For the Gram-positive B. subtilis inhibition bioassay, 80 µl of stock solution was suspended in 20 ml Müller-Hinton Broth and incubated overnight at 37°C. Before usage, the cell number was determined using a spectrophotometer (M501, Camspec, Garforth, United Kingdom) at 600 nm. At an optical density (OD₆₀₀) between 0.8 and 1.1, the culture was ready to use for EDA. An aliquot of the bacteria suspension (2 ml) was sprayed on the planar chromatogram (red nozzle, level 6) (Morlock et al., 2021b). The plate was incubated at 37°C for 2 h. As substrate solution (2 mg/ml), MTT was freshly prepared in phosphate-buffered saline. After the application of 250 µl substrate solution (blue nozzle, level 6), the plate was incubated again for 30 min at 37°C. Inhibitory zones appeared colorless (white) on a formazan-purple background. The positive control was tetracycline (10 μg/ml in ethanol, 0.4, 0.8, and 1.2 μl/band).

The bioluminescent marine Gram-negative bacteria A. fischeri were cultured according to DIN EN ISO 11348-1, Section 5 (European Committee for Standardization, 2009). Therefore, 200 µl of cryostock were suspended in 20 ml medium. The cultivation was performed overnight (18–24 h) in a 100 ml Erlenmeyer flask at room temperature by shaking at 75 rpm. Once the culture showed brilliant blue fluorescence by shaking in the dark, it was ready for use. An aliquot of the bacteria suspension (3 ml) was sprayed on the plate (blue nozzle, level 6) and directly recorded (BioLuminizer 2, CAMAG) (Morlock et al., 2021a; Morlock et al., 2021b). The native bioluminescence (depicted as a greyscale image) was documented in ten images at time intervals of 3 min. Exposure time was set to 120 s. Antibacterial components were detected as dark zones, whereas metabolism-enhancing substances appeared as bright zones on the bioluminescent background. The positive control was caffeine (1 mg/ml in methanol, 0.5, 1.5, and 3 µl/band).

The initial AChE and BChE inhibition assays (Marston et al., 2002) were modified (Hage and Morlock, 2017; Morlock et al., 2021b). The plates were pre-wetted with 0.5 ml TRIS-HCl buffer (7.55 mg/ml TRIS, pH 7.8 adjusted with HCl, green nozzle, level 6). Then, 1.5 ml of enzyme solution (AChE 6.66 U/ml, BChE 3.34 U/ml, and each 1 mg/ml BSA in TRIS-HCl buffer) were applied (green nozzle, level 6) and the chromatogram was subsequently incubated at 37°C for 30 min. For detection, 0.5 ml substrate mixture (1 mg/ml 1-naphthyl acetate, 2 mg/ml Fast Blue B salt) was sprayed (red nozzle, level 6) onto the plate to obtain colorless (white) inhibition zones on a purple background. The positive control was rivastigmine (0.1 mg/ml in methanol, 2, 4, and 8 μl/band).

An improved version of Simões-Pires et al. (Simões-Pires et al., 2009) was used to detect α- and β-glucosidase inhibitors. The substrate solution (12 mg 2-naphthyl-α-d-glucopyranoside or 2-naphthyl-β-d-glucopyranoside in 9 ml ethanol and adding 1 ml of 10 mM NaCl solution) was sprayed (1 ml, red nozzle, level 6) onto the plate, followed by drying in a stream of cold air. Pre-wetting was carried out by spraying 0.5 ml sodium acetate buffer (41 mg/ml, pH 7.5 adjusted with 0.1 mM acetic acid, green nozzle, level 6). An aliquot of the respective enzyme solution (α-glucosidase 10 U/ml, β-glucosidase 1,000 U/ml in sodium acetate buffer) was applied (1 ml; green nozzle, level 6) and the plate was subsequently incubated at 37°C for 30 min. The antidiabetic effect was visualized by Fast Blue B salt staining (2 mg/ml in water, 0.5 ml, red nozzle, level 6), resulting in colorless (white) inhibitory zones on a purple background. The positive controls were acarbose (3 mg/ml in ethanol, 1, 3, and 6 μl/band) for the α-glucosidase assay and imidazole (1 mg/ml in ethanol, 3, 5, and 7 μl/band) for the β-glucosidase assay.

The β-glucuronidase inhibition assay was run as described recently (Mahran et al., 2020) The chromatogram was pre-wetted with potassium phosphate buffer (0.5 ml; 9.34 mg/ml K₂HPO₄ and 6.31 mg/ml KH₂PO₄; green nozzle, level 6). Then, 750 µL enzyme solution (25 U/ml in potassium phosphate buffer with 1 mg/ml BSA) were sprayed onto the chromatogram (green nozzle, level 6). Incubation followed for 15 min at 37°C. As substrate, 750 µl of a 2 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronide sodium salt solution was sprayed. The plate was incubated again for 60 min for producing colorless (white) inhibitory zones on a blue background. The positive control was d-saccharolactone (0.1 mg/ml in water, 1, 1.5, and 2 µl/band).

The tyrosinase inhibitor potential was investigated according to an improved (Morlock et al., 2021b) workflow (Taibon et al., 2015). To prepare the substrate solution, 45 mg levodopa, 25 mg CHAPS, and 75 mg PEG 8000 were dissolved in 10 ml of phosphate buffer (1.4 mg/ml K₂HPO₄, 1.68 mg/ml Na₂HPO₄, pH 6.8) and stored at 4°C until use. The levodopa substrate solution was sprayed onto the chromatogram (1 ml, blue nozzle, level 6) and subsequently dried for 2 min in a stream of cold air. Then, 1 ml of enzyme solution (400 U/ml in phosphate buffer) was sprayed onto the plate (blue nozzle, level 6), followed by incubation at room temperature for 20 min. After incubation, the plate was immediately dried and documented. Tyrosinase inhibition activity was apparent as colorless (white) zones on a greyish-brown background. The positive control was kojic acid (0.1 mg/ml in ethanol, 1, 3, and 6 μl/band).

