## 2 Materials and Methods
### Quantitative Determination of Proanthocyanins
#### 2.2.6 Testing of the Hydroalcoholic Extracts Included in the CILTAG Product  
The following parameters on the extracts were tested: the organoleptical characteristics, the composition (by HPLC), and the quantitative determination of ascorbic acid (by HPLC). The Compositional Analysis  
To determine the compounds in the solubilized phyto-complexes from the hydroalcoholic solution, HPLC was performed on each solution. In the case of the _Cynosbati fructus_ extractive solution, from the HPLC method we could not identify the chromatographic peaks. For this reason, the constituents were determined by a UV-vis spectrophotometric method using a Cary 50 UV-Vis spectrophotometer (Varian Inc., California, USA). The determinations were made in quartz cuvettes (with a 10 mm transmitted path length), the hydroalcoholic _Cynosbati fructus_ extractive solution was diluted to 1:10 (_v/v_) with 95% (_v/v_) ethanol (Fluka(r) Analytical, Switzerland), and the spectra were registered in the domain of \(\lambda\) = 220-500 nm. The extractive hydroalcoholic solutions of _Medicaginis herba_, _Trifoli pratense flores_, _Ginkgo blobane folium_, _Myrtilli fructus_, and _Cynosbati fructus_ were obtained by dissolving 4 mg of extract in ethanol and diluting the solution to 25 mL. The solutions were filtered through a 0.45 \(\upmu\)m Millipore filter. The standard solution was prepared by dissolving 3 mg of each reference substance in ethanol and diluting the solution to 25 mL. As a stationary phase, a C\({}_{18}\) (Discovery C\({}_{18}\) 250 \(\times\) 4.6 mm i.d., 5 \(\upmu\)m particles) column was used. The mobile phase was formed by Solution A (water and phosphoric acid (3 mL of 85% phosphoric acid was diluted with 1000 mL of water)) and Solution B (acetonitrile). The linear gradient from 0 to 100% is presented in Table S1 of the Supplementary Materials File. A mobile phase flow rate of 1 mL/min was used, and the detection was registered at 335 nm. The spectra were obtained at wavelengths of 200-400 nm, the injection volume  
Figure 1: The extraction process for the CILTAG product. was 20 \(\mu\)L, and the temperature was the ambient one. The peaks were identified by a comparison of the retention times between samples and references. The Quantitative Determination of Ascorbic Acid  
The samples were analyzed immediately after preparation and at 60 days. The HPLC data cited in the literature suggest that the determination be made by reversed-phase HPLC [51]. A VARIAN ProStar HPLC system with a quaternary pump and a photodiode array detector (PDA) produced by Varian, Inc., USA was used. The parameters for the chromatographic system were an Inertsil 5 OD2-2 column, 250 x 4.6 min (i.d.) with 5 \(\mu\)m particles, mobile phase: A = 0.1% H\({}_{3}\)PO\({}_{4}\) in water with CH\({}_{3}\)OH 1% and B = CH\({}_{3}\)OH, 95A:5B, the flow rate was 0.6 mL/min with isocratic elution at 25 \({}^{\circ}\)C, the separation period was 20 min, and the injection volume was 20 \(\mu\)L. The reagents used were ascorbic acid, a reference substance (BASF, Ludwigshafen, Germany), CH\({}_{3}\)OH for HPLC (Sigma-Aldrich, SA Darmstadt, Germany), H\({}_{3}\)PO\({}_{4}\) 89% p.a. (Sigma-Aldrich, SA Darmstadt, Germany), and ultrapure H\({}_{2}\)O. The used ascorbic acid standard solutions were _Solvent A_: 0.1% H\({}_{3}\)PO\({}_{4}\) in water with CH\({}_{3}\)OH 1%; _Solution A_: 2 \(\times\) 10\({}^{-3}\) g/mL (0.1 g of ascorbic acid dissolved in solvent A to 50 mL); _Solution E_1: 2 \(\times\) 10\({}^{-5}\) g/mL (0.5 mL of solution A diluted with solvent A to 50 mL); _Solution E_2: 3 \(\times\) 10\({}^{-5}\) g/mL (1.00 mL of solution A diluted with solvent A to 50 mL); _Solution E_4: 5 \(\times\) 10\({}^{-5}\) g/mL(1.25 mL of solution A diluted with solvent A to 50 mL); _Solution E_5: 6 \(\times\) 10\({}^{-5}\) g/mL (1.50 mL of solution A diluted with solvent A to 50 mL); and _Solution E_6: 8 \(\times\) 10\({}^{-5}\) g/mL (2.00 mL of solution A diluted with solvent A to 50 mL). The sample solution was obtained by diluting 0.10 mL of CILTAG with 25 mL of solvent A.