A static maceration method was used to obtain the plant extracts. Ten grams of dried and crushed plant material was subjected to extraction with 50 mL of 96% ethyl alcohol at room temperature in the dark for a period of 7 days to prevent the degradation of light-sensitive compounds.

The thermogravimetric analysis of plant samples was performed using a Sartorius thermobalance, a device that allows real-time monitoring of the dehydration process by continuously weighing the sample during heating. This method provides accurate information on the mass loss associated with water removal, which is essential for the preliminary characterization of the plant material used in phytochemical analyses. The measurement accuracy of the equipment is 0.1% for samples with a mass greater than 1 g and 0.02% for samples with a mass greater than 5 g [10]. The determination was performed by weighing 1 g of crushed plant material from each sample. The thermobalance was set to a maximum temperature of 110 °C, which was sufficient for complete water evaporation without significant thermal degradation of sensitive organic compounds. The dehydration process was controlled, and continuous weighing allowed automatic recording of mass variation over time. The evolution of weight loss, correlated with the gradual increase in temperature, provided information on the thermal stability and moisture content of the plant material.

A starting line was drawn on each chromatographic plate with a pencil and a ruler 1 cm from the base of the plate. The samples were applied to this line using a 1 µL microsyringe or a fine capillary tube in the form of small circular spots to avoid overlap during migration. The samples analyzed were the alcoholic extract of Trifolium pratense (red clover) and the alcoholic extract of Capsella bursa-pastoris (shepherd’s purse). After applying the samples, the plates were placed in a vertical developing chamber, previously saturated with solvent vapors, to ensure uniform migration of the mobile phase. To optimize separation, several elution systems were tested, as shown in Table 1, containing solvents of different polarities, to obtain the clearest possible chromatographic profile of the compounds present.

After completion of the migration process (when the solvent front reached approximately 1 cm from the upper edge of the plate), the plates were removed from the developing chamber using tweezers and dried for several minutes in an oven at 100 °C. After complete drying, the chromatograms were analyzed by UV irradiation using a Fisher Bioblock Scientific spectrophotometer at wavelengths of 256 nm for viewing aromatic, phenolic, and flavonoid compounds, and 365 nm for identifying flavonoids and other naturally fluorescent compounds. The results obtained were photographed and interpreted qualitatively by comparing the intensity and position of the spots between the different elution systems. These data provide an overview of the phytochemical composition of the tested extracts, which is useful in the preliminary stage of evaluating their anticancer potential.

The total antioxidant capacity was determined using the CUPRAC (Cupric Reducing Antioxidant Capacity) method, which is based on the ability of antioxidant compounds in plant extracts to reduce cupric ions (Cu²⁺) to cuprous ions (Cu⁺) in the presence of the ligand neocuproine, forming a colored complex measurable spectrophotometrically at 450 nm. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), an antioxidant soluble in both water and fats, was used as a reference standard. To determine antioxidant activity, 1 mL of 0.01 M copper chloride solution, 1 mL of alcoholic neocuproine solution (7.5 × 10⁻³ M), and 1 mL of acetate buffer solution (pH 7.0) were mixed. To this mixture, 1.1 mL of the sample, standard (Trolox), or blank solution was added. The solutions were homogenized and kept at room temperature for 30 min, after which the absorbance was determined at 450 nm using a UV-Vis spectrophotometer. The total antioxidant capacity was expressed in μmol Trolox equivalents per gram of sample (μmol TE/g).

The total polyphenolic compound content was determined using the Folin–Ciocalteu colorimetric method based on the reducing properties of polyphenols. These polyphenols reduced the Folin–Ciocalteu reagent, forming a blue-colored complex with maximum absorption at 750 nm. The intensity of the color was proportional to the total concentration of polyphenols in the sample. The alcoholic extracts obtained from the plant material were diluted accordingly. From each sample, 0.5 mL of extract was taken, to which 2.5 mL of Folin–Ciocalteu reagent (1:10, v/v) and 2 mL of 7.5% sodium carbonate solution (m/v) were added. The mixture was homogenized and incubated at room temperature for 30 min, after which the absorption was measured spectrophotometrically at 750 nm. Gallic acid was used as a standard in concentrations of 0–0.7 μM/mL to construct the calibration curve. The results were expressed as mg gallic acid equivalents (GAE) per 100 g sample.

