Effect-Directed, Chemical and Taxonomic Profiling of Peppermint Proprietary Varieties and Corresponding Leaf Extracts

During the development of novel, standardized peppermint extracts targeting functional applications, it is critical to adequately characterize raw material plant sources to assure quality and consistency of the end-product. This study aimed to characterize existing and proprietary, newly bred varieties of peppermint and their corresponding aqueous extract products. Taxonomy was confirmed through genetic authenticity assessment. Non-target effect-directed profiling was developed using high-performance thin-layer chromatography–multi-imaging–effect-directed assays (HPTLC–UV/Vis/FLD–EDA). Results demonstrated substantial differences in compounds associated with functional attributes, notably antioxidant potential, between the peppermint samples. Further chemical analysis by high-performance liquid chromatography–photodiode array/mass spectrometry detection (HPLC–PDA/MS) and headspace solid-phase microextraction–gas chromatography–flame ionization/MS detection (headspace SPME–GC–FID/MS) confirmed compositional differences. A broad variability in the contents of flavonoids and volatiles was observed. The peppermint samples were further screened for their antioxidant potential using the Caenorhabditis elegans model, and the results indicated concordance with observed content differences of the identified functional compounds. These results documented variability among raw materials of peppermint leaves, which can yield highly variable extract products that may result in differing effects on functional targets in vivo. Hence, product standardization via effect-directed profiles is proposed as an appropriate tool.


Introduction
Plants from Mentha species (Lamiaceae) are globally widespread and broadly consumed worldwide for its pleasant flavor, alone or mixed with other herbs [1]. Mentha is derived from the Greek Mintha, the name of a mythical nymph who metamorphosed into this plant [2] and its species name piperita is from the Latin piper meaning pepper, which indicates its aromatic and pungent taste [3]. Mentha × piperita L. is the natural hybrid from M. aquatica and M. spicata [4], and is widely identified as peppermint. It is a perennial herb native to Europe, naturalized in the North of America, and cultivated in many parts of the world [5]. Table 1. Different peppermint products from Europe and the USA studied, i.e., the minced dried leaf samples L1-L8 and the respective powdered extract samples E1-E8 ( Figure S1). Eriocitrin, eriodictyol-7-O-glucoside, luteolin-7-O-glucuronide, luteolin-7-O-rutinoside, luteolin-7-O-glucoside, isorhoifolin, caffeic acid, rosmarinic acid, naringenin were purchased from Merck, Phytolab, and Extrasynthese, Genay, France. Formic acid, dimethyl sulfoxide (DMSO), acetonitrile, methanol and water of chromatographic quality were purchased from VWR.

Caenorhabditis elegans Strain and Maintenance
Caenorhabditis elegans strain N2 (C. elegans var. Bristol, wild-type) was obtained from the Caenorhabditis Genetics Center, University of Minnesota, Minneapolis, MN, USA, and maintained at 20 • C on Nematode Growth Medium (NGM) plates with Escherichia coli strain OP50 as normal diet for nematodes.

Genetic Assessment
ADM Mentha germplasm collection, Corvallis, OR, USA, is a valuable source of diversity for genetic and functional ingredient studies and for mint breeding. As plant materials, 24 mint varieties belonging to M. x piperita, M. canadensis or M. arvensis were from ADM Mentha germplasm collection including several proprietary varieties employing the Tajima-Nei model [22] (Table S1).

HPLC-PDA/MS
Three standard solutions (1 mg/mL each) of eriocitrin, rosmarinic acid and luteolin-7-O-glucoside in DMSO were prepared by ultrasonication for 5 min. External calibration curves were assessed with at least five different calibration points (r > 0.99) for the quantification of the main groups of components in peppermint leaves and the corresponding extracts, respectively flavanones, flavones and caffeic acid derivatives.

HPLC-PDA/MS
The peppermint leaves and corresponding extracts ( Table 1) were extracted/dissolved in DMSO (10 mg/mL) in 10-mL centrifuge tubes, followed by shaking via an orbital shaker (Grant bio PSU-10i, VWR) at maximum velocity for 30 min, and filtered (0.45 µm syringe nylon filter, VWR). For MS analysis, samples were extracted/dissolved as mentioned, but in methanol instead of DMSO.

Headspace SPME-GC-FID/MS
Each peppermint leaf (0.5 g/5 mL) or extract (0.2 g/2 mL; Table 1) sample was extracted/dissolved in water (100 mg/mL) and placed into a 20-mL headspace vial sealed with a silicone septum. Water blanks were analyzed in the beginning of the sequence and between samples.

Caenorhabditis elegans Method
Powdered peppermint extracts E1-E7 were dissolved in water and respective stock solutions were prepared. Then, serial dilutions were performed. Different doses were added on NGM medium surface to get the final doses in the plate (0.1-10 mg/mL).

Genetic Assessment
Total genomic DNA was isolated from 200 mg fresh leaves following a modified cetyltrimethylammonium bromide (CTAB) procedure [23].