The hormonal-effective bioassays were run as recently described (Klingelhöfer et al., 2020). Cryogenic YAS or YES cell culture (1 ml each) was suspended in 39 ml or 29 ml medium, respectively. The suspensions were cultivated by shaking at 70–75 rpm and 30°C overnight (20–22 h). The cell number was determined with a hemocytometer after diluting 50 µl culture in 950 µl 0.9% NaCl solution. The required cell count of 0.8 × 10⁸ cells/ml was adjusted via centrifugation (2,500 × g, 5 min) of 5 ml yeast cell culture and resuspension in the required amount of medium plus 50 µl copper sulfate. This suspension was sprayed on the plate (1.4 ml, red nozzle, level 6), followed by incubation for 4 h (pYAS) or 3 h (pYES) at 30°C. Substrate solution (2 mg 4-methylumbelliferyl-β-d-galactopyranoside, 100 µl DMSO, 3 ml citrate buffer) was sprayed onto the chromatogram (1.5 ml, yellow ultra-nozzle, level 2). Subsequently, the plates were incubated for 1 h at 37°C. Bioautograms were recorded at FLD 366 nm. Endocrine agonists appeared as 4-methylumbelliferone-blue fluorescent zones on a dark blue background. As a positive control, testosterone (for pYAS: 0.5 µL, 1.5 μg/ml in methanol) or 17-β-estradiol (for pYES: 5 μl, 100 ng/ml in ethanol) were applied.

Potential estrogens resulting from liver metabolism were investigated by adding the S9 enzyme mixture (500 µl) and respective cofactors (166 µl NADP, 42 µl glucose 6-phosphate, 958 µl phosphate buffer) to 3,334 µl Saccharomyces cerevisiae cell culture (0.8 × 10⁸ cells/ml). The assay was performed as described above.

To screen the antagonistic activity, the pYAS or pYES bioassays were extended by overspraying along the middle of each track a 1 mm × 70 mm area of testosterone (4 μl, 1.5 μg/ml in methanol) or 17-β-estradiol (5 μl, 2 ng/ml in ethanol), respectively, with the Freemode option of winCATS (Klingelhöfer et al., 2020). Endocrine antagonists appeared as fluorescence-reducing bands in the 4-methylumbelliferone-blue fluorescent testo-sterone or 17-β-estradiol track part.

The planar SOS-Umu-C bioassay was run on HPTLC silica gel plates without a fluorescence indicator. After development, the plates were additionally scanned at 546/>580 nm using the TLC Scanner 3 (CAMAG). Salmonella typhimurium cells (50 µl cryostock) were suspended in 35 ml Lysogeny broth (20 mg/ml with 1 mg/ml d-(+)-glucose and 106 mg/L ampicillin sodium salt) and incubated overnight at 75 rpm and 37°C for 16 h. Before use, the cells were centrifuged (3,000 × g, 10 min). The pelleted cells were re-suspended in fresh medium to obtain the required OD₆₆₀ of 0.2 (Meyer et al., 2020). The chromatogram was sprayed with Salmonella suspension (2.8 ml, yellow nozzle, level 3) and incubated at 37°C for 3 h. Substrate solution [15 µl resorufin-β-d-galactopyranoside solution (20 mg/ml in DMSO) in 2.1 ml phosphate buffer and 0.9 ml glycerol] was sprayed onto the plate (2.5 ml, red nozzle, level 6). Incubation followed at 37°C for 1 h. The plates were documented at white light illumination and 366 nm. The generated resorufin fluorescence was measured at 546/>580 nm. Genotoxic substances were detectable either as pink zones on the colorless background at white light illumination or as pink fluorescent zones on a brown-reddish background at 254 nm or 366 nm. The positive control was 4-nitroquinoline-1-oxide (1 μg/ml in methanol, 1 µl/spot).

The latest α-amylase inhibition method, which used immersion of the plate into enzyme and substrate solutions (Agatonovic-Kustrin and Morton, 2017; Agatonovic-Kustrin et al., 2019), was adjusted and transferred to a piezoelectric spraying procedure, in which the enzyme solution (62.5 U/mL in sodium acetate buffer) was sprayed onto the chromatogram (1 ml, red nozzle, level 5), followed by 30 min incubation at 37°C. As substrate 2%-soluble starch solution was sprayed onto the wet plate (0.5 ml, red nozzle, level 5). After 20 min incubation at 37°C, Gram’s iodine solution was sprayed (250 μl, yellow nozzle, level 5) for visualization. The α-amylase inhibition activity was observed as violet zones on a colorless background. The positive control was acarbose (0.1 mg/ml in methanol, 0.3, 0.6, and 0.9 µl/band).

In traditional medicines, plant-based extracts are used to assist in the treatment of bacterial infections (Brantner and Grein, 1994; Palombo and Semple, 2001). Antibacterial activities can be so effective that plant extracts are also used as preservatives in food products. For example, the ingredients carnosol and carnosic acid of rosemary extract are marketed as preservative E 392. Rosemary (no. 46) is also screened here, using non-pathogenic bacterial representatives which are easier to handle in the laboratory. The Gram-positive B. subtilis bioassay is based on an oxidoreductase enzyme reaction. Intact enzymes of viable B. subtilis cells reduce the tetrazolium salt MTT to the insoluble purple formazan (Marston, 2011). Cell death is visualized as colorless zones indicating antibacterial compounds. Most antibacterials detected were located at hR _F values ≥ 90 (Figures 2–4C). In eucalyptus (no. 9), marjoram (no. 35), yerba mate green (no. 37), Siberian ginseng (no. 56), thyme (no. 57), hawthorn leaves (nos. 63 and 64), and cinnamon bark (no. 66) additional antibacterials were detected in the lower hR _F range. Essential oils from herbs such as oregano and thyme are known for their antimicrobial activity, especially against Gram-positive bacteria (Soković et al., 2010).

