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

Abstract

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.

Keywords: Caenorhabditis elegans; HPLC–PDA/MS; HPTLC–UV/Vis/FLD–EDA; antioxidant; flavonoid; functional food; headspace SPME–GC–FID/MS; high-performance thin-layer chromatography; planar bioassay; planar enzyme assay.

Figure 1

Bioprofiling of 14 peppermint products for 9 different bioactivity mechanisms: HPTLC–Vis/UV/FLD chromatograms of the various leaf samples L1–L7 (Table 1; all extracted/dissolved with water—ethanol—ethyl acetate 1:1:1; 10 µL/band each) and respective powdered extracts E1–E7 (2 µL/band each) along with standard mixture (M; eriocitrin, luteolin-7-O-glucoside, rosmarinic acid, and apigenin, 1.5 µg/band each) and the respective solvent blank (B) separated on HPTLC plate silica gel 60 F₂₅₄ s with 7 mL ethyl acetate—toluene—formic acid—water 8:2:1.5:1 and detected at UV 254 nm, FLD 366 nm and after the respective (bio)assay at white light illumination (bioluminescence depicted as greyscale image for A. fischeri). The respective PC was applied on the upper right plate edge (cropped for some assay images). Derivatization with natural product reagent A was performed after the assay on the tyrosinase inhibition autogram.

Figure 2

Bioprofiling for endocrine active and genotoxic compounds: HPTLC–FLD bioautograms of peppermint products (Table 1) extracted with water—ethanol—ethyl acetate 1:1:1 (0.1 mg/mL each; 15 µL/band or 1.5 mg for L1–L7, and 3 µL/band or 0.3 mg for E1–E8; L5/E5 skipped) on HPTLC plate silica gel 60 along with standard mixture (M; eriocitrin, luteolin-7-O-glucoside, rosmarinic acid, and apigenin, 2.3 µg/band each) and the respective solvent blank (B) separated with 7 mL ethyl acetate—toluene—methanol—water 4:1:1:0.4 and detected at 254 nm via the duplex pYAES and pYAAS bioassays as well as the planar SOS-Umu-C bioassay. Green fluorescent estrogenic compounds (*) were detected. The green fluorescence of the respective agonist stripe (applied along each sample track) or of the genotoxin 4-nitroquinoline 1-oxide (applied on the upper right plate edge) were considered as positive controls.

Figure 3

HPLC–PDA profile at 280 nm exemplarily for the peppermint leaf sample L2 from Europe, showing caffeic acid (9.01 min), eriocitrin (12.31 min), luteolin-7-O-rutinoside (12.72 min), eriodictyol-7-O-glucoside (13.39 min), luteolin-7-O-glucuronide (13.64 min), luteolin-7-O-glucoside (13.92 min), isorhoifolin (14.98 min), and rosmarinic acid (17.83 min).

Figure 4

HPLC–PDA profile at 280 nm of the corresponding peppermint extract E2 from Europe.

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.

Figure 6

Percentage of survival of Caenorhabditis elegans N2 treated with the different peppermint extracts E1–E7 and infected with the pathogen Staphylococcus aureus (ATCC 25923) at day 4 and 5 post-infection. **** Significant p-value < 0.0001 in comparison to S. aureus condition at every corresponding day post-infection. A two-way ANOVA test was applied. Data are the average of two independent experiments.