HPTLC-UV/Vis/FLD-EDA
HPTLC plates silica gel 60 F 254 s and HPTLC plates silica gel 60, the latter for the SOS-Umu-C, pYAES and pYAAS bioassays, were prewashed via development with methanolwater 4:1 (V/V; Simultan Separating Chamber, biostep, Burkhardtsdorf, Germany), dried at 120 • C for 20 min (Heating Oven, Memmert, Schwabach, Germany), covered by a clean glass plate and wrapped in aluminum foil for storage in a desiccator [24]. HPTLC instruments (all CAMAG, Muttenz, Switzerland) were operated and data processed with visionCATS version 3.0 software. Application parameters (Automatic TLC Sampler 4) were as follows: 8 mm band, track distance 11 mm, distance from lower edge 8 mm and from left edge 17.5 mm, dosage speed 200 nL/s, filling vacuum time 1 s, rinsing vacuum time 6 s, rinsing cycles 3, filling cycle 1, and return unused sample into vial.
For effect-directed assay detection, 12 analogous chromatograms were prepared with adaptations for some assays as mentioned. The respective PC for each assay was applied on the upper right plate edge (some PCs were not cut off and evident as three increasing bands in Figure 1), or for duplex assays, as agonist stipe along each separated sample track. Assay solutions/suspensions were piezoelectrically sprayed (level 5, Derivatizer) using different nozzles as specified. In case of the acidic development, the plate was neutralized by spraying (yellow nozzle) with disodium hydrogen phosphate buffer (8 g in 60 mL water, citric acid 0.1 M added to reach pH 7.5, filled up to 100 mL), followed by plate drying for 4 min (hair dryer). Note that plate neutralization was not needed for the DPPH• assay. For incubation, the plate was placed horizontally in a premoistened polypropylene box (27 cm × 16 cm × 10 cm, KIS, ABM, Wolframs-Eschenbach, Germany) as described [15][16][17][18][19]21]. The resulting (bio)autograms were dried (4 min, cold airstream, hairdryer) and documented (TLC Visualizer/BioLuminizer). For DPPH• assay and B. subtilis bioassay, the response signals increase over time and should be compared after one day.
Still on the same assay plate (e.g., after tyrosinase inhibition assay and A. fischeri bioassay), microchemical derivatization was performed as reagent sequence using two different derivatization reagents (3 mL each, Derivatizer), i.e., natural product reagent A (1% in methanol, green nozzle), and after plate drying for 2 min and documentation, anisaldehyde sulfuric acid reagent (2% in sulfuric acid-glacial acetic acid-methanol 1:1:9, V/V/V, blue nozzle; followed by heating at 110 • C on plate heater for 5 min), both detected at Vis and FLD 366 nm. in methanol, green nozzle), and after plate drying for 2 min and documentation, anisaldehyde sulfuric acid reagent (2% in sulfuric acid-glacial acetic acid-methanol 1:1:9, V/V/V, blue nozzle; followed by heating at 110 °C on plate heater for 5 min), both detected at Vis and FLD 366 nm.

HPLC-PDA/MS
The analyses of flavanones, flavones and caffeic acid derivatives were performed according to [29]. The HPLC equipment used for the analysis consisted of a Shimadzu Nexera XR UHPLC (70 MPa) coupled to a PDA detector (SPD-M40, Izasa Scientific, Spain). Separation was performed on the octadecyl silane column Zorbax Eclipse Plus C18 (length 250 mm, ID 4.6 mm, particles 5 µm) protected by a corresponding precolumn (Agilent Technologies, Barcelona, Spain). The temperature of the column oven was 25 • C, the flow rate 1.0 mL/min, and the injection volume 2 µL. The binary gradient consisted of (A) water and (B) acetonitrile, both acidified with formic acid (0.2%, V/V), starting with A/B 90:10, increased to 25% B in 15 min, to 35% B in 17 min, and to 70% B in 19 min. For 9 min, the column was equilibrated to the initial gradient conditions. The total analysis time took 60 min. For quantification, PDA detection was performed at 280 nm using an external calibration curve with at least 5 different calibration points (r > 0.99). The results were expressed in percent (%, dry basis). The sum of flavanones (eriocitrin and eriodictyol-7-O-glucoside) was calculated as eriocitrin equivalents, flavones (luteolin-7-O-glucuronide, luteolin-7-O-rutinoside, luteolin-7-O-glucoside, and isorhoifolin) as luteolin-7-O-glucoside equivalents, and caffeic acid derivatives (caffeic acid and rosmarinic acid) as rosmarinic acid equivalents. For identification, the flow was set to 0.6 mL/min using a Triple TOF 5600 LC/MS/MS with software PeakView (AB SCIEX, Atlanta, GA, USA). Full mass spectra from m/z 80-1300 were recorded via electrospray ionization (ESI) in the negative ion mode using capillary voltage 3 kV, extractor voltage 4.0 V, cone voltage 30 V, ion source temperature 150 • C, desolvation temperature 300 • C, and desolvation gas flow 600 L/h.

