The Influence of Selected Factors on the Aqueous Cryptotanshinone Solubility

The application of cryptotanshinone (CT), a diterpenoid obtained from the root of Salviae miltiorrhiza, is significantly hindered due to its poor aqueous solubility. The aim of the present research was to develop an optimal solvent for analytical and preparative procedures of prospective dermal hydrogel formulations with CT. The influence of pH, temperature, and cosolvent presence on the solubility of CT was examined. Various components were applied to increase CT solubility, i.e., ethanol, 2-amino-2-methyl-1,3-propanediol, 2-amino-2-(hydroxymethyl)-1,3-propanediol, 2,2′,2″-nitrilotriethanol, and triisopropanoloamine. The concentration of CT was analyzed by spectral and chromatographic methods, including UV–vis and HPLC methods. The increased solubility of CT was demonstrated in alkaline solvents with ethanol as a cosolvent. CT solutions doped with alcoholamines are more stable compared to CT solutions doped with NaOH.


Introduction
Solubility is an important factor determining the pharmaceutical availability and bioavailability of a drug. It influences the optimal form of the preparation administered to the patient and has a significant effect on the blood concentration of the drug and the expected pharmacological response [1]. Drugs of weak aqueous solubility, presumed for circulatory absorption, often require higher doses than water-soluble medicines. More than 40% of newly developed pharmaceuticals have very low solubility; therefore, the problem is significant in the context of formulation and pharmacokinetics [2]. Many biologically active substances of natural origin are characterized by low water solubility. One of them is cryptotanshinone (CT) [3].
In the last two decades, the dissolution and bioavailability of CT has be including very modern approaches, such as solid dispersion [14,27,29,30], etho niosomal [32], cerasomal [33], and solid lipid nanoparticles [30], and cyclode plexation [22,34]. Studies of the influence of pH on CT solubility revealed struc ifications of the compound above pH 11.0 and even destruction below pH 1.0, discovered during research on fluorescence properties [35], whereas the highe of CT structure was recorded between pH 8.0 and pH 12.0 in absorbance test UV-vis method [34]. The tetrahydrofuran ring of CT collapsed in strongly alka tions [28,36] with an enol form of tanshinone V has been established ( Figure 1 The alkaline environments may be induced both by strong inorganic hydro NaOH, or by alcoholamines, which are bifunctional components, combining ties of alcohols and amines [37]. Aminomethyl propanediol (AMPD), tro (TRIS), triethanolamine (TEOA), and triisopropanolamine (TIPA) are compone interest due to potential application in pharmaceutical and cosmetic industr acid binders [37][38][39] (Figure 2). TEOA is applied as antiacne preparation in t salicylate. AMPD may react with the methyl esters of the organic acids form [40]. TRIS and TIPA may be used in the synthesis of surface-active agents as e agents and emulsion stabilizers in pharmaceutical applications [41,42]. TIPA idly with acids forming amine salts [41]. AMPD and TIPA enable pH neutra cosmetic preparations, e.g., lotions, aerosols, and tonics [40,41]. TRIS is admin the treatment of salicylates, barbiturates, and methanol intoxications, and it m for plasma alkalization [42]. NaOH and alcoholamines are used in pharmace could serve as potential ingredients of the preparation for external applicatio tion of selected alkaline components may enhance the aqueous solubility of C maceutical preparation.
In the last two decades, the dissolution and bioavailability of CT has been studied, including very modern approaches, such as solid dispersion [14,27,29,30], ethosomal [31], niosomal [32], cerasomal [33], and solid lipid nanoparticles [30], and cyclodextrin complexation [22,34]. Studies of the influence of pH on CT solubility revealed structural modifications of the compound above pH 11.0 and even destruction below pH 1.0, which was discovered during research on fluorescence properties [35], whereas the highest stability of CT structure was recorded between pH 8.0 and pH 12.0 in absorbance tests using the UV-vis method [34]. The tetrahydrofuran ring of CT collapsed in strongly alkaline conditions [28,36] with an enol form of tanshinone V has been established ( Figure 1b).
The alkaline environments may be induced both by strong inorganic hydroxides, e.g., NaOH, or by alcoholamines, which are bifunctional components, combining the properties of alcohols and amines [37]. Aminomethyl propanediol (AMPD), tromethamine (TRIS), triethanolamine (TEOA), and triisopropanolamine (TIPA) are components of high interest due to potential application in pharmaceutical and cosmetic industries as fatty acid binders [37][38][39] (Figure 2). TEOA is applied as antiacne preparation in the form of salicylate. AMPD may react with the methyl esters of the organic acids forming amides [40]. TRIS and TIPA may be used in the synthesis of surface-active agents as emulsifying agents and emulsion stabilizers in pharmaceutical applications [41,42]. TIPA reacts rapidly with acids forming amine salts [41]. AMPD and TIPA enable pH neutralization in cosmetic preparations, e.g., lotions, aerosols, and tonics [40,41]. TRIS is administered for the treatment of salicylates, barbiturates, and methanol intoxications, and it may be used for plasma alkalization [42]. NaOH and alcoholamines are used in pharmaceuticals and could serve as potential ingredients of the preparation for external application. Application of selected alkaline components may enhance the aqueous solubility of CT in pharmaceutical preparation.
The aim of this work was to evaluate the aqueous solubility of CT in an alkaline environment and the selected factors influencing the process of dissolution.