The Gram-negative A. fischeri bacteria are able to convert metabolic energy into turquoise bioluminescence via luciferase. A change in bioluminescence intensity is correlated with substances enhancing or reducing the cell metabolism. Such effects are visualized as lightened or dark zones on the bioluminescent background of the bioautogram (depicted as greyscale image). Almost all botanical extracts showed antimicrobial activity against A. fischeri (Figures 2–4D). Intense antimicrobial zones were detected in yerba mate green (no. 37), passionflower (no. 43), peppermint (no. 44), rooibos (no. 45), licorice (no. 55), Siberian ginseng (no. 56), and cinnamon bark (no. 66). Most samples showed at least one dark antimicrobial zone, while more than half of all samples had two or more. The more universally and sensitively detecting A. fischeri bioassay proved to be a good starting assay to investigate complex mixtures.

In traditional or ayurvedic medicine, plants and their phytoconstituents are used to assist in the treatment of Alzheimer´s disease (Azadniya et al., 2021). The pathophysiology of Alzheimer’s disease is often associated with cholinergic system dysfunction. Therefore, many synthetic drugs target the inhibition of cholinesterases. Both AChE and BChE catalyze the hydrolysis of the neurotransmitter acetylcholine into acetic acid and choline. While AChE is highly selective for acetylcholine, BChE can also convert other substrates, e.g., butyrylcholine, succinylcholine, or organophosphates. Besides synthetic drugs, also natural compounds are able to inhibit this enzyme mechanism. Particularly polyphenols interact with amino acid residues of the active side of the enzymes terminating the splitting from acetylcholine into acetic acid and choline and thus maintaining colinergic neurotransmission and improve cognition of Alzheimer’s patients (Jabir et al., 2018). The enzyme-inhibiting potential is revealed as colorless zones on a purple background. Acerola fruit (no. 1) showed a remarkably strong inhibition zone at hR _F 41 (Figures 2E,F). While roots and branches from the acerola tree are known to have anti-cholinesterase activity through the norfriedelins A-C (Liu et al., 2013), only cytotoxic, anti-HIV, antioxidant, antihyperglycemic, skin whitening, and antimicrobial activities are described for extracts from the fruits (Motohashi et al., 2004; Belwal et al., 2018a). This zone showed not only a strong response in the AChE and BChE assays, but also in most other assays (Figure 2). Other inhibitory zones were detected in ginger (no. 25, hR _F 99), kola (no. 31, hR _F 68), marjoram (no. 35, hR _F 99), yerba mate green (no. 37, hR _F 88), lemon balm (no. 38, hR _F 32), peppermint and rooibos (nos. 44 and 45, both hR _F 34), Siberian ginseng (no. 56, hR _F 93), and hawthorn (nos. 62–64, hR _F 60 and 85) (Figures 2–4E,F). Some typical traditional medicines used in the treatment of Alzheimer´s disease, such as Panax notoginseng, Ginkgo biloba (no. 14), Melissa officinalis (no. 38), and Salvia officinalis (no. 47), are also screened here. Among these, Melissa officinalis (no. 38) possess cholinesterase-inhibiting potential, the others operate according to a different mechanism to treat the neurodegenerative disease (Sharma et al., 2019).

The detoxification mechanism via glucuronidation can be reversed with β-glucuronidase from opportunistic Enterobacteriaceae such as Escherichia coli, resulting in gastrointestinal malfunction. This can be prevented by inhibiting the microbial β-glucuronidase, where such inhibitors address the extra loop in the bacterial enzyme (in contrast to the mammalian one) (Mahran et al., 2020). The enzyme inhibitors do not cleave the chromogenic substrate and are therefore detected as colorless zones on an indigo-blue background. In each eucalyptus (no. 9), guarana (no. 16), marjoram (no. 35), yerba mate green (no. 37), oregano (no. 42), Siberian ginseng (no. 56), and cinnamon bark (no. 66), the whole sample track appeared white on the indigo-blue background due to the comparatively high abundance of β-glucuronidase inhibitors (Figures 2–4G). If the application volume is reduced by a factor of 4 for these botanicals, the individual inhibitors become evident (Supplementary Figure S2). All botanical extracts showed β-glucuronidase inhibitory potential at least in the solvent front (hR _F 99). For such screening results, repetition using a mobile phase of reduced solvent strength is recommended in order to better differentiate the individual inhibitors (Supplementary Figure S3C,D). Some isolated flavonoid standards, i.e., isorhamnetin, kaempferol, liquiritigenin, daidzein, etc., already proved to be active against β-glucuronidase (Sun et al., 2020). Their activity and also that of additional flavonoids have been confirmed by our study directly in herbs and spices.

