3.5.1. Extraction

For the selection of the extraction solvent, 400 mg each of pineapple (PTW) or mango (NDM) puree were placed in a 1.5 mL centrifuge tube, mixed and extracted with 1 mL solvent (methanol, acetone, ethanol, ethyl acetate, and acetonitrile) in the ultrasonic bath for 10 min. Each suspension was centrifuged (5 min, 11,600× *g*) and the supernatant transferred into an autosampler vial (1.8 mL). For effect-directed profiling, the puree (2 g) and juice samples (2 mL) were extracted with 10 mL ethyl acetate, mixed (vortexed) and centrifuged (5 min, 3000× *g*). The supernatant was removed with a Pasteur pipette, was transferred to a 10 mL glass vial and stored at −18 ◦C until use.

#### 3.5.2. Sample Application and Development

Instrumentation was used from CAMAG. Plates were cut to smaller pieces using the smartCUT Plate Cutter. Solutions were sprayed as a band (8 mm) for sugar analysis or as area (6.0 × 3.0 mm for 10 µL or 8.0 × 6.0 mm for 100 µL) for other analyses on HPTLC plates with the Automatic TLC Sampler (ATS) 4. Up to 8 tracks were applied onto an HPTLC plate, 10 cm × 10 cm, with a distance of 10 mm from the lower edge, 15 mm distance from the left edge and 10 mm distance between the bands. Sample volumes ranged 10 µL for extraction solvent selection and 100 µL for effect-directed profiling. Development was performed with a mixture of with toluene–ethyl acetate–methanol–formic acid 2.4:1.8:0.7:0.06, *v*/*v*/*v*/*v* [31] up to a migration distance of 60 mm from the lower plate edge in a twin-through chamber. The chromatogram was dried in a stream of warm air (hair dryer) for 2 min.

#### 3.5.3. Derivatization and Documentation

For derivatization, the HPTLC plate was immersed into the anisaldehyde sulfuric acid reagent (5 mL concentrated sulfuric acid was carefully added to a mixture of 500 µL anisaldehyde, 10 mL acetic acid, and 100 mL methanol) or diphenylamine aniline *o*-phosphoric acid reagent (mixture of 70 mL aniline solution, 70 mL diphenylamine solution, 2% each in acetone, and 10 mL *o*-phosphoric acid, 85%) using the TLC Chromatogram Immersion Device. The immersion speed was 3 cm/s and the immersion time 2 s. The plates were heated at 110 ◦C on the TLC Plate Heater for 5 min. The HPTLC chromatograms were documented at UV 254, FLD 366 nm and white light illumination (reflection and transmission mode) using the TLC Visualizer. All data obtained was processed with the software winCATS, version 1.4.7.2018.

#### 3.5.4. Effect-Directed Detection

The HPTLC plates were immersed into the respective assays using the TLC Chromatogram Immersion Device (immersion speed 3 cm/s), heated using the TLC Plate Heater, and if not stated otherwise, documented using the TLC Visualizer or DigiStore 2 Documentation System. Before application of the biological assays, the chromatogram was neutralized with ammonia vapor for 5 min and freed from any excess vapor or acidic mobile phase traces (Automatic Developing Chamber 2; relative humidity control glass flask filled with dry molecular sieve) for 25 min.

Radical scavenging assay: The chromatogram was dipped in a 0.02% methanolic DPPH• solution [32] for 5 s (immersion time). The chromatogram was dried in the dark at room temperature for 90 s, heated at 60 ◦C for 30 s and documented at white light illumination (reflectance mode), repeated after a day.

*A. fischeri* bioassay: The nutrient medium for the bacterial suspension was prepared as described [33]. The dried, neutralized chromatogram was immersed into the suspension for 3 s. Bioluminescence images (bioautograms) were recorded and processed with the BioLuminizer software, version 1.0.2.6107. Ten images were recorded over 30 min at time intervals of 3 min, each over an exposure time of 50 s.

*B. subtilis* bioassay: The dried, neutralized chromatogram was immersed in the bacterial suspension for 5 s and incubated at 37 ◦C for 2 h [31,34]. For visualization, the plates were immersed into a 0.2% PBS-buffered MTT solution for 1 s. During the incubation for 30 min at 37 ◦C, the MTT was reduced to a purple formazan dye, stopped by drying the plate at 50 ◦C for 5 min. Bioautograms were documented at white light illumination (reflection mode).

AChE inhibition assay: The dried, neutralized plate was immersed in the enzyme solution (AChE 666 units and 100 mg BSA in 100 mL 0.05 M TRIS buffer, pH 7.8) for 5 s. The plate was incubated for 25 min at 37 ◦C according to Akkad and Schwack [35]; Hage and Morlock [36]. For visualization, the chromatogram was immersed in the substrate solution (25 mg α-naphthyl acetate and 50 mg Fast Blue salt B in 90 mL ethanol-water, 1:2) and documented at white light illumination (reflectance mode).

Tyrosinase inhibition assay: The dried, neutralized plate was immersed in the enzyme solution (400 U/mL mushroom tyrosinase in 0.02 M phosphate buffer, pH 6.8) for 5 s [37]. The plate was dried for about 2 min and immersed in the substrate solution (L-DOPA, 18 mM in phosphate buffer, pH 6.8) for 3 s, followed by incubation for 15 min at room temperature. The dried plate was recorded at white light illumination (reflectance mode).

#### **4. Conclusions**

The bioactivity patterns revealed multiple bioactive compounds of mango and pineapple purees and juices, self-produced from self-harvested authentic fruits. The bioactivity assessment can be used to valorize and add value to the plant-based products. It can also be used with regard to distinct health claims, e.g., based on antioxidative compounds. Some of the bioactive compounds were not detected by UV/Vis/FLD, but first with the planar biochemical or biological assays. Effect-directed profiling thus makes product control more powerful. Since it is a non-targeted method, also unknown effective compounds, which are not in the focus of current analysis, can be detected. This is of high importance due to the global food processing chain. Compared to the status quo, product changes caused by contamination, adulteration, processing, treatments, etc. can be more comprehensively and easily detected through multi-imaging exploiting an array of different detection techniques.

**Supplementary Materials:** The following are available online, Table S1: Investigated mobile phases 1–6, Table S2: Chromatograms at UV 254 nm and FLD 366 nm before the respective assay detection.

**Author Contributions:** Conceptualization, methodology, formal analysis, data curation, writing review and editing, all; investigation, N.W. (harvest and production); writing—original draft preparation, G.E.M.; supervision, resources, project administration, G.E.M. and D.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are available from the authors on request.

**Acknowledgments:** Thanks are owed to Anja Rheinberger, Anja Giehl, Claus-Dieter Patz (Department of Beverage Research) and Birgit Krause (Department of Soil Science and Plant Nutrition), all of Hochschule Geisenheim University, for their assistance with the analysis of certain parameters (Table 1) as well as to Stefanie Krüger, Food Science, JLU Giessen, for performing HPTLC experiments.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are not available from the authors.

### **References**

