AChE assay, PIP was by a factor of 1.7 more active than iturin A, but almost half as active than surfactin (Table 3 and Figure 3F).

Table 2. Mean contents (µg/mg, n = 5) of surfactin and iturin A in five dried Bacillus culture extracts (ca. 25 mg/mL each, 1 µL/band applied) Strain

Surfactin mean (µg/mg)

Intermediate precision (%RSD)

Ex1 vs. Ex2

10.7 12.6 13.1 27.2 38.4

60 vs. 56 67 vs. 44 55 vs. 37 67 vs. 61

Iturin A mean (µg/mg)

Intermediate precision (%RSD)

Ex1 vs. Ex2

4.1 vs. 5.4 2.5 vs. 8.6 6.1 vs. 4.7 3.5 vs. 3.8 -

70 vs.50 20 vs. 42 vs. 36 80 vs. 50

1.4 vs. 5.9 4.3 vs. 3.8 vs. 2.9 2.6 vs. 4.1

Inhibitor Equivalency in situ. The densitometric measurement of active zones after the HPTLC-assays allowed a quantitative analysis of active compounds directly in the (bio)autogram according to their effect-directed responses.20-25 The surfactin (0.1–1.5 µg/zone) and iturin A (0.5–4 µg/zone) standard mixture as well as the Ex2 sample of strain SS-12.6 were applied on five different HPTLC plates and days for each assay. After development, positive control patterns of PIP for AChE inhibition and KA for tyrosinase inhibition were applied on the edge track of the same plate at five

Figure 4. Effect-directed profiling of Ex2 sample of SS-27.2 (S, 1.5 µL applied) after tyrosinase (A) and AChE (B) assays showing inhibitor zones a-f, a mixture of surfactin (0.1, 0.2, 0.5, 1 and 1.5 µg/zone) and iturin A (0.5, 1, 2, 3 and 4 µg/zone) as well as positive control patterns of KA and PIP applied on the edge tracks (0.4, 0.8, 1.0, 1.2 and 1.4 µg/zone) on the HPTLC autograms at white light illumination; respective 3D densitograms by inverse measurement at 530 nm after tyrosinase (C) and AChE (D) assays and calibration curves of surfactin and iturin A in reference to KA for tyrosinase inhibition (E) or to PIP for AChE inhibition (F).

Table 3. LOD, LOQ and inhibitor equivalency of surfactin and iturin A in reference to KA or PIP obtained by inverse


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Analytical Chemistry measurement at 530 nm after HPTLC-tyrosinase/AChE assays

Substance

Surfactin Iturin A KA Surfactin Iturin A PIP

LOD/LOQ (µg/zone) Equivalent amount Calibration S/N (µg/zone) curve method HPTLC-tyrosinase assay 0.6/1.3 0.5/1.0 3.2 0.5/1.4 0.5/1.0 1.8 0.5/0.7 0.2/0.4 1.0 HPTLC-AChE assay 0.4/0.8 0.2/0.5 0.6 1.0/2.6 1.0/2.0 1.7 0.5/0.9 0.4/0.8 1.0

Intermediate precision (%RSD, n = 5) 14.8 12.9 10.5 14.2 17.2 8.1

The inhibitor equivalency calculation allowed the parallel investigation of the equivalent contents of not identified active compound zones in a sample. By reference to known inhibitors, their importance was rated. In the tyrosinase assay, the peak area of the unknown active zone a supposedly assigned as fengycin by HRMS in the Ex2 sample of SS-27.2 (1.5 µL applied) was more than 3 times the highest working range concentration of surfactin. Thus, the peak area of zone a was calculated via the enzymatic inhibition curves of iturin A and KA and calculated as equivalent to 2.3 and 0.9 µg/zone of iturin A and KA, respectively (Table S-2). The active zone c detected in the AChE assay was calculated as equivalent to 0.6, 1.2 and, 0.7 µg/zone of surfactin, iturin A and PIP, respectively. The activity of the AChE inhibitor zone f was by a factor 1.6 above the highest working range concentration of PIP, but it was calculated as equivalent to 1.4 and 3.1 µg/zone of surfactin and iturin A, respectively. The overall reproducibility (%RSD, n = 5) of the equivalency estimation of all unknown zones over 5 days and 5 HPTLC plates for both assays was 21% (Table S-2).

Conclusion A quantitative bioanalytical tool was demonstrated to collect comprehensive data on natural inhibitors. After finding a new, unknown inhibitor (not identified or no reference compound available), the question of its importance was solved by a straightforward workflow that demonstrated the equivalency estimation of inhibitors directly in the autogram. For the first time, HPTLC-tyrosinase/AChE assays were demonstrated as a reliable approach for inhibitory equivalency studies, shown for five Bacillus strains extracted via two different methods. In contrast to previous study, an alternative for mobile phase including chloroform was developed for separation and the primuline reagent was used for detection. After effect-directed profiling, the inhibiting zones found were characterized by HPTLC-HRMS. For inhibitory equivalency calculation, an inverse densitometric measurement was used. The inhibition activities of the identified surfactin and iturin A were quantified, whereas unknown (not-identified) inhibitors in a strain extract were calculated as equivalents to surfactin, iturin A and a common natural inhibitor for each assay (positive control). If compared to iturin A, surfactin showed a higher AChE inhibition (by factor of ca. 3) but lower tyrosinase inhibition (by factor of ca. 0.5). These equivalency data were obtainable in two hours (chromatography and assay) and on one HPTLC plate. Thus, it takes only 7 min per sample for 18 analyses on a plate in parallel. This straightforward strategy can contribute in natural products development for targeted screening of natural inhibitors.

ASSOCIATED CONTENT SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website. Table S-1: Mobile phases investigated for optimization of a green mobile phase for the Bacillus strain separation on an HPTLC plate silica gel 60; Table S-2: Surfactin, iturin A, KA and PIP equivalency calculation for the unknown inhibitors a, c and f in Ex2 sample of SS-27.2; Figure S-1. Schematic representation of the HPTLC-EDA-HRMS workflow to reduce the whole complexity of different samples to comprehensive information on few bioactive compounds; Figure S-2. HPTLC chromatograms at UV 366 nm of the optimization of a green mobile phase for the separation of the Bacillus LPs; Figure S-3: Overlaid densitograms at 190 nm of five Bacillus strains (all Ex2) as well as standard mixtures of surfactin and iturin A; Figure S-4: HPTLC-UV/vis spectra of the assay background colors generated. (PDF)

AUTHOR INFORMATION Corresponding Author *Phone: +49-641-99-39141. Fax: +49-641-99-39149. E-mail: [email protected] ACKNOWLEDGMENT The authors thank Professor. Dr. Slaviša Stanković and Dr. Ivica Dimkić (Faculty of Biology, University of Belgrade, Serbia) for the shipment of sample aliquots of their previous publications35,36. Thanks are owed to Ebrahim Azadniya and Dr. Salim Hage for support with regard to assays, and Merck, Darmstadt, Germany for plates.

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Analytical Chemistry

TOC


Fast Equivalency Estimation of Unknown Enzyme Inhibitors in

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