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. 2023 May 2;19(5):48.
doi: 10.1007/s11306-023-02008-8.

Chemical and cytotoxicity profiles of 11 pink pepper (Schinus spp.) samples via non-targeted hyphenated high-performance thin-layer chromatography

Fernanda L B Mügge  1 , Gertrud E Morlock  2
Affiliations

Chemical and cytotoxicity profiles of 11 pink pepper (Schinus spp.) samples via non-targeted hyphenated high-performance thin-layer chromatography

Fernanda L B Mügge et al. Metabolomics. .

Abstract

Introduction: Pink pepper is a worldwide used spice that corresponds to the berries of two species, Schinus terebinthifolia Raddi or S. molle L. (Anacardiaceae). Toxic and allergic reactions by ingestion or contact with these plants were reported, and classical in vitro studies have highlighted the cytotoxic properties of apolar extracts from the fruits.

Objectives: Perform a non-targeted screening of 11 pink pepper samples for the detection and identification of individual cytotoxic substances.

Methods: After reversed-phase high-performance thin-layer chromatography (RP-HPTLC) separation of the extracts and multi-imaging (UV/Vis/FLD), cytotoxic compounds were detected by bioluminescence reduction from luciferase reporter cells (HEK 293 T-CMV-ELuc) applied directly on the adsorbent surface, followed by elution of detected cytotoxic substance into atmospheric-pressure chemical ionization high-resolution mass spectrometry (APCI-HRMS).

Results: Separations for mid-polar and non-polar fruit extracts demonstrated the selectivity of the method to different substance classes. One cytotoxic substance zone was tentatively assigned as moronic acid, a pentacyclic triterpenoid acid.

Conclusion: The developed non-targeted hyphenated RP-HPTLC-UV/Vis/FLD-bioluminescent cytotoxicity bioassay-FIA-APCI-HRMS method was successfully demonstrated for cytotoxicity screening (bioprofiling) and respective cytotoxin assignment.

Keywords: Bioassay; Bioprofiling; HPTLC; Schinus molle; Schinus terebinthifolia; Triterpene.

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Conflict of interest statement

There are no competing interests to declare that are relevant to the content of this article.

Figures

Fig. 1
Fig. 1
Physicochemical profiles of eleven commercial pink pepper samples. Extracts were prepared either with a mixture of ethyl acetate/ethanol/water 1/1/1 (V/V/V) or n-hexane, applied on RP-18 W HPTLC plates (10 µL/band, 100 mg/mL extracts, 7 mm bands), separated with toluene/ethyl acetate/methanol/acetic acid 30/5/2/1 (V/V/V) up to 7 cm, and detected at (a) FLD 366 nm, (b) UV 254 nm, (c) at white light illumination after derivatization with the p-anisaldehyde sulfuric acid reagent, (d) FLD 366 nm after the vanillin sulfuric acid reagent, and (e) FLD 366 nm after the natural product A reagent and polyethylene glycol 6000 (NP/PEG) (*sample ID 11 n-hexane extract was not applied due to an error)
Fig. 2
Fig. 2
Cytotoxicity screening and dose–response dependency of the n-hexane extract of pink pepper sample ID 4 (100 mg/mL) analyzed as in Fig. 1 in increasing amounts (10, 20, 30, and 40 µL; 1, 2, 3, and 4 mg/band), and detected (a) at FLD 366 nm, (b) under white light illumination, and (c) via the bioluminescence (depicted as greyscale) of HEK 293 T-CMV-ELuc reporter cells incubated on the plate for 6 h or 24 h, dried for 5 min under cold air and immersed in lysis buffer containing D-luciferin; bioluminescence reduction indicates cytotoxic substance zones (NT: not treated cells used as negative control)
Fig. 3
Fig. 3
Physicochemical profiles of 11 commercially available pink pepper ethyl acetate/ethanol/water versus n-hexane extracts applied (10 µL/band, 100 mg/mL, 7 mm bands) on the RP-18 W HPTLC plate, separated with n-hexane/acetone 4/1 (V/V), and detected at (a) FLD 366 nm, (b) UV 254 nm, and (c) FLD 366 nm after derivatization with the p-anisaldehyde sulfuric acid reagent
Fig. 4
Fig. 4
Confirmation of the cytotoxic zone in the n-hexane extract of sample ID 4 (40 µL, 7 mm band) analyzed as in Fig. 3 and detected at (a) FLD 366 nm, (b) white light illumination, and (c) via the bioluminescence reduction after incubation with HEK 293 T-CMV-ELuc reporter cells for 24 h (NT: not treated cells used as negative control)
Fig. 5
Fig. 5
RP-HPTLC−FIA-APCI-HRMS spectra in the (a) negative and (b) positive ionization mode and respective signal assignments (Table S4) of the eluted cytotoxic substance zone in sample ID 4 analyzed as in Fig. 3 and tentatively assigned as moronic acid
Fig. 6
Fig. 6
Cytotoxicity comparison of pink pepper ID 4 n-hexane extract (10 and 20 µL, 1 and 2 mg), the eluted cytotoxic substance zone (EZ, 100 µL), and the eluted blank plate background (100 µL), analyzed as in Fig. 3, and detected under white light illumination (a) and via the bioluminescence reduction after incubation with HEK 293 T-CMV-ELuc reporter cells for 24 h (b), as well as of (c) oleanolic acid (OA), ursolic acid (UA), and moronic acid (MA), 5 µg/band each, detected either at white light illumination using the p-anisaldehyde sulfuric acid reagent or (d) at FLD 366 nm using the berberine reagent, and (e) via the bioluminescence reduction after incubation with HEK 293 T-CMV-ELuc reporter cells for 24 h (MA, 10 µg/band; NT: not treated cells used as negative control)
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