Caenorhabditis elegans Method
For oxidative stress measurement, Caenorhabditis elegans strain N2 was egg-synchronized in the NGM plates (control medium) and NGM plates supplemented with different doses of peppermint extract (1, 2, 5 and 10 mg/mL, and in addition, 0.1 and 0.5 mg/mL for the peppermint extract E4) for an initial screening. Then, the optimal dose of each peppermint extract was performed by duplicate. Vitamin C (10 µg/mL) was used as an internal positive control. Nematodes survival was counted after oxidative stress (2 mM H 2 O 2 ) according to [31]. Survival data were analyzed by One Way ANOVA test using a Tukey's multiple comparison pots-test with GraphPad Prism 4 software (GraphPad Software, San Diego, CA, USA).
For Staphylococcus aureus infection, age-synchronized nematodes of the wild-type N2 were obtained and maintained in NGM plates, partially supplemented with a doseresponse of each powdered mint extract (0.1, 0.5 and 1 mg/mL), and incubated at 20 • C. When worms reached young adult stage were transferred to the infection plates containing a lawn of S. aureus ATCC 25923 strain. NGM plates with the E. coli strain OP50 (condition without infection) and with the pathogen (infected condition) were included. Survival was scored at 25 • C during 5 days. Worms were counted as alive or dead by gentle touching with a platinum wire. Once determined the dose with the highest positive effect, a second assay was performed to confirm result at this optimal dose. Survival curves were analyzed using the log Rank T-test significance test (GraphPad Prism 4 software).

Genetic Assessment
Both the quality and quantity of genomic DNA were evaluated by agarose gel electrophoresis and by UV/Vis spectrophotometry (NanoDrop 2000c, Thermo Fisher Scientific, Pittsburgh, PA, USA). RAD seq was performed as reported [32] with the exception that the restriction enzyme ApeKI (New England Biolabs, Ipswich, MA, USA) was used. Adapterligated DNA fragments were pooled and sheared to a mean size of 500 bp. The RAD-Seq libraries were enriched by polymerase chain reaction amplification and sequenced on an Illumina HiSeq 2000 (BGI, Shenzhen, China) using single-ended reads (50 bp) for each peppermint variety. For SNP calling and data analysis, the Illumina sequence reads were quality-filtered by removing the adapter sequences and reads containing greater than 50% low-quality bases. These processed reads were mapped on the reference peppermint genome using BWA-MEM (version 2, GitHub, Hobro, Denmark). Then, the comparison results filtered and SNPs were called by SAMTOOLS (version 1.16, Genome Research, Cambridgeshire, UK). High quality SNPs were used for the estimate of pairwise genetic distances and the reconstruction of phylogenetic trees based on maximum parsimony using MEGA 11 (Mega Software Technologies, Philadelphia, PA, USA) [33].

Development of the Effect-Directed Profiling (HPTLC-UV/Vis/FLD-EDA)
Industrial peppermint products from Europe and the USA which differ regarding producer and batch (Table 1, Figure S1) were selected, i.e., 7 minced green dried leaf samples (L1-L7) and 7 brown crystalline powdered extract samples (E1-E7). The powdered extract samples were the dried water extracts industrially produced from the corresponding dried leaf samples. Both related sets of 7 samples each were compared to detect differences caused by the extraction and drying process with regard to the composition of valueadding compounds, such as bioactive compounds, flavonoids, and volatiles, important for targeting functional applications. A further powdered native extract sample E8 obtained from European peppermint leaves at industrial scale, standardized by HPLC into caffeic acid derivatives (≥1%, dry basis), flavanones (≥4%, dry basis), and flavones (≥2%, dry basis), was provided later by ADM, and in some bioassays, analyzed instead of L5 (due to limited parallel analyses per plate). The leaf samples required homogenization due to the different structural parts contained, whereas the extract samples were obtained homogenously powdered ( Figure S1).
For the development of the non-target effect-directed profiling, there are no preselected target compounds in the analytical focus. The intention was a spreading of the compounds along the whole migration distance. The mobile phase system of a previous project on botanicals (ethyl acetate-toluene-formic acid-water 8:2:1.5:1) [25] was tested and found suited also for the bioprofiling of peppermint products. The separation took ca. 33 min, which was acceptable. For extraction of polyphenols, normally water or methanol were used [29]. However, another extractant, i.e., water-ethanol-ethyl acetate 1:1:1, was tested due to good experience in other projects [34]. The comparison of the different extractants for the same peppermint leaf samples L1-L7 showed that water-ethanol-ethyl acetate 1:1:1 extracted comparatively more and a broader range of polar to apolar compounds than methanol ( Figure S2). The comparison of the same separation on two different plates, i.e., HPTLC plate silica gel 60 with the regular fluorescence indicator F 254 versus the acidstable F 254 s, proved the latter to be more stable against the acidic mobile phase system and superior with regard to zone sharpness ( Figure S3, especially evident at UV 254 nm and FLD 366 nm). As a starter assay, the A. fischeri bioassay was applied during the development of the bioprofiling ( Figures S2 and S3), since it provided the most response in many other studies. Although the patterns were almost comparable, obviously the different fluorescence indicator or plate pH had an influence on the bioluminescence signal, i.e., the brilliance of the plate background was higher for the plate with F 254 . Still on the same A. fischeri bioassay plate (also shown after the tyrosinase inhibition assay later, Figure S6), optional derivatization was performed using two different derivatization reagents as reagent sequence, first the natural product reagent A and then anisaldehyde sulfuric acid reagent ( Figures S2 and S3). The application order is explained by the increasing pH of the reagents. Including this reagent sequence, 6 different detection mechanisms were performed on the same plate, i.e., (1) Vis, (2) UV, (3) FLD, (4) effect-directed assay, (5) natural product A reagent, and (6) anisaldehyde sulfuric acid reagent.