Polyphenoloxidases are responsible for the browning of cut or injured fruits or plant tissues. In mammalian cells, the corresponding tyrosinase controls melanogenesis by catalyzing the hydroxylation of phenols with subsequent oxidation to quinones. An overproduction of melanin induces pigmentary abnormality, freckles, or age spots. Preferably, the cosmetics industry is interested in naturally derived tyrosinase inhibitors (Taibon et al., 2015). In this context, ethnobotanicals are brought into focus. In South Africa, herbal extracts are traditionally used as skin care products to treat burns, abcesses, wounds and acne (Lall and Kishore, 2014). Chinese herbal medicines with anti-tyrosinase activity are traditionally used as folk skin whiteners. Among the studied botanicals, Ginkgo biloba (no. 14), Panax ginseng (no. 15), and Zingiber officinale (no. 25) were reported to inhibit mushroom tyrosinase (Ye et al., 2010; Hu et al., 2020). Our screening results showed that ginseng (no. 15) and ginger (no. 25) played a minor role in tyrosinase inhibition compared to other botanicals. However, differences in effects can be caused for example by plant subspecies, climate, soil, environmental and agricultural conditions as well as extraction mode. In the planar assay, tyrosinase inhibitors are shown as colorless zones on a greyish-brown background. Many botanical extracts showed multiple tyrosinase inhibitors (Figures 2–4H). While acerola (no. 1) (Belwal et al., 2018a), ginkgo (no. 14) (Shu et al., 2020), licorice (no. 55) (Li et al., 2017), and hawthorn (nos. 62–64) (Rocchetti et al., 2020) are known for their anti-tyrosinase activity, the screening results proved similarly potent tyrosinase inhibitors in artichoke (no. 4), plantain (no. 35), yerba mate green (no. 37), rosemary (no. 46), and yarrow (no. 50).

The enzymes α- and β-glucosidase hydrolyze the saccharide dimers and oligomers, as well as glucosides dependent on the anomeric glycosidic bond, into resorbable monomers such as glucose and into aglycones. In the treatment of hyperglycemic blood levels of type 2 diabetes patients, enzyme inhibitors are of therapeutic interest by reducing postprandial glucose uptake (Simões-Pires et al., 2009; Turkiewicz et al., 2019). Screening results showed several α- and β-glucosidase inhibitors as a specific pattern (at hR _F 42, 48, 62, and 80) for artichoke (no. 4) in both assays (Figures 2I,J). Although the α- (Turkiewicz et al., 2019) and β-glucosidase (Morlock et al., 2021a) inhibitory potential and chemical composition of extracts of different artichoke cultivars have already been described, the distinct bioactive components have been scarcely assigned in literature. In both glucosidase inhibition assays, samples of blackberry leaves (no. 8), yellow fruit tea (no. 11), red fruit tea (no. 12), elderflower (no. 22), yerba mate green (no. 37), horsetail (no. 49), plantain (no. 53), Siberian ginseng (no. 56), and lemon verbena (no. 68) showed a positive response at hR _F 42 (Figures 2–4I,J). In all of them, the same bioactive compound was assumed. Another remarkably similar active compound was observed at hR _F 94 for basil (no. 5), ginkgo (no. 14), European blueberry (no. 18), elderberry (no. 21), hop (no. 24), yerba mate green (no. 37), lemon balm (no. 38), oregano (no. 42), peppermint (no. 44), rosemary (no. 46), star anise (no. 54), Siberian ginseng (no. 56), thyme (no. 57), and cinnamon bark (no. 66). All of them showed both α- and β-glucosidase inhibitory activities. Worldwide more than 1,000 herbal remedies were traditionally deployed for the maintenance and treatment of high blood glucose levels and thus diabetes. Both, the ethnobotanicals used and their mode of application (as tincture or extract, orally or as infusion) differ between local communities (Cock et al., 2021). Since western medical treatment methods for type 2 diabetes focus on hypoglycemic drugs, such as insulin, ethnopharmacological remedies are considered to be safe and to have less toxic side effects. According to the theory of traditional Chinese medicine, flavonoids (Bai et al., 2019) and polyphenols (Umeno et al., 2016) are attributed to have antidiabetic effects via several mechanisms. Glycyrrhizic acid from Glycyrrhiza glabra (no. 55), apigenin and its derivates as well as quercetin, found in many botanicals (Table 2), are known to target α-glucosidase (Bai et al., 2019).

Disrupting endocrine signaling pathways can have a severe impact on hormonal balance and cause feminization/masculinization, infertility, acne, and menstrual cycle disorders. Also phytochemicals present in food (Morlock and Klingelhöfer, 2014) or commodities in daily use (Klingelhöfer et al., 2020) can affect the human hormone system. HPTLC hyphenated to the planar yeast androgen/estrogen screens (pYAS/pYES) and their antagonistic versions (pYAAS/pYAES) showed positive reactions to a limited extent (Figures 2–4K–N). In the pYAS bioassay, a few 4-methylumbelliferone-blue and thus androgenic responses were detected in acerola (no. 1, hR _F 16), elderberry (no. 21, hR _F 72), chamomile (no. 28, hR _F 99), orange peel (no. 41, hR _F 67 and 98), horsetail (no. 49, hR _F 49 and 61), lemon peel (no. 67, hR _F 99), and for several samples at the application zone (nos. 4, 15, 25, 30, and 56). In the pYES bioassay, 15 botanicals showed estrogen-like activity. In some samples, phytoestrogens were known and expected, e.g., 8-prenylnaringenin in hop (no. 24) (Prencipe et al., 2014; Mbachu et al., 2020), (iso-)liquiritigenin in licorice (no. 55) (Boonmuen et al., 2016), or pesticide residues from fruit surfaces (Schulte-Oehlmann et al., 2011) such as orange peel (no. 41), grape peel (no. 59), or lemon peel (no. 67). However, in acerola (no. 1, hR _F 15 and 27), galangal (no. 13, hR _F 99), chamomile (no. 28, hR _F 63 and 95), lovage (no. 34, hR _F 97), marjoram (no. 35, hR _F 62), oregano (no. 42, hR _F 45), juniper (no. 60, hR _F 89), grape leaves (no. 61, hR _F 92), and hawthorn (nos. 62–64, hR _F 94), estrogen-like responses were also detected. Since steroid hormone-like compounds are known to have a greater affinity to hERα, but several phytoestrogens (e.g., daidzein or genistein) to hERβ (Mbachu et al., 2020), the pYES bioassay was also performed via the hERβ for the first time. The assay was analogously run for both receptors, but no remarkable difference was observed in the results obtained by both (Figure 5A versus Figure 5B). The hERα seemed to be less selective. However, a significant difference in the estrogenic pattern was observed after metabolization with the S9 mixture (Figure 5C). The use of genetically modified yeast cells containing the hERβ for the pYES bioassay and the simulated metabolization via the S9 liver enzyme system were reported here for the first time.

