Scanning of chicoric acid in different parts of Cichorium intybus by high-performance thin-layer chromatography with quantitation by image analysis

2.1 Reagents and chemicals

All reagents used in the study were of analytical grade, as specified by the suppliers. Ethanol (96%), glacial acetic acid (≥ 99.7%), hydrochloric acid (37%), 1,1-diphenyl-2-picrylhydrazyl radical, (DPPH·,  ≥ 98.0%), polyethylene glycol 400 (macrogol, tested according to Ph. Eur.), 2-aminoethyl diphenylborinate (97.0%), and chicoric acid (≥ 95.0%) were purchased from Merck (Darmstadt, Germany). Cellulase was purchased from Pangbo Enzyme (Guangxi, China). The solvents used for chromatographic analysis were purchased from Millipore (Bedford, MA, USA) with the following purities: methanol (≥ 99.8%), ethyl acetate (≥ 99.5%), and formic acid (≥ 98.0%).

2.2 Plant material

The plant material was collected from the village of Strâmbu, Cluj County, during July and August of 2022 and 2023. It was certified at the Alexandru Borza Botanical Garden in Cluj-Napoca under identification number 61134. The fresh plants were air-dried in the dark at room temperature (22–24 °C) in a well-ventilated space. Afterward, the material was finely grounded for 5 min at 29 Hz to achieve a uniformly granulated powder using Retsch Mixer Mill MM 400.

2.3 Preparation of the extracts

The preparation of the extracts followed the same procedure for each extraction method. Separate extracts were prepared from roots, leaves, flowers, and aerial parts using each extraction method (Table 1).

Table 1 Plant materials, extraction methods, and codification

The ratio of plant material to extraction solvent was consistent across all methods, with 1 g of plant material to 10 mL of solvent. The solvent used for maceration was 70% ethanol and the samples were left in the dark for 10 days and shaken daily. The UAE were performed in Emmi-20 HC (EMAG, Germany) ultrasonic bath at 30 °C temperature for 30 min using 70% ethanol as solvent. For enzyme-assisted UAE, the amount of cellulase added to each sample was calculated to represent 3% relative to plant material weight [13]. The extractions were performed in the ultrasonic bath at 55 °C for 40 min, using 55% ethanol as solvent.

After each extraction, the supernatants were separated from the solid residue by centrifugation using a Centurion Scientific centrifuge C2006 (Centurion Scientific Limited, Bosham, UK) and the extracts were kept at 4 °C for further analysis.

2.4 Quantification of chicoric acid by HPTLC–IA

For the quantitative analysis and determination using high-performance thin-layer chromatography, normal-phase chromatographic plates (HPTLC Silica Gel 60 F254, 20 × 10 cm, 1,056,420,001) were used. To quantify chicoric acid in the extracts, a calibration curve was first established. The calibration curve was established on 0.1–3 µg/band domain by applying different volumes of standard ethanolic solution of chicoric acid (0.1 mg/mL) on the chromatographic plates as 8 mm bands with application speed of 70 nL/s. In the case of extracts, aliquots of 5 μL of flower and leaf extracts and 10 μL of root and aerial parts extracts were applied on plates as 8 mm bands with application speed of 70 nL/s. All bands were applied at 1.5 cm from the bottom on the plate using CAMAG Linomat 5 applicator (Muttenz, Switzerland). The optimum mobile phase is a mixture of ethyl acetate–formic acid–acetic acid–water (8:0.2:2:1.8, v/v). The plates were developed over a distance of 7.5 cm in a twin-through chromatographic chamber presaturated for 30 min with mobile phase. After developing with the mobile phase, to enhance fluorescence, the plates were immersed first in NP natural product (NP) reagent and then in polyethylene glycol solution (PEG). The NP reagent was prepared by weighing 1 g of 2-aminodiphenylborinate and dissolving it in 200 mL of ethyl acetate with agitation until fully dissolved. PEG was prepared by dissolving 10 g of polyethylene glycol in 200 mL of dichloromethane [14]. Before immersion, the plates were heated at 100 °C for 3 min and then the hot plates were immersed in NP solution. After drying, the plates were derivatized with PEG. Immersion was performed using an automatic immersion device (CAMAG) with a vertical speed of 35 mm/s, for 3 s. In all instances, the final treated plates were documented at 366 nm with a TLC visualizer device (Digistore 2, CAMAG) and the images were stored as JPEG files. winCats software controlled all CAMAG instruments. The ImageJ sotware was used to create the densitograms and integrate the peaks [14].

The calibration curve was constructed by using ImageJ software to process the image of the original plate and after splitting in red, blue and green channels to obtain the peak areas after subtracting the background. The LODs and LOQs were calculated using the formulas:

$$} = \sigma /s} = 0 \, \sigma /s$$

where σ represents the standard deviation of the residuals and s denotes the value of the slope of the curve.

2.5 Screening for antioxidant activity: DPPH assay

To assess the antioxidant activity of chicoric acid separated by thin-layer chromatography from the extracts, the DPPH· radical was used. DPPH· is a stable organic radical with a violet color that turns yellow in the presence of antioxidant compounds. The DPPH· solution was prepared at a concentration of 0.03% by dissolving 0.0750 g of DPPH· in 250 mL of ethanol.

The chromatographic plates were immersed in the preprepared DPPH· solution using the automatic immersion device for 1 s. Afterward, the plates were dried for 30 min in the dark, resulting in plates with yellowish bands on violet–blue background, indicating the presence of antioxidant compounds [13, 14].

2.6 Statistics

Results for the calculated concentrations of chicoric acid in original, red, green, and blue channels were analyzed with a one-way analysis of variance (ANOVA) at a 95% confidence level with the statistical software for MS Excel. The difference between the methods is considered significant if the calculated p-value is lower than 0.05 and the F-value is higher than F critical.

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