Preliminary Evaluation of the CT Solubility Performance in Various Solvents
The visual appearance of the CT samples in a variety of solvents was evaluate solvents presented in Table S1 (Supplementary Materials) were used. A 1 mg amo CT was weighed and placed in 2 mL of solvent in a test tube, as described in T (Supplementary Materials). Observations were made after 24 h and after 8 day tested samples were protected from light at 25 °C.
For microscopic evaluation, samples of 0.2 mg/mL CT concentration in 96% e (S1), aqueous solution of 8% ethanol (S2), and aqueous solution of 8% ethanol wit mol/L solution of NaOH were prepared (S3) and assessed with an optical micr (Delta Optical Evolution 100) with photo processing software (DLT-Cam PRO 18MP Optical, Poland).
The influence of temperature and sonification was further evaluated on the s

Preliminary Evaluation of the CT Solubility Performance in Various Solvents
The visual appearance of the CT samples in a variety of solvents was evaluated. The solvents presented in Table S1 (Supplementary Materials) were used. A 1 mg amount of CT was weighed and placed in 2 mL of solvent in a test tube, as described in Table S1 (Supplementary Materials). Observations were made after 24 h and after 8 days. The tested samples were protected from light at 25 • C.
For microscopic evaluation, samples of 0.2 mg/mL CT concentration in 96% ethanol (S1), aqueous solution of 8% ethanol (S2), and aqueous solution of 8% ethanol with a 0.1 mol/L solution of NaOH were prepared (S3) and assessed with an optical microscope (Delta Optical Evolution 100) with photo processing software (DLT-Cam PRO 18MP, Delta Optical, Poland).
The influence of temperature and sonification was further evaluated on the selected solvent: 0.1 mol/L NaOH. The system of 5 mg of CT and 100 mL of 0.1 mol/L NaOH was sonicated at 40 kHz (Sonic-5, Polsonic, Warsaw, Poland) for 1, 5, or 10 min at temperatures of 30 ± 1.5, 40 ± 1.5, 50 ± 1.5, and 60 ± 1.5 • C. The resulting change in color was recorded as present or absent.

Absorbance Studies of CT in Varied Alkaline Solvents
A standard curve for CT was prepared in methanol at a wavelength of 271 nm [19], according to available literature [43]. The achieved standard curve prepared by the methanol solution equation was y = 0.052x, and the linear determination index R 2 was 0.9998 (n = 3). The UV-vis spectra of CT in Samples 2 and 8-11 were evaluated using a UV-vis spectrophotometer (Halo DB-20, Dynamica, Poland) in the range of 245-300 nm. The determination of the content of alkaline groups that could potentially bind acidic groups of cryptotanshinone was based on the modified pharmacopoeial method for the acid number determination [44]. In the modified method, the absorbance of Samples 2 and 8-11 after sonication for 10 min in 60 ± 1.5 • C was measured at 271 nm. Solvent 2 or 8-11 was added in 1 mL portions until a volume of 8 mL was obtained, and the absorbance was measured after every 1 mL.