Densitometric analysis was performed using the CAMAG TLC Scanner 3 densitometer, controlled by WinCATS software. Densitometry enables the quantification of substances separated on a chromatographic plate by measuring the intensity of the optical signal (absorption or fluorescence) at each position on the plate. This method provides an accurate and reproducible assessment of the relative content of bioactive compounds, complementing the visual observations obtained by thin-layer chromatography. To identify and characterize the separation zones, the Rf (retention factor) values were determined for each detected compound. The Rf value expresses the ratio between the distance traveled by a substance on the plate (Xs, the distance between the start line and the center of the spot) and the distance traveled by the solvent front (XD):

The Rf values obtained provide specific information about the migration of the compounds and allow for comparison between the analyzed extracts (Trifolium pratense and Capsella bursa-pastoris) to identify similarities or differences in phytochemical composition.

From the analysis of the data presented in Table 2, it was found that the dehydration process took place in a shorter time for Capsella bursa-pastoris (approximately 9 min) compared to Trifolium pratense, for which complete drying took approximately 14 min. Both species have a high water content of over 90%, which indicates an advanced degree of hydration of plant tissues (Figure 1), as evidenced by the thermogravimetric curves (Figure 2). The differences in dehydration time can be explained by the morphological and structural characteristics of the plant material, such as tissue thickness and density, hygroscopic substance content, or the proportion of different cell fractions.

The total antioxidant capacity (TAC) and total polyphenol content (TP) were determined for extracts of Trifolium pratense and Capsella bursa-pastoris using hydroalcoholic solutions of different concentrations and varying extraction times. The CUPRAC method was applied to determine total antioxidant activity, and total polyphenol content was determined using the Folin–Ciocalteu method. The obtained results are presented in Table 3.

The analysis of the values obtained highlights notable differences between the two plant species regarding both solvent type and extraction duration. It shows that, at an alcohol concentration of 20% and an extraction time of 24 h, Trifolium pratense exhibits the highest values for total antioxidant capacity (3110 µmol Trolox/g DM) and polyphenol content (10,710 mg/L gallic acid/100 g DM), surpassing the corresponding values for Capsella bursa-pastoris (3010 µmol Trolox/g DM and 8470 mg/L gallic acid/100 g DM). In the case of extended extractions (144–1440 h) and solvents with high alcohol concentrations (96°), a gradual decrease in antioxidant activity and total polyphenol content is observed, a phenomenon attributed to thermal degradation and oxidation of phenolic compounds sensitive to prolonged extraction conditions [11]. The differences between the two plants can be explained by their unique phytochemical profiles. Trifolium pratense contains isoflavones (genistein, daidzein, formononetin, biochanin A), anthocyanins, and phenolic acids, all known for their potent antioxidant activity [12]. Conversely, Capsella bursa-pastoris contains peptides, glycosides, and unsaturated fatty acids, which serve different biological functions and contribute less to free radical neutralization [13]. The findings confirm that moderate solvent polarity (20% ethanol) and a short extraction duration (24 h) are optimal for producing an extract rich in phenolic compounds with high antioxidant activity.

Following the described TLC method, distinct chromatographic profiles were obtained for the alcoholic extracts of Trifolium pratense and Capsella bursa-pastoris(Figure 3). The qualitative analysis aimed to achieve the preliminary identification of phenolic, flavonoid, and isoflavonoid compounds, based on their chromatographic behavior and fluorescence under UV light. Among the tested elution systems (Table 1), the best results were obtained with the following combinations: for Capsella bursa-pastoris, ethyl acetate:methanol:water (8:1:1, v/v/v), and for Trifolium pratense, ethyl acetate:hexane (9:1, v/v). In the case of Capsella bursa-pastoris, UV analysis at 256 nm revealed three visible spots, and after spraying the plate with ammonium molybdate and sulfuric acid, four to five distinct spots appeared, confirming the presence of phenolic and glycosidic compounds. The color reaction (blue–brown) indicated that most compounds were reduced oxidants with moderate antioxidant potential. For the Trifolium pratense extract, the ethyl acetate:hexane system allowed clear separation of apolar and semipolar compounds. Under UV radiation at 256 nm, three spots were observed, and at 365 nm, there were five fluorescent areas, corresponding to isoflavones and other polyphenolic compounds. The fluorescence intensity and contrast corroborate a higher concentration of conjugated aromatic compounds, supporting the high antioxidant activity previously determined in Section 3.2.

Comparative analysis of chromatographic profiles reveals greater phytochemical complexity in Trifolium pratense compared to Capsella bursa-pastoris. The higher number of spots and increased fluorescence intensity suggest a high content of isoflavones, flavonoids, and phenolic derivatives, known for their role in reducing oxidative stress and inhibiting skin tumor processes [12]. Capsella bursa-pastoris extracts showed a simpler profile with a predominance of polar compounds (glycosides, peptides), indicating a moderate but complementary antioxidant potential [4].