Effect-Directed Profiles via HPTLC-UV/Vis/FLD-EDA
For the application of the non-target effect-directed profiling, 14 peppermint samples were applied at two different adjusted sample volumes, i.e., 10 µL/band for the leaf samples L1-L7 and 2 µL/band for the powdered extract samples E1-E7. The required lower volume was expected for the extract samples which are enriched in compounds due to the extraction/drying step and release/dissolve more compounds due to the high sample surface [34]. A respective solvent blank (B) was treated and analyzed like a sample. In the middle of the plate, a standard mixture (M) was applied with example reference compounds, such as eriocitrin, luteolin-7-O-glucoside, rosmarinic acid, and apigenin with increasing hR F values ( Figure S4), reported to be present as polyphenols with antioxidative activity in peppermint and related species [29]. All in all, the 16 applications filled the plate format and the samples were analyzed using the previously selected chromatographic system. The resulting chromatogram was evaluated at UV/Vis/FLD, which proved the good spread of the compounds along the migration distance ( Figure 1). For the application of the 12 effect-directed assays (2 duplex bioassays included), the preparation of such a chromatogram was repeated with few adaptations for some assays as mentioned. All (bio)assay responses were repeated several times and confirmed.
After the assay application, the respective PCs showed a response in all assays, whereas the solvent blank treated like a sample did not ( Figure 1). This was an important precondition for evaluation of the (bio)autograms. The applied 5-fold higher volume of the leaf samples L1-L7 had to be considered for the comparison with the powdered extract samples E1-E7. Thus, stronger intensities were given for the fine powdered crystalline extracts. The profile comparison of each minced dried leaf sample with the respective dried and powdered water extract sample showed, despite smaller differences in the response intensity, a similar profile in each pairing case (Table 1). This proved that the applied industrial leaf processing (aqueous extraction of the leaf materials) did not lose important bioactive compounds.
The DPPH• radical scavenging assay (Figures 1 and S5) revealed in the 14 peppermint samples up to a dozen antioxidative compounds detected as yellow bands on a purple background. The DPPH• response was more intense after one day ( Figure S5). All four reference compounds showed an antioxidative activity as expected. At the given sample amount applied, eriocitrin at hR F 29 was visually assigned to be present (at higher amounts) in L1, L2, L5, and L7 as well as in the corresponding powdered extract samples E1, E2, E5, and E7. Rosmarinic acid at hR F 85, though varying in the response intensity, was present in all samples. Clear differences between the samples were observed, e.g., L4 or L6 contained less and more apolar antioxidative compounds if compared to L1 or L5, which both were strongest in the overall response.
For the tyrosinase inhibition assay (Figures 1 and S6), colorless (white) inhibition bands were revealed on a grey background. One prominent tyrosinase inhibitor at hR F 85 was observed in all samples and also in the standard mixture, thus assigned as rosmarinic acid. All leaf samples L1-L7 contained another tyrosinase inhibitor at hR F 95. Only few samples (L4-L6 and the corresponding E4-E6) showed a third tyrosinase inhibitor at hR F 27. The given incubation at room temperature was compared with incubation at 37 • C, which was also reported as an optimal temperature for the enzyme [35], however, incubation at room temperature resulted in a superior response intensity ( Figure S6). It was studied whether an autogram can be used for post-assay derivatization. The tyrosinase inhibiting plate was already 5 days old and then derivatized with the natural product A reagent detected at FLD 366 nm and white light illumination. Still sharp colorful zones were obtained (Figures 1 and S6). This highlighted the potential for on-surface storage of separated samples and for multi-detection.
For the β-glucuronidase inhibition assay (Figures 1 and S7), colorless (white) inhibition bands were observed on an indigo-blue colored background. Depending on the sample, up to 10 inhibition bands were revealed along the whole polarity range. All leaf samples L1-L7 contained similar to the tyrosinase inhibiting assay an additional β-glucuronidase inhibitor at hR F 95. At the applied amounts, all four reference compounds showed β-glucuronidase inhibition, too.
For the acetylcholinesterase inhibition assay (Figures 1 and S8), colorless (white) inhibition bands were detected against a purple background. One prominent acetylcholinesterase inhibitor at hR F 32 was evident in all 14 samples. For plate neutralization, two different buffers, i.e., sodium hydrogen carbonate buffer (2.5 g in 100 mL water) versus disodium hydrogen phosphate buffer, were compared. The resulting autograms showed less interfering color formation for the disodium hydrogen phosphate buffer ( Figure S7), which was preferred despite the comparatively three-fold higher salt load on the layer. The butyrylcholinesterase inhibition response was comparatively weaker and more in the apolar compound range (Figure 1).