Potentiometric and Conductometric Studies
Samples P1-P2 presented in Table S2 (Supplementary Materials) were titrated with 0.1 mL portions of 0.001 mol/L NaOH until a volume of 10.0 mL was obtained in a TitraLab AT 1000 Series titrator potentiometer (Hach Lange, Düsseldorf, Germany), with a CC-505 conductivity sensor (Elmetron, Zabrze, Poland) and a PHC 805 pH-electrode (Hach Lange, Düsseldorf, Germany), to provide an insight into the initial phase of CT neutralization. In P2, 1.2 mL of 96% ethanol as a cosolvent was added to 5.0 mg of CT and left for 20 h, then 35 mL of distilled water was provided. The solutions without CT, P1r and P2r, were used as blank samples.

HPLC Analysis
The HPLC method was used to overcome the limitations of the UV-vis spectrophotometric method due to the standard curve. The analytical methods of tanshen and tanshinones were previously been performed [5,8,43]. The method was modified to analyze the concentration of CT in the studied solutions. The method was set as follows: a flow rate of 0.4 mL/min, a column temperature of 40 • C, and a wavelength of 280 nm were applied. The mobile phase consisted of distilled water, 0.1% formic acid (phase A) and acetonitrile, and 0.1% formic acid (phase B). The gradiented A/B was set, respectively, as 98:2-1:99 and 1:99-98:2 v/v% for 28 min. Powdered CT of purity 99.26%, dissolved in methanol, was used as a standard. To adapt to the concentration range, two standard curves were prepared: one for alcoholamine solutions (S1) and the other for the NaOH solutions (S2).
The HPLC system (Ultimate 3000 model, Thermo Fisher Scientific, Schwerte, Germany) consisted of a pump (LPG-3400SD), an autosampler (WPS-3000TSL), a column thermostat (TCC-3000SD), a UV detector (UV, DAD-3000), and a column (Kinetex 2.6 µm C18 100Å, Phenomenex, Torrance, CA, USA). The solutions were filtered through 0.22 µm pores filters. The mixtures were prepared using an ultrasonic washer for 10 min at 40 ± 1.5, 50 ± 1.5, and 60 ± 1.5 • C and ethanol as a cosolvent at an amount of 8% w/w. Various concentrations of NaOH and different temperatures of 1% alcoholamine exposure to ultrasound waves were tested (Table S3, Supplementary Materials). The chromatographic measurements of CT in ethanol (E) and water (W) were executed as standards. The alkaline solvents were added to obtain a saturated solution of CT (approximately 1.0 mg of CT in 5.0 g of solvents). HPLC measurements were performed after preparation and after 1, 2, 3, 6, and 8 weeks. The pH measurements of the samples designed for HPLC assays were carried out on the first day of sample preparation at a temperature of 25.0 ± 0.5 • C.

Visual Appearance of CT-Solvent Systems
The color intensity in the test tubes after 24 h and after 8 days in the respective solvents doped with CT is presented in Figure 3 (left panel). The most intense color was observed in the case of methanol, ethanol, and isopropanol, which was derived from dissolved CT. The aqueous samples, acidic samples, and alcoholamine samples remained colorless with The 96% ethanolic solutions were transparent and colorful directly after the implementation of CT and after 5 min (S1a, S1b), with separated particles of undissolved CT in the observation field (S1c), whereas the CT aqueous dispersion doped with 0.5 mL of ethanol (8% ethanol) provided a turbid system, with observable, floating, insoluble CT particles (S2a, S2b) of longitude in the range of 25 µm (S2c). The solution was colorless. The system with a low ethanol content (8%), but with a 0.1 mol/L solution of NaOH, showed turbidity; however, the entire solution was colored, which indicated the partial dissolution of CT. The solid particles also floated, and their dimensions were in the range of 5 µm (Figure 3, right panel).
A further evaluation of CT in the presence of 0.1 mol/L NaOH revealed color in the selected samples, whereas the others were colorless, as presented in Table 1. For the summarized factors overcoming a time of 5 min and a temperature of 40 • C, the color appeared what was accepted as CT solubilization in the respective fluid.
ceutics 2021, 13, x exception was the NaOH solution, where, in addition to floating particles of C color was observed.
The 96% ethanolic solutions were transparent and colorful directly after t mentation of CT and after 5 min (S1a, S1b), with separated particles of undissolv the observation field (S1c), whereas the CT aqueous dispersion doped with 0.5 m anol (8% ethanol) provided a turbid system, with observable, floating, insolubl ticles (S2a, S2b) of longitude in the range of 25 µm (S2c). The solution was colo system with a low ethanol content (8%), but with a 0.1 mol/L solution of NaOH turbidity; however, the entire solution was colored, which indicated the partia tion of CT. The solid particles also floated, and their dimensions were in the rang ( Figure 3, right panel).  (6), isopropanol (7), AMPD (8), TRIS (9), TEOA (10), and TIPA (11). Middle panel: m illustration of CT in 96% ethanol (S1), 8% ethanol (S2), and 8% ethanol with a 0.1 mol/L NaOH (S3). Illustration of the influence of ethanol on the solubility of CT in the 0.1 mol/ of NaOH immediately (a) and after 5 min (b), and a microscope image of the powdered C in the air. Right panel: microscope images of a drop taken from the surface of the sam test tubes S1b, S2b, and S3b, respectively. The bar represents the same longitude (200 µm tails are in the text.
A further evaluation of CT in the presence of 0.1 mol/L NaOH revealed co selected samples, whereas the others were colorless, as presented in Table 1. For marized factors overcoming a time of 5 min and a temperature of 40 °C, the color what was accepted as CT solubilization in the respective fluid.

