2.2. Extraction of Plant Material

The moisture content of the milled red clover flowers was determined using a KERN MLB apparatus (KERN & Sohn GmbH, Balingen, Germany). A total of 0.3 ± 0.01 g grams of the material was placed in the apparatus and heated to 105 °C. At the end of the operation, the device provided a calculated moisture content of the material [27]. The moisture of the red clover plant material humidity ranged from 7% to 7.4%.

Maceration extraction was carried out using a modified method of Krähmer et al., 2013 [28]. A total of 0.3 ± 0.001 g of dried and milled flower heads were macerated in 10 mL ethanol (70 or 50% v/v). The samples were centrifuged for 10 min at 3382 g, followed by the decantation of the supernatant. The extracts were hydrolyzed using alkaline hydrolysis and then filtered through PVDF syringe filters (pore size 0.22 μm) for further HPLC analysis. The extraction conditions are displayed in Table 1 and decoding of the samples are provided in Figure 2.

Decoding of the samples provided in Table 1.

Extraction conditions used for the experiment.

* U—Ultrasound-assisted extraction; H—Heat-reflux extraction; M—maceration.

Ultrasound-assisted extraction was performed using an ultrasound bath (frequency 38 kHz) (Cambridge, UK, Grant Instruments™ XUB12 Digital). A total of 0.3 ± 0.001 g of dried and milled flower heads was macerated in 10 mL of solvent. The extraction of isoflavones was performed by employing different extraction conditions—solvent (70 or 50% ethanol and purified water v/v) and extraction time: 10 to 30 min, processing temperature 40 ± 2 °C [17,29]. The samples were centrifuged for 10 min at 3382 g, followed by the decantation of the supernatant. The extracts were hydrolyzed and then filtered through PVDF syringe filters (pore size 0.22 μm) for further HPLC analysis. The extraction conditions are displayed in Table 1 and decoding of the samples are provided in Figure 2.

For easier comprehension, the samples are coded according to their conditions (Figure 2). The first letter of the sample indicates extraction method; the second, the hydrolysis method; and the third, the solvent and ultrasound processing time (if ultrasound was not applied, no additional number was added).

Some of the samples were modified and prepared with vinylpyrrolidone-vinyl acetate copolymer, croscarmellose sodium, sodium carboxymethyl starch or magnesium aluminometasilicate. The extracts were made in the same conditions, which were listed earlier. Sample preparation conditions are listed in Table 2 and decoding of the samples are provided in Figure 3.

Decoding of the samples provided in Table 2.

Extraction conditions using excipients for the experiment.

* U—ultrasound-assisted extraction; H—heat-reflux extraction; M—maceration.

The samples in Table 2 are coded according to their conditions (Figure 3). The first letter of the sample indicates the extraction method; the second, the hydrolysis method; the third, the solvent; the fourth, the excipient; and the fifth, the excipient concentration and the number show ultrasound processing time (if ultrasound was not applied, no additional number was added).

Purified water or 50% of ethanol (v/v) was used as the solvent and the excipient was added to the extraction mixture. The excipients concentration in the extract were 1% (v/w) (0.1 ± 0.001 g was added to the extraction mixture of 10 mL); for vinylpyrrolidone-vinyl acetate copolymer, it was from 1 to 5% (v/w) (0.1 ± 0.001–0.5 ± 0.001 g were added to the extraction mixture of 10 mL). The excipient amount was based on solvent quantity. The samples were centrifuged for 10 min at 3382 g, followed by the decantation of the supernatant. The extracts were filtered through PVDF syringe filters (pore size 0.22 μm) prior to HPLC analysis.

A total of 0.3 ± 0.001 g of dried and milled flower heads were mixed with 10 mL of used solvent (70%, 50% ethanol or purified water v/v) in a 250 mL round bottom flask and it was refluxed in the sand bath at 100 °C for 1 h. Consequently, the mixture was left to cool at 25 ± 2 °C temperature. The samples were centrifuged for 10 min at 3382 g, followed by decantation of the supernatant. The extracts were filtered through PVDF syringe filters (pore size 0.22 μm) prior to HPLC analysis. The extraction conditions are displayed in Table 1.