In the respective antagonistic assays, only a few zones were detected. For acerola (no. 1, hR _F 99), fenugreek (no. 6, hR _F 99), eucalyptus (no. 9, hR _F 5 or 89), ginseng (no. 15, hR _F 96), guarana (no. 16, hR _F 60), kola (no. 31, hR _F 73), orange peel (no. 41, hR _F 25 or 32), licorice (no. 55, hR _F 27), thyme (no. 57, hR _F 24 or 50), and lemon verbena (no. 68, hR _F 99), the possibly antiandrogenic zones were also investigated using an overlayed area of the fluorescent 4-methylumbelliferone (Supplementary Figure S4) to exclude any false-positive response as observed for the physicochemical fluorescence reduction by pigments or dyes (Klingelhöfer et al., 2020). Seven botanical samples (nos. 6, 15, 16, 31, 41, 55, and 68) showed truly antiandrogenic activities. The same verification test was run for possible antiestrogens in galangal (no. 13, hR _F 99), guarana (no. 16, hR _F 72 or 91), garlic (no. 30, hR _F 99), kola (no. 31, hR _F 99), orange peel (no. 41, hR _F 34), licorice (no. 55, hR _F 31 or 43), thyme (no. 57, hR _F 31), grape seeds/leaves (nos. 58/61, hR _F 48, 61 or 92), and lemon verbena (no. 68, hR _F 99). Seven botanicals (nos. 16, 30, 41, 55, 57, 58, and 68) revealed true antiestrogenic properties (Supplementary Figure S5). In the antiestrogenic assay, synergistic effects were evident. The overlapping 17-β-estradiol area (10 pg/70 mm area) is partially enhanced on sample tracks, apparent as a cotton swab shape if compared to the solvent blank (Figures 2–4N). This observation revealed synergistic effects between distinct botanical ingredients in most samples (except for nos. 7, 29, and 31) and 17-β-estradiol, resulting in fortified estrogenic activities (Figure 6). This overlapped experimental setup can be transferred to all other assays to identify synergy. Such a synergistic effect detection strategy was reported here for the first time.

In recent decades, there has been a steady trend away from industrial medical care towards phytotherapy based on medicinal herbs, which have been used in traditional medicine for years. The common belief that phytochemicals are gentler than synthetic medicines may prove to be a fallacy, as most toxic compounds originate in nature (Efferth and Kaina, 2011). The SOS-Umu-C assay is a reporter gene assay indicating genotoxicity based on a genetically modified test organism Salmonella typhimurium TA1535 [pSK1002]. Under genotoxic stress, the SOS-DNA repair mechanism is induced. The SOS-Umu response activates the lacZ gene, encoding for β-galactosidase, which enables substrate cleavage into detectable products (Meyer et al., 2020). As the analyzed botanicals are commonly used as spices, in herbal medicine, or as tea, no genotoxic substances were expected. Nevertheless, pyrrolizidine alkaloids are known for genotoxic properties and their occurrence in herbal formulations (Habs et al., 2017), e.g., only a few micrograms per liter of tea (6 μg/L) (Mulder et al., 2015). The European Food and Safety Authority calculated a margin of exposure to 237 g/kg body weight per day for pyrrolizidine alkaloids and their N-oxides (Knutsen et al., 2017; Kaltner et al., 2020). No genotoxicity was detected as pink fluorescent zone for the 68 botanical extracts applied at 400 µg/band (Figures 2–4O). This bioassay was repeated to prove for the absence of genotoxic effects at a 2.5-fold (Supplementary Figure S6) and 12.5-fold higher amount applied, which latter at 5 mg/band was closest to overloading the chromatographic system (Supplementary Figure S7).

In the context of hypoglycemic drugs from nature, not only the mentioned α- and β-glucosidase inhibitors, but also α-amylase inhibitors play a role in ethnopharmacological remedies with less toxic side effects. Hyperglycemic blood levels could be reduced by inhibiting α-amylases of the saliva and pancreas. The inhibition reduces the cleavage of starch into oligo- and disaccharides, and so the release of glucose molecules absorbed postprandially into the blood. Plant-derived flavones as luteolin present in celery, parsley, broccoli, carrot, peppers, cabbage, and apple peel can inhibit α-amylase (Bai et al., 2019). HPTLC coupled to α-amylase assay (Figures 2–4P) showed only a few inhibiting signals in white horehound (no. 2, hR _F 43, 83, 91, and 93), artichoke (no. 4, hR _F 94), fenugreek (no. 6, hR _F 81), ginkgo (no. 14, hR _F 46), licorice (no. 55, hR _F 59 and 68), and lemon peel (no. 67, hR _F 93 and 95). An α-amylase inhibitory activity has already been described for artichoke extracts from bracts (Turkiewicz et al., 2019), but it was not assigned to any single compound. In Ginkgo biloba extracts the compound sciadopitysin (C₃₃H₂₄O₁₀) was found to potentially inhibit α-amylase (Petersen et al., 2019).