UV-Vis Spectrophotometric Evaluation of CT Solubility
In order to evaluate the neutralization of acidic groups of CT via alkaline components, the CT solution was combined with increasing volumes of NaOH solution or alcoholamine solutions, as presented in Figure 4. The equilibrium was observed at 6 mL of 0.001 mol/L NaOH added to 5 mg of CT, whereas the equilibrium point for alcoholamines was in the range of 4.5-6 mL.

UV-Vis Spectrophotometric Evaluation of CT Solubility
In order to evaluate the neutralization of acidic groups of CT via alkal nents, the CT solution was combined with increasing volumes of NaOH solu holamine solutions, as presented in Figure 4. The equilibrium was observed 0.001 mol/L NaOH added to 5 mg of CT, whereas the equilibrium point for alc was in the range of 4.5-6 mL. The resulting molar proportion of NaOH/CT was ca. 27, where amounts of CT solved in various alcoholamine solutions provided varied prop ble 2). The equilibrium state in the case of the AMPD solution as a solvent for C a molar ratio of AMPD/CT 889, and for the TRIS solution, the molar ratio of TR equilibrium state was 1514. Similar ratios were observed, as when TEOA was TEOA/CT molar ratio reached a value of 1089. The lowest molar ratio was reco TIPA solution, where the value was 737 in the equilibrium state. The mean p the overtitrated systems at 8 mL are presented in Table 2   The resulting molar proportion of NaOH/CT was ca. 27, whereas the same amounts of CT solved in various alcoholamine solutions provided varied proportions ( Table 2). The equilibrium state in the case of the AMPD solution as a solvent for CT provided a molar ratio of AMPD/CT 889, and for the TRIS solution, the molar ratio of TRIS/CT in the equilibrium state was 1514. Similar ratios were observed, as when TEOA was applied, the TEOA/CT molar ratio reached a value of 1089. The lowest molar ratio was recorded in the TIPA solution, where the value was 737 in the equilibrium state. The mean pH values of the overtitrated systems at 8 mL are presented in Table 2 and were 10.07, 10.08, 9.16, 9.46, and 9.18.

Titration Studies
The addition of the 0.001 mol/L solution of NaOH resulted in a respective increase in pH and conductivity (Figure 5a,b). The range of the pH of CT in water titrated by 0.001 mol/L NaOH was 6.37-10.09, and a plateau phase was established. In the case of conductivity, the range was 0.00045-0.00516 S/m. The range of the pH of CT in 3.3% ethanolic solution titration with 0.001 mol/L NaOH was 6.21-10.18, and a plateau phase was established, as in the previous case (Figure 5c). The range of conductivity was 0.00069-0.00663 S/m (Figure 5d). Theoretical potentiometric titration plot was added to Figure 5a,c to reflect the influence of CO 2 from the atmosphere in a concentration of ca. 0.004 mmol/L, which could interfere with the results.

HPLC Studies
Equations of standard plots for the evaluation of CT concentration in the HPLC assays are presented in Table 3.