For the α-glucosidase inhibition assay, colorless (white) inhibition bands on a purple background were revealed (Figures 1 and S9). Up to 9 different inhibiting bands were detected, whereby the more prominent responses were observed in the middle to apolar compound range (upper autogram part). At the given amounts, three reference compounds (except for luteolin-7-O-glucoside) showed β-glucuronidase inhibition, too. Similar to the butyrylcholinesterase inhibition response, the β-glucosidase inhibition was comparatively weaker.
Up to 9 different antibacterial compounds acting against Gram-negative Aliivibrio fischeri bacteria in the samples (Figures 1 and S3) were detected as dark bands (or brightened at the start zone) on the instantly bioluminescent plate background (depicted as greyscale image). However, the most prominent response was close to the solvent front. This response of at least 2 different apolar compounds was observed in all samples. All four reference compounds revealed a response at the given amount applied.
The respective sample profiles indicating antibacterial compounds acting against Gram-positive Bacillus subtilis bacteria were different (Figure 1), although few zones acted against both bacteria types. Up to 11 different antibacterial compounds were detected, although very different in the response intensity. Very similar to the tyrosinase inhibition autogram, one prominent sharp zone at hR F 95 was observed in all leaf samples L1-L7. Due to the characteristic zone profile in both autograms, it was assumed to be the same, thus multi-potent, compound. All four reference compounds revealed a response at the given amount applied, whereby apigenin was strongest and clearly present in all leaf samples L1-L7.
For the SOS-Umu-C, pYAES and pYAAS bioassays (Figures 2 and S10-S12), HPTLC plates without the fluorescence indicator were used to avoid measurement signal interference since the fluorescein, which is the enzyme-substrate reaction end-product, was also detected at 254 nm. Since for all three bioassays, the same substrate was used, i.e., fluorescein di(β-D-galactopyranoside), the same green fluorescence (due to the formed fluorescein) was revealed for any agonist. Note that substrates can differ in costs by a factor of 10, and there is room for costs reduction since the substrate used here is very expensive. The mobile phase was adjusted to be acid-free (ethyl acetate-toluene-methanol -water 4:1:1:0.4) to skip the neutralization step and thus additional buffer load on the layer since the duplex bioassays required additional zone fixation. The late-delivered sample E8 (Table 1) was analyzed instead of E5. The sample volumes were increased to a maximal sample load (15 µL/band for L1-L7, 3 µL/band for E1-E8, and 2.3 µg/band each for M) to detect even traces of any estrogen, antiestrogen, androgen, antiandrogen and genotoxin. The respective green fluorescent agonist stripe was considered as the PC for the duplex assays. Antagonistic effects were observed via the signal reduction of the respective green fluorescent agonist stripe, which was applied along each sample track before the bioassay application. The detection of agonistic and antagonistic effects in the same analysis made it a duplex bioassay. However, the observation of antagonists needs further verification (V) via the pYAVES [15] and pYAVAS [17] bioassays through an additionally applied end-product stripe, which can differentiate true versus false-positive antagonistic effects.
Bioprofiling for estrogenic and anti-estrogenic compounds via the duplex pYAES bioassay (Figures 2 and S10) revealed up to two apolar green fluorescent estrogenic compounds in the samples, whereby the lower one was assigned to rosmarinic acid. The strong response for the given rosmarinic acid amount in the standard mixture and the missing acid in the mobile phase explained its tailing. Some anti-estrogenic compound zones were observed as signal reduction of the green fluorescent estrogen stripe. However, these antagonistic responses were not so strong and seemed to be caused by reaction with the buffer ( Figure S3) since the same horizontal pattern was evident in all assays, in which the color reaction interfered (not the case for the DPPH• assay and the Aliivibrio fischeri bioassay). Therefore, the confirmatory pYAVES bioassay was not performed as it was not deemed necessary to do so.
Bioprofiling for androgenic and antiandrogenic compounds (Figures 2 and S11) via the duplex pYAAS bioassay studied the presence of any androgens and anti-androgens. However, there were no androgens detected as green fluorescent band despite the high sample load. The proper bioassay functioning was verified by the green fluorescent testosterone stripe (applied along each sample track and considered as the PC). As already discussed for the pYAES bioassay, the same horizontal anti-androgenic compound zone pattern was observed as signal reduction of the green fluorescent testosterone stripe, which underlines our hypothesis. Also here, the respective pYAVAS bioassay was not performed.
Bioprofiling for genotoxic compounds (Figures 2 and S12) via the planar SOS-Umu C bioassay investigated the presence of any genotoxins in the industrial peppermint prod ucts. There was no green fluorescent genotoxin band revealed in the samples despite the high sample load of the dried leaf (1.5 mg for L1-L7) and powdered extract (0.3 mg fo E1-E7) samples. The proper bioassay functioning was verified by the green fluorescence of the genotoxin 4-nitroquinoline 1-oxide, applied as the PC on the upper right plate edge