For orange peel, manifold positive responses across the different assays were observed at hR _F 34 (Figure 7A, marked*). Using the pYEAS bioautogram, this zone was heart-cut eluted directly to RP-HPLC−DAD−HESI-MS. In the DAD chromatogram, three compound peaks were evident in the range of RT 6.87–7.07 min, apart from the 4-methylumbelliferone background signal at RT 5.71 min (Figure 7B). The corresponding mass spectral data led to the tentative identification of naringin (Figure 7C), hesperidin (Figure 7D), and meranzin (Figure 7E, coeluting with an unknown marked blue) and is discussed as follows. The total ion current (TIC) peak at RT 6.87 min revealed mass signals in the positive ionization mode at m/z 581 [M + H]⁺, 603 [M + Na]⁺, and 619 [M + K]⁺. Corresponding mass signals in the negative ion mode were detected at m/z 579 [M−H]⁻, 615 [M + Cl]⁻, and 642 [M + NO₃]⁻ (Figure 7C). The resulting neutral mass of 580 Da together with the absorption spectrum and maximal wavelengths (λ_max = 220 and 284 nm) suggested naringin, which was confirmed via co-chromatography against a bought standard (Supplementary Figure S8E). The extracted mass spectra of the TIC peak at RT 6.99 min showed positive ions at m/z 611 [M + H]⁺, 633 [M + Na]⁺, and 649 [M + K]⁺ and negative ions at m/z 609 [M−H]⁻, 645 [M + Cl]⁻, and 672 [M + NO₃]⁻ (Figure 7D). The absorption spectrum revealed a maximum wavelength at 284 nm. Based on this data, hesperidin was assumed. The third peak at RT 7.06 min had a major response in TIC-HESI⁺ and a minor response in TIC-HESI⁻ and DAD chromatograms. The UV absorbance spectrum and respective mass spectra suggested two different constituents. The HESI⁺ mass signals at m/z 261 [M1+H]⁺ and 278 [M1+NH₄]⁺ potentially belong to the coumarin derivate meranzin (Figure 7E). The HESI⁺ mass signals at m/z 441 [M2+H]⁺, 458 [M2+NH₄]⁺, 463 [M2+Na]⁺, and 479 [M2+K]⁺ and corresponding HESI⁻ mass signals at m/z 475 [M2+Cl]⁻, 485 [M2+HCOO]⁻, and 499 [M2+H₃C-COO]⁻ were assigned to a component with the neutral mass of 440 Da, which is not yet known in orange peel. The UV maxima at 258 and 325 nm provided additional evidence for meranzin (Fan et al., 2012). Other UV maxima at 223 and 288 nm were possibly induced by the unknown.

Citrus sinensis is rich in beneficial secondary metabolites and therefore traditionally used in the treatment of gastrointestinal malfunction, diseases of the upper respiratory tracts, or menstrual disorders (Favela-Hernández et al., 2016). Both naringin and hesperidin have been shown to bind to the antagonist pocket (3ERT) of hERα, and thereby cause an antiestrogenic effect (Puranik et al., 2019). This effect was verified for both via co-chromatography of standards and samples (Supplementary Figure S8E, Supplementary Table S1), whereby an estrogenic activity was not observed for the applied amount (4 µg/band). Furthermore, naringin was reported to slightly bind to the androgen receptor (Fang et al., 2003). The pronounced antiandrogenic effects observed in the pYAAS bioautogram confirmed this (Figure 3M, no. 41). Tyrosinase activity attributed to naringin (Itoh et al., 2009) and hesperidin (Zhang et al., 2007) has already been demonstrated. The only bioactivity reported for meranzin was no effect (Smyth et al., 2009) or a minor (Rosselli et al., 2007) antibacterial effect against B. subtilis. After NP-HPTLC–FLD comparison to a standard, meranzin was located at hR _F 99 (Supplementary Figure S8G), where antibacterial activity against B. subtilis and A. fischeri was detected in orange peel (no. 41). Hence, the assumption that meranzin was co-eluting with hesperidin and naringin at hR _F 34 was discarded.

In licorice (no. 55) a few bioactive analytes were found in the zone at hR _F 31 with antibacterial, tyrosinase, and β-glucuronidase inhibitory, antidiabetic, and endocrine-antagonistic properties (Figure 8A, marked*). These results illustrate the diverse pharmacological activities of this root, which has long been used in traditional medicine as remedy to treat gastrointestinal problems (β-glucuronidase inhibition) and respiratory infections (antibacterial activity). Moreover, Glycyrrhiza glabra extracts were subject of many pharmacological studies showing neuroprotective, antimicrobial, estrogenic and skin-whitening activity (Pastorino et al., 2018). The second chromatography produced five individual signals with pure mass spectra (Figures 8B–E). The mass spectral data extracted from the peaks at RT 6.52/6.63 min (colored blue) were identical, indicating a single analyte in different configurations (Figure 8C). The ESI⁻ signals at m/z 549 [M−H]⁻, 585 [M + Cl]⁻, and 612 [M + NO₃]⁻ were correlated to the highly plant-specific liquiritin apioside with a neutral mass of 550 Da. In positive ion mode, the signals at m/z 573 and 589 were identified as sodium and potassium adducts, respectively. The ESI⁺ mass signals at m/z 257 [M−C₁₁H₁₈O₉+H]⁺ and 419 [M−C₅H₈O₄+H]⁺ could be assigned to fragments with a loss of carbohydrates. Additionally, the experimental UV absorbance spectra were consistent with the ones of liquiritin apioside found in literature (Wong et al., 2018). The isomeric isoliquiritin apioside probably caused the second peak at RT 6.63 min. ESI⁺ mass signals at RT 7.10 min were m/z 563 [M + H]⁺, 585 [M + Na]^+, and 601 [M + K]⁺ (Figure 8D). (Iso)liquiritin apioside was found to inhibit capsaicin-induced cough, confirming its traditional use (Pastorino et al., 2018). In negative ion mode, signals were detected at m/z 561 [M−H]⁻, 597 [M + Cl]⁻, 621 [M + H₃C-COO]⁻, and 624 [M + NO₃]⁻. The less abundant DAD signal showed a UV absorption maximum at 250 nm. The spectral data could indicate glycyroside found in licorice root extracts (Montero et al., 2016), which is not known for any bioactivity. The third signal at RT 7.86 min (colored orange) was identified as glycyrrhizic acid against a standard (Supplementary Figure S8E). The ESI⁻ and ESI⁺ ions at m/z 821 [M−H]⁻, 843 [M−2H + Na]⁻, 859 [M−2H + K]⁻, 845 [M + H]⁺, and 861 [M + K]⁺ originated from the neutral molecule of 822 Da. Lacking a π-electron system and conjugated double bonds, the UV absorbance spectrum showed background absorbance maxima at 251 nm. Glycyrrhizic acid was reported to have anti-inflammatory effects (Yu et al., 2015) similar to those of glucocorticoids (Pastorino et al., 2018), antitussic activity through increased tracheal mucus secretion (Sharma et al., 2018), and neuroprotective (Kao et al., 2009) properties.