HPLC Studies
Equations of standard plots for the evaluation of CT concentration in the HPLC assays are presented in Table 3. The quantification of CT in the assessed solutions was performed by HPLC, with respective CT retention times in different solvents ( Figure 6). The highest concentration of CT was observed in NaOH solutions of 1.0, 0.1, and 0.01 mol/L, respectively (Figure 7a), whereas the concentration of CT in 0.001 mol/L NaOH was close to the concentration of CT in 1% AMPD solution (Figure 7b). The CT concentrations in other alcoholamines, TRIS, TEOA, and TIPA, were ca. ten-fold lower than the CT concentrations in NaOH of 1.0, 0.1, and 0.01 mol/L concentrations. An increase in the values of CT concentrations was observed with an increase in the temperature of the solution prepared with alcoholamines (Figure 7b-d). Moreover, a decrease in the concentration of CT in NaOH solutions was noticed after 21 days, whereas in the case of CT in alcoholamine solutions, a decrease was observed after 42 days (Figure 7). The highest concentration of CT was observed in NaOH solutions of 1.0, 0.1, and 0.01 mol/L, respectively (Figure 7a), whereas the concentration of CT in 0.001 mol/L NaOH was close to the concentration of CT in 1% AMPD solution (Figure 7b). The CT concentrations in other alcoholamines, TRIS, TEOA, and TIPA, were ca. ten-fold lower than the CT concentrations in NaOH of 1.0, 0.1, and 0.01 mol/L concentrations. An increase in the values of CT concentrations was observed with an increase in the temperature of the solution prepared with alcoholamines (Figure 7b-d). Moreover, a decrease in the concentration of CT in NaOH solutions was noticed after 21 days, whereas in the case of CT in alcoholamine solutions, a decrease was observed after 42 days (Figure 7). was close to the concentration of CT in 1% AMPD solution (Figure 7b). The CT concentrations in other alcoholamines, TRIS, TEOA, and TIPA, were ca. ten-fold lower than the CT concentrations in NaOH of 1.0, 0.1, and 0.01 mol/L concentrations. An increase in the values of CT concentrations was observed with an increase in the temperature of the solution prepared with alcoholamines (Figure 7b-d). Moreover, a decrease in the concentration of CT in NaOH solutions was noticed after 21 days, whereas in the case of CT in alcoholamine solutions, a decrease was observed after 42 days (Figure 7).

Discussion
The stability of the CT molecule depends on the pH value. Below pH 2, CT tends to be degraded, whereas over pH 11, structural changes are observed [36]. The pH range of 2-8 mildly influences the solubility, whereas pH 10 to 12 strongly favors the aqueous dissolution of CT [4]. In our study, an alkaline environment between pH values of 10.0 and 12.70 was chosen to establish optimal conditions for CT solubility and stability in the solution. Concentrations of 0.001, 0.01, 0.1, and 1.0 mol/L sodium hydroxide were selected with pH values of 10.07, 11.70, 12.55, and 12.66, respectively. The solutions of 1% of aminomethyl propanediol (AMPD), tromethamine (TRIS), triethanolamine (TEOA), and triisopropanolamine (TIPA) were characterized by the following pH values: 10.08, 9.16, 9.46, and 9.18 ( Table 2). The relationship between the aqueous CT solubility and pKa of selected solvents was confirmed. The variability of the pKa of alcoholamines (Figure 8) was reflected in the solubility pattern, where the AMPD with the highest pKa provided the best results in terms of CT solubility compared with Figure 9 (inserted panel).

Discussion
The stability of the CT molecule depends on the pH value. Below pH 2, CT tends to be degraded, whereas over pH 11, structural changes are observed [36]. The pH range of 2-8 mildly influences the solubility, whereas pH 10 to 12 strongly favors the aqueous dissolution of CT [4]. In our study, an alkaline environment between pH values of 10.0 and 12.70 was chosen to establish optimal conditions for CT solubility and stability in the solution. Concentrations of 0.001, 0.01, 0.1, and 1.0 mol/L sodium hydroxide were selected with pH values of 10.07, 11.70, 12.55, and 12.66, respectively. The solutions of 1% of aminomethyl propanediol (AMPD), tromethamine (TRIS), triethanolamine (TEOA), and triisopropanolamine (TIPA) were characterized by the following pH values: 10.08, 9.16, 9.46, and 9.18 ( Table 2). The relationship between the aqueous CT solubility and pKa of selected solvents was confirmed. The variability of the pKa of alcoholamines (Figure 8) was reflected in the solubility pattern, where the AMPD with the highest pKa provided the best results in terms of CT solubility compared with Figure 9 (inserted panel).