Results of HPLC-PDA/MS Analysis
Identification of flavanones, flavones and caffeic acid derivatives were performed by comparing retention time, PDA and MS/MS spectra with the corresponding reference standards ( Table 2). A typical HPLC-PDA chromatogram at 280 nm is exemplarily illus Bioprofiling for genotoxic compounds (Figures 2 and S12) via the planar SOS-Umu-C bioassay investigated the presence of any genotoxins in the industrial peppermint products. There was no green fluorescent genotoxin band revealed in the samples despite the high sample load of the dried leaf (1.5 mg for L1-L7) and powdered extract (0.3 mg for E1-E7) samples. The proper bioassay functioning was verified by the green fluorescence of the genotoxin 4-nitroquinoline 1-oxide, applied as the PC on the upper right plate edge.

Results of HPLC-PDA/MS Analysis
Identification of flavanones, flavones and caffeic acid derivatives were performed by comparing retention time, PDA and MS/MS spectra with the corresponding reference standards (Table 2). A typical HPLC-PDA chromatogram at 280 nm is exemplarily illustrated for peppermint leaf sample L2 from Europe ( Figure 3) and the corresponding extract E2 (Figure 4). Depending on the phenolic components, such as flavones, flavanones or caffeic acid and its derivatives, the recorded UV-Vis spectra differed. trated for peppermint leaf sample L2 from Europe ( Figure 3) and the corresponding extract E2 (Figure 4). Depending on the phenolic components, such as flavones, flavanones or caffeic acid and its derivatives, the recorded UV-Vis spectra differed.     trated for peppermint leaf sample L2 from Europe ( Figure 3) and the corresponding extract E2 (Figure 4). Depending on the phenolic components, such as flavones, flavanones or caffeic acid and its derivatives, the recorded UV-Vis spectra differed.    The HPLC-QTOF-MS/MS recording in the negative ionization mode allowed the identification of the major phenolic compounds in the peppermint samples ( Table 2). The individual composition of the major bioactive components of selected peppermint leaf samples and their corresponding extracts (all %dry basis) was determined by HPLC-PDA ( Table 3). The composition of the peppermint leaf samples from USA highly fluctuated within different proprietary varieties. The content of eriocitrin ranged between 0.06% and 1.16%, whereas the European peppermint leaf samples were almost similar (1.30% to 1.43%). The highest flavanone contents ( Figure S13) were observed for USA peppermint L7 (1.20%) and European peppermint L8 (1.64%). Rosmarinic acid in the peppermint leaf samples from USA (0.22% to 0.75%) also varied more than from Europe (0.27% to 0.49%). The highest flavone content ( Figure S13) was observed for USA peppermint L4 (4.23%) and European peppermint L2 (1.48%). Isohoifolin and luteolin-7-O-glucoside showed a higher content in the USA varieties than European peppermint leaves. For the corresponding extracts obtained from the USA and European peppermint leaves, the contents of bioactive components were significantly increased, i.e., for eriocitrin, luteolin-7-O-glucoside, luteolin-7-O-glucuronide and isohoifolin about two-fold, for rosmarinic acid more than three-fold and for eriodictyol-7-O-glucoside more than four-fold ( Table 3). The highest flavanone contents ( Figure S14) were observed for USA peppermint extract E7 (3.98%) and European peppermint extract E2 (6.79%). Also regarding the flavones, the highest contents were obtained for USA peppermint extract E7 (4.35%) and European peppermint extract E2 (6.84%).

Antioxidant Activity in Caenorhabditis elegans Assay
Previous work has been demonstrated C. elegans as a good model to assess antioxidant properties of oils from three Mentha species [36]. To determine this capacity of the peppermint extract samples E1-E7, a dose-response of each sample was performed to establish the dose with the best antioxidant activity ( Figure S19). The optimal dose for each peppermint extract is indicated ( Figure 5). When nematodes were fed with the different peppermint extracts and subject to acute oxidative stress, worms were more resistant to oxidative stress than control-fed nematodes (p-value < 0.0001). Moreover, worms treated with peppermint extract E6 showed the highest survival increase in comparison to control condition NGM (37%), while those fed with peppermint extracts E1 and E4 exhibited the lowest increase (14% and 13%, respectively).

Antioxidant Activity in Caenorhabditis elegans Assay
Previous work has been demonstrated C. elegans as a good model to assess antioxidant properties of oils from three Mentha species [36]. To determine this capacity of the peppermint extract samples E1-E7, a dose-response of each sample was performed to establish the dose with the best antioxidant activity ( Figure S19). The optimal dose for each peppermint extract is indicated ( Figure 5). When nematodes were fed with the different peppermint extracts and subject to acute oxidative stress, worms were more resistant to oxidative stress than control-fed nematodes (p-value < 0.0001). Moreover, worms treated with peppermint extract E6 showed the highest survival increase in comparison to control condition NGM (37%), while those fed with peppermint extracts E1 and E4 exhibited the lowest increase (14% and 13%, respectively). Figure 5. Percentage of Caenorhabditis elegans N2 fed with control condition (OP50) or with each peppermint extract E1-E7 at the respective optimal dose. Data correspond to the average of two independent assays. A one-way ANOVA test with a Tukey's multiple comparison pots-test was applied. **** Significant at p-value < 0.0001 in comparison to NGM condition.