Galangal extract (no. 13) responded in almost all assays at hR _F 99 (Figure 9A, marked*). After separating this zone with RP-HPLC-DAD-HESI-MS, multiple signals were observed (Figure 9B). The spectral details and tentative identifications are listed in the table below (Figure 9C). Traditionally, Alpinia officinarum is used against cold, which antibacterial effects were confirmed by the Gram-negative A. fischeri and Gram-positive B. subtilis bioassays. Other traditional applications were described for gynecological disorders, diabetes treatments, and skin washing (Abubakar et al., 2018). All these bioactivities were verified by the respective assays. To assign the bioactivity to one single component out of the coeluting substances from HPTLC, fractionation after column separation is necessary. The fractions can be applied again on a new plate followed by EDA and MS characterization. Alternatively, the mobile phase for planar chromatography has to be optimized in order to separate the previously coeluting substances during HPTLC analysis.

Biologically active isomers were also separated and detected, shown for example in yerba mate green (no. 37). A multipotent bioactive compound zone (hR _F 40) was observed in AChE, tyrosinase, and α-/β-glucosidase inhibition autograms (Figure 10A, marked*). This zone was separated into three distinct signals via RP-HPLC (Figure 10B), all providing the same absorbance and mass spectra (Figure 10D) and assigned to chlorogenic acid isomers, which differed only in quinic acid positioning (Figure 10C). The most common isomers of mono-caffeoylquinic acid are 3-O-caffeoylquinic acid (neochlorogenic acid), 4-O-caffeoylquinic acid (cryptochlorogenic acid), and 5-O-caffeoylquinic acid (5-QCA, chlorogenic acid). The mass signals at m/z 353 [M−H]⁻ and 707 [2M−H]⁻ in the negative ionization mode and at m/z 355 [M + H]⁺, 377 [M + Na]⁺, 393 [M + K]⁺ and 747 [2M + K]⁺ in the positive ionization mode matched to the neutral mass of mono-caffeoylquinic acids of 354 Da. The UV absorbance spectra with maxima at 218 and 325 nm that we obtained were also consistent with literature (Velkoska-Markovska et al., 2020). While chlorogenic acids are reported to have antioxidant (Huang et al., 2017), anti-inflammatory (Willems et al., 2016; Huang et al., 2017), and anti-HIV (Tamayose et al., 2019) properties, this screening revealed even more bioactive potential for these phenolics. For instance, the assigned bioactive compound zone also showed anti-Alzheimer, antidiabetic, and skin-whitening effects in this study. Co-chromatography against standards (Supplementary Figure S8A,B and D, Supplementary Table S1) confirmed that these beneficial health effects of yerba mate green (no. 37, Figure 10) come from the mono-caffeoylquinic acids. The various effects of Ilex paraguariensis traditionally consumed as herbal beverage qualify this botancial for its new role as functional food (Cardozo Junior and Morand, 2016).

The characterization of additional multipotent bioactive zones, partially plant-specific, is shown in Figure 11. Artichoke (no. 4) showed bioactivity at hR _F 42 in the tyrosinase and α-/β-glucosidase inhibition assays. Transferring this zone to RP-HPLC-DAD-HESI-MS provided five signals (Table 2), one of which is specific for artichoke. At RT 6.30 min, cynarascoloside C was assumed (Figure 11A). Spectral data showed no UV absorbance, referring to structural properties of cynarascoloside C which possess neither a π-electron system nor conjugated double bonds. The mass signals at m/z 444 [M + NH₄]⁺, 449 [M + Na]⁺, 465 [M + K]⁺, 461 [M + Cl]⁻, 471 [M + HCOO]⁻, 485 [M + H₃C-COO]⁻, and 488 [M + NO₃]⁻ indicated a neutral mass of 426 Da. Cynarascoloside C was not suspected to have bioactive effects. The bioactivity was probably attributed to the coeluting derivates of chlorogenic acid as in yerba mate green (no. 37, Figure 10). Nevertheless, Cynara scolymus has been used since the 4th century B.C. as medicinal product due to its health benefits and bioactive constituents, responsible for the hypoglycemic, anti-inflammatory, antimicrobial, and antioxidant properties (Turkiewicz et al., 2019).