Visual Results
Despite the numerous therapeutic properties of CT, a diterpene compound, its practical application encounters hindrances, resulting from low water solubility and low bioavailability [3]. A visual examination of the impact of the series of solvents on the solubility of CT illustrated high solubility in alcohols, i.e., methanol, ethanol, and isopropanol. Lower solubility was observed in sodium hydroxide solutions, whereas CT was practically insoluble in distilled water, sulfuric acid (VI) solution, hydrochloric acid solution, and alcoholamine solution (Figure 3). In alcoholic solvents and in sodium hydroxide solution, CT dissolved to form yellow-colored solutions, which may be regarded as a confirmation of CT dissolution. Alcohols with a low molecular mass favor the solubility of CT, which was availed in solubility studies [45,46]. However, the presence of long hydrocarbon chains destroys the association between water and alcohol. We observed an increase in the color of the alcoholic solution of CT (Figure 3, left panel). Our observation showed that the addition of water to the solution of CT dissolved in alcohol caused the precipitation of CT, whereas the addition of 0.1 mol/L NaOH solution triggered better CT dissolution. Different dispersions and particle sizes of CT were observed under 10× microscope magnification. There were many differences between the crystal form of CT and that dissolved in ethanol. It was noticed that under the influence of the addition of 0.11 M NaOH solution, the CT particles were broken down and dispersed to a greater extent compared to the sample in pure ethanol. The addition of water resulted in the formation of elongated and thin structures (Figure 3, right panel). The varied behavior of the CT alcoholic solution after the addition of water or NaOH solution should be further examined to elucidate the possible intermolecular interactions, resulting from the slightly acidic nature of alcohols.

Discussion
The stability of the CT molecule depends on the pH value. Below pH 2, CT t be degraded, whereas over pH 11, structural changes are observed [36]. The pH r 2-8 mildly influences the solubility, whereas pH 10 to 12 strongly favors the aque solution of CT [4]. In our study, an alkaline environment between pH values of 1 12.70 was chosen to establish optimal conditions for CT solubility and stability in lution. Concentrations of 0.001, 0.01, 0.1, and 1.0 mol/L sodium hydroxide were with pH values of 10.07, 11.70, 12.55, and 12.66, respectively. The solutions of 1% nomethyl propanediol (AMPD), tromethamine (TRIS), triethanolamine (TEOA triisopropanolamine (TIPA) were characterized by the following pH values: 10.0 9.46, and 9.18 ( Table 2). The relationship between the aqueous CT solubility and selected solvents was confirmed. The variability of the pKa of alcoholamines (F was reflected in the solubility pattern, where the AMPD with the highest pKa p the best results in terms of CT solubility compared with Figure 9 (inserted panel).

Visual Results
Despite the numerous therapeutic properties of CT, a diterpene compound, its prac-

The Approximated Concentrations of CT under the Influence of Various Alkaline Solutions
The variability of concentrations obtained from the spectrophotometric assays indicated that the solubility of CT in alkaline solutions was limited, and saturation could be deduced when the volume of the alkaline component was in the range of 4.5-6 mL ( Figure 4). The concentration of the alkaline component of ca. 0.05-0.1 mol/L for the alcoholamines resulted in CT dissolution of ca. 15-30 mg/L, whereas the NaOH solution of a much lower concentration of 0.001 mol/L provided comparable solubility of ca. 10 mg/L. The obtained saturation values were of preliminary guidance quality data due to the high volume of added solutions. However, the calculation of the CT/alkaline component ratio provided an assumption to the statement that the CT saturation state in the assessed aqueous systems is possible in the presence of ca. 27 particles of NaOH per one particle of CT, whereas in the case of alcoholamines, the ratio was in the range of ca. 737 to 1514 particles per one particle of CT. The difference may be connected to the lower alkalinity of alcoholamines compared to NaOH; however, the pattern is disturbed by the higher pH in the case of the AMPD solution compared to the NaOH solution ( Table 2). Other factors, such as the logP of the alkaline components, may also influence the solubility of CT. The logP may be of special interest for the apparent permeability coefficient in cellular monolayers [3].