Enhanced Resistance of Caenorhabditis elegans to Staphylococcus aureus Pathogen Infection
It is known that essential oils from Mentha species have antimicrobial and antiviral activities [5]. A dose-response study of each peppermint extract E1-E7 was carried out in C. elegans on pathogen infection to determine the protection of each sample ( Figure S20).
All peppermint extracts increased significantly worm's survival during pathogen challenge (p-value < 0.0001) in comparison to infected control condition at day 4 and 5 post-infection ( Figure 6 and Figure S21). Peppermint extracts E5-E7 provided the highest pathogen resistance, followed by E2, E1 and E4. The peppermint extract E3 showed the lowest protection.

Enhanced Resistance of Caenorhabditis elegans to Staphylococcus aureus Pathogen Infection
It is known that essential oils from Mentha species have antimicrobial and antiviral activities [5]. A dose-response study of each peppermint extract E1-E7 was carried out in C. elegans on pathogen infection to determine the protection of each sample ( Figure S20). All peppermint extracts increased significantly worm's survival during pathogen challenge (p-value < 0.0001) in comparison to infected control condition at day 4 and 5 postinfection (Figures 6 and S21). Peppermint extracts E5-E7 provided the highest pathogen resistance, followed by E2, E1 and E4. The peppermint extract E3 showed the lowest protection.

Genetic Assessment
The sequencing produced over 36,000,000 reads with an average of 2.5 million reads for each variety. After quality assessment and adapter trimming approximately 33,500,000 reads were used for mapping. An initial pool of 38,543 SNPs was first identified. After a quality filtering process, 15,000 SNPs were used for the data analysis.
The estimates of pairwise genetic distances among the peppermint varieties based on Tajima-Nei model [22] were obtained (Table S1). Overall, genetic distances among different varieties of M. x piperita were between 0.10-0.17. The peppermint leaf samples L5, L7, and MP15CS28 are genetically similar but unique to M. x piperita Black Mitcham. The genetic distances among different varieties of M. canadensis were between 0.12-0.17. L4 was a M. canadensis species but different from other varieties. The genetic distances between the two species were above 0.21. The genetic distances of the peppermint leaf sample L6 to all mint varieties were under 0.21 with one exception to MP981 at 0.24.

Discussion
The peppermint samples investigated (Table 1) for 14 different effects using 12 different non-target planar effect-directed assays, whereof the pYEAS and pYAAS were duplex bioassays, differed very much in their functional properties (Figures 1 and 2). Hence,

Genetic Assessment
The sequencing produced over 36,000,000 reads with an average of 2.5 million reads for each variety. After quality assessment and adapter trimming approximately 33,500,000 reads were used for mapping. An initial pool of 38,543 SNPs was first identified. After a quality filtering process, 15,000 SNPs were used for the data analysis.
The estimates of pairwise genetic distances among the peppermint varieties based on Tajima-Nei model [22] were obtained (Table S1). Overall, genetic distances among different varieties of M. x piperita were between 0.10-0.17. The peppermint leaf samples L5, L7, and MP15CS28 are genetically similar but unique to M. x piperita Black Mitcham. The genetic distances among different varieties of M. canadensis were between 0.12-0.17. L4 was a M. canadensis species but different from other varieties. The genetic distances between the two species were above 0.21. The genetic distances of the peppermint leaf sample L6 to all mint varieties were under 0.21 with one exception to MP981 at 0.24.