Eucalyptus (no. 9) is known for its beneficial health properties against infections of the upper respiratory tracts or as antiseptic and is therefore widely used in the pharmaceutical industry (Hasegawa et al., 2008; Ács et al., 2018). The species Eucalyptus is closely associated with herbal medicine and traditional health care in various human cultures. Its leaf extracts are administered to fight against cold and cough, bacterial infections, high blood glucose levels, and to boost the immune system and skin health (Salehi et al., 2019). Planar bioanalytical screening confirmed most of those traditional uses, showing many positive responses through the assays in a wide hR _F range (Figure 11B). Focusing on hR _F 57, highly plant-specific eucaglobulin was identified with UV (λ_max = 222 and 261 nm) and mass spectral data (Boulekbache-Makhlouf et al., 2010). Both the positive ion species at m/z 521 [M + Na]⁺ and 537 [M + K]⁺, and the deprotonated molecule [M−H]⁻ confirmed this suspicion. Anti-melanogenesis activity was attributed to eucaglobulin (Hasegawa et al., 2008) and proved with a planar tyrosinase bioassay. Extracts of eucalyptus fruits were demonstrated to have antibacterial effects against B. subtilis (Boulekbache-Makhlouf et al., 2013), which so far have not been directly correlated to the monoterpene conjugate eucaglobulin. The anti-cholinesterase activity was only described for the whole methanolic extract of Eucalyptus globulus (Amat-ur-Rasool et al., 2020) but not directly correlated to eucaglobulin.

Extracts of Ginkgo biloba seeds and leaves were applied as herbal remedies all over the globe. Originating from traditional Chinese medicine, it is used to treat bacterial skin diseases, cognitive decline (Chassagne et al., 2019), and cardiovascular diseases (Shu et al., 2018). Affirming the ethnopharmacological usage, EDA of ginkgo leaf extract (no.14) demonstrated a variety of bioactivity at hR _F 93, i.e., antibacterial and antidiabetic effects, as well as β-glucuronidase and tyrosinase inhibition, and antiandrogenic activity (Figure 11C). RP-HPLC-DAD-HESI-MS analysis revealed the three ginkgolides A–C incorporated in this bioactive zone. Ginkgolide C eluted earlier from RP column (RT 6.42 min). Despite the second chromatography, ginkgolides A and B were not separated (both RT 6.86 min) and were thus detected in the same UV and mass spectra. Structurally they only differ in one additional hydroxy group of ginkgolide B. The high abundance of different ion species describing the two compounds is listed in Table 2 and displayed in Figure 11C. The wide bioactive spectrum of these two diterpenes was confirmed against standards (Supplementary Figure S8B, Supplementary Table S1).

As an alternative to coffee, the leaves of Ilex paraguariensis are widely consumed as beverage in Latin America. Due to its chemical composition, mainly alkaloids and polyphenols, yerba mate exhibits many bioactive effects, e.g., antibacterial, cardiovascular-protective, neuroprotective, and antidiabetic activities (Gan et al., 2018). In yerba mate green (no. 37), a multipotent bioactive analyte zone was detected at hR _F 20 (Figure 11D). Showing signals at m/z 609 [M−H]⁻ in ESI⁻ and at m/z 611 [M + H]⁺, 633 [M + Na]⁺, and 649 [M + K]⁺ in ESI⁺ at an RT of 6.84 min, the flavonoid rutin was assumed. Its antibacterial (Orhan et al., 2010) potential against B. subtilis, anti-tyrosinase activity (Kishore et al., 2018), and glucosidase inhibition (Li et al., 2009) have already been demonstrated. The assumption was verified against a standard (Supplementary Figure S8A and D, Supplementary Table S1).

Salvia rosmarinus, predominantly growing in Mediterranean regions, is independently used in Mexican and Spanish ethnopharmacology. As medicinal plant, it was used to fight bacterial skin diseases, colds, intestinal parasites, and headaches (Heinrich et al., 2006). A very broad spectrum of bioactivities was confirmed by the presented study. The comprehensive NP-HPTLC-EDA-heart-cut RP-HPLC-DAD-HESI-MS analysis of rosemary (no. 46) showed antibacterial activity in the A. fischeri bioassay and antidiabetic properties in the β-glucosidase assay at hR _F 25. Several ions at m/z 387 [M−H]⁻, 406 [M + NH₄]⁺, 411 [M + Na]⁺, and 427 [M + K]⁺ in positive and negative ion mode were correlated to the neutral molecular weight of 388 Da (Figure 11E). The mass spectrometric data substantiate the suspicion that this bioactivity is related to medioresinol. In rosemary extracts, various bioactive compounds have been described. Carnosic acid and carnosol are known to inhibit the growth of human cancer cell lines and operate as anti-inflammatory agents (Bai et al., 2010; Wang et al., 2018) or antioxidants (Loussouarn et al., 2017). Rosmarinic acid and rosmanol were found to have antioxidant potential (Vallverdú-Queralt et al., 2014), but to our knowledge, there is no bioactivity reported for medioresinol.

Many more examples could be explained in detail. All have in common that the effect profiles we obtained explain why consuming green tea and using fresh herbs and spices as seasoning can reduce the risk of diabetes by inhibiting α- and β-glucosidases, why healthy nutrition can protect the intestinal flora from severe impairment caused by β-glucuronidases from Enterobacteriaceae, why plant-derived cosmetics can reduce skin abnormalities via its tyrosinase-inhibiting potential, and why Alzheimer’s disease can be prevented by daily intake of chlorogenic acid or rosmarinic acid from herbs such as artichoke, lemon balm, peppermint, thyme and rosemary, and so on. The wealth of effect information obtained inspires the mind and could fuel further studies. An enormous diversity in bioactivity was revealed in the effect-profiles of the 68 botanicals, contributing to human health by drug-like properties. It clearly shows the potential and spectrum of nature as basis for alternative medicines.