Discussion
The peppermint samples investigated (Table 1) for 14 different effects using 12 different non-target planar effect-directed assays, whereof the pYEAS and pYAAS were duplex bioassays, differed very much in their functional properties (Figures 1 and 2). Hence, the developed non-target effect-directed profiling was found very informative with regard to the quality control of functional peppermint samples. Product standardization via effectdirected profiles is proposed as an appropriate tool to control all functional properties and not only targeted marker compounds, as is the case with the currently prevailing quality control.
The quantification of bioactive components by HPLC-PDA analysis showed a broad range of flavonoid contents, which confirms the fluctuations in value-added compounds. European and proprietary peppermint varieties from USA presented similar chromatographic profiles obtained by HPLC-PDA (Figure 3). Main differences were due to the individual content of the bioactive compounds (Table 3), which apart from the variety, are affected by different factors such as pedoclimatic and agricultural conditions [38]. The respective water extracts obtained from the different peppermint leaf samples from Europe and USA showed a similar chromatographic profile to the corresponding leaf sample (Figures 3 and 4). The major bioactive components ( Table 2) were extracted by the greenest solvent which is water [6]. Therefore, a preselection of compliant peppermint leaves is key to standardize the extracts into objective and primary quality markers such as flavanones, flavones and hydroxycinnamic acids as main bioactive components.
Different analytical techniques such as NMR, LC-MS, GC-MS, etc. have been employed to study water-soluble polyphenols from peppermint [6,29,39]. Nevertheless, to the best of our knowledge, the current research work covers a complete analytical study of identification and quantification by different liquid and gas chromatography techniques, together with functionality evaluation, on-surface of the separation layer and in vivo (C. elegans) of several Mentha piperita leaves and extracts, with different quality and origin.
Traditionally, the aroma composition of peppermint is identified or related with the essential oil [4][5][6], but the volatile profile is also useful to authenticate powdered extracts [30]. Therefore, the volatile profile was also analyzed in the peppermint leaf samples from USA and Europe and the corresponding powdered extracts, and showed characteristic volatile components [6,40]. Thus, additional valuable information was obtained to avoid fraud and adulteration ( Figures S15-S17). Especially, the presence of characteristic volatile components in the peppermint extracts add value to the products. Several functional effects have been associated to those characteristic peppermint volatiles [5,6].
Main bioactive components in peppermint leaves are flavonoids, phenolic acids, lignan and stilbenes, and volatile components [6,29]. However, considering water-soluble (polar) polyphenols, major bioactive compounds identified in the peppermint samples were flavonoids and phenolic acids. The flavonoids were grouped into flavones (luteolin-7-Orutinoside, luteolin-7-O-glucuronide, luteolin-7-O-glucoside, and isohoifolin), flavanones (eriodictyol-7-O-glucoside, and eriodictyol), which together with rosmarinic acid, were the major active phytochemical constituents in the leaves and the corresponding extracts. For industrial mint extracts, such as the E8, it is fundamental to evaluate the quality, and to exclude a possible fraud and adulteration. Hence, working on mint leaves (no spent mint), together with the non-drastic conditions employed during the aqueous extraction process at laboratory and industrial scales, allowed standardization of the Mentha piperita extracts into primary quality markers, and their comprehensive characterization by bioactivity profiles and volatiles' fingerprints.
Phylogeny reconstruction using the 15,000 SNPs revealed genetic relationships and relatedness among the studied 24 mint varieties ( Figure S22). Most mint varieties formed a distinct and well supported clade at 100%. These varieties included the peppermint leaf samples L5 and L7. Most M. canadensis varieties formed 2 or 3 distinct but less supported clades at below 67%. It indicated large genetic variations among these varieties. The peppermint leaf sample L6 formed a clade with mint varieties but only supported at 61%, which indicated that it might be a hybrid or cybrid between peppermint and M. canadensis.
The use of molecular markers for genotyping is an effective tool in peppermint breeding and variety identification and protection [41]. In this study, RAD-Seq technology was used for a rapid and cost-effective discovery of genomic SNPs from 24 mint varieties including several proprietary varieties. These SNPs have not only provided more molecular markers for peppermint genetic identification but also provided comprehensive data about the genetic diversity, divergency, and relatedness at a genome-wide scale. The RAD-Seq technology has been used for other applications ranging from QTL mapping, genome-wide association studies, and marker-assisted selective breeding. The genomic SNPs related to functional genes, especially those involved in secondary metabolic processes, can be identified and experimentally validated that will support future research on the molecular mechanism of specific traits of peppermint varieties for commercial bioactive productions.
The antioxidant capacity was in vitro evaluated by spectrophotometry via the DPPH• assay, which is linearly correlated with other antioxidant capacity indexes such as ABTS, Total Polyphenols, FRAP, and Raci [42,43]. One of the major drawbacks of in vitro assays, widely applied to evaluate antioxidant capacity, is that the oxidizing compounds used for the analyses are not present in the living being [44]. In the current research work, also in vivo studies were performed with the C. elegans model, which is closer to the real conditions since samples were metabolized by the living being, and the results were monitored throughout their entire life. The C. elegans animal model has already been used to evaluate the antioxidant properties and pathogen resistance activity of botanical extracts, among them essential oils from Mentha species [37,45,46]. The peppermint extracts from USA and Europe evaluated in this study enhanced worm's survival after acute oxidative stress and S. aureus infection. These results are in accordance with the antioxidative and antibacterial compounds detected in the peppermint extracts in the HPTLC-UV/Vis/FLD-EDA bioprofiling. It is well known that peppermint species have benefits as natural antioxidants [36,47] and have antimicrobial properties against Gram-positive bacteria, such as S. aureus [48]. These properties are mainly due to phenolic compounds, such as flavonoids, and volatile compounds [49,50].

Conclusions
For quality control and standardization of plant-based products, the use of orthogonal and thus complementary analytical tools and methods (HPLC-PDA/MS, headspace SPME-GC-FID/MS, HPTLC-UV/Vis/FLD-EDA and C. elegans model) was crucial to cope with the sample complexity and to obtain the full picture on the 16 peppermint leaf/extract samples. Substantial differences in compounds associated with functional attributes such as flavonoids and volatiles were revealed between the peppermint samples. Especially, the side-by-side bioactivity profiles in form of an image were worth a thousand words. Nontarget effect-directed analysis provided a deep understanding on the bio-functionalities of such multicomponent mixtures. Using it for industrial quality control, the costs and time per sample analysis are affordable (Euro 0.5-1.2 and 5-20 min) but vary depending on the (bio)assay, with costs for HPTLC plate and enzyme substrate being the highest expenses. The DPPH• assay and A. fischeri bioassay were fastest and cheapest, whereas the duplex bioassays took longest and were most expensive. Due to the step-based instrumentation, the next plate was already started when the application of the first plate was finished. Hence, for a plate handling shifted by 30 min, about 200 samples (on 12 plates) can be screened per day. New initiatives devoted to the challenges of sample complexity (www.vielstoffgemische.de) provide a good platform for exchange of knowledge.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.