Titration Studies
Evaluation of the influence of increasing concentrations of NaOH on the pH and conductivity of CT dispersions revealed that only slight differences were observed when CT was present in the system. The pH plot for the titration of CT was adjacent to the plot for the titration of water (Figure 5a). The course of the dependence was compared to the case of the neutralization of CO 2 , usually present in water; however, we used deionized water. The conductivity data provided more insight into the CT-water-NaOH interaction (Figure 5b). In the presence of CT, the conductivity increased faster compared to the aqueous system. This increase was effectively observed after CO 2 neutralization, which was confirmed by plotting the pH with the experimental and theoretical curves. CO 2 neutralization took place at approximately 1 mL of NaOH 0.001 mol/L. The higher conductivity values (black dots) reflected the dissolution of CT under the influence of NaOH solution. Interestingly, when the ethanol-water system was applied in the same conditions, the pH variability between the sample with CT and without CT was less pronounced, supposedly due to the lower content of ionizable water generating the protons (Figure 5c). The conductivity pattern suggests that ethanol supported the dissolution of CT, which is visible when comparing the plots in Figure 5b,d. The conductivity at the initial point was higher when the ethanol was present in the sample, and also, an increasing conductivity tendency was observed. Ethanol has low conductivity compared to water [47,48], and the presence of alcohol should favor a decrease in conductivity; however, in the presence of NaOH, a reduction in conductivity was not noticed (Figure 10).

HPLC Studies
The HPLC method was widely applied to qualitatively and quantitatively assay CT in several compositions [5,49], as well as the active substances of Salvia miltiorrhiza [27] or the CT release processes [32]. Our HPLC studies confirmed the correlation between the temperature conditions of the dissolving procedure. The increased temperature favored the higher solubility of CT in the solutions of alcoholamines ( Figure 10 values (black dots) reflected the dissolution of CT under the influence of NaOH solution. Interestingly, when the ethanol-water system was applied in the same conditions, the pH variability between the sample with CT and without CT was less pronounced, supposedly due to the lower content of ionizable water generating the protons (Figure 5c). The conductivity pattern suggests that ethanol supported the dissolution of CT, which is visible when comparing the plots in Figure 5b,d. The conductivity at the initial point was higher when the ethanol was present in the sample, and also, an increasing conductivity tendency was observed. Ethanol has low conductivity compared to water [47,48], and the presence of alcohol should favor a decrease in conductivity; however, in the presence of NaOH, a reduction in conductivity was not noticed (Figure 10).

HPLC Studies
The HPLC method was widely applied to qualitatively and quantitatively assay CT in several compositions [5,49], as well as the active substances of Salvia miltiorrhiza [27] or The molar ratios of alcoholamine/CT achieved the lowest values for the AMPD solution, which confirms its highest efficacy in terms of the CT solubility increase in aqueous solutions of the selected alcoholamines-AMPD, TRIS, TEOA, and TIPA (Figure 11a). The stability of CT was higher in the solutions of alcoholamines compared to the NaOH solutions, which is very interesting from the practical point of view. The stable content of CT was retained two-fold longer in the case of alcoholamines, i.e., for 42 days, whereas the NaOH solutions retained the initial level of CT only for ca. 21 days. the CT release processes [32]. Our HPLC studies confirmed the correlation between the temperature conditions of the dissolving procedure. The increased temperature favored the higher solubility of CT in the solutions of alcoholamines ( Figure 10, inserted plot). The NaOH solutions enabled an increase in CT concentrations up to the range of 0.056-0.320 mmol/L, depending on the NaOH concentrations. When compared to NaOH solutions, the 1% solutions of TRIS, TEOA, and TIPA provided a moderate increase in CT aqueous solubility. The resulting CT concentrations were in the range of 0.01-0.03 mmol/L compared to the aqueous solubility of CT of ca. 0.003 mmol/L. The 1% AMPD solution was distinguished out of the alcoholamine solutions and enabled the highest concentrations of CT in the range of 0.03-0.06 mol/L, and CT solubilization varied depending on the temperature of the dissolution process ( Figure 9). The molar ratios of alcoholamine/CT achieved the lowest values for the AMPD solution, which confirms its highest efficacy in terms of the CT solubility increase in aqueous solutions of the selected alcoholamines-AMPD, TRIS, TEOA, and TIPA (Figure 11a). The stability of CT was higher in the solutions of alcoholamines compared to the NaOH solutions, which is very interesting from the practical point of view. The stable content of CT was retained two-fold longer in the case of alcoholamines, i.e., for 42 days, whereas the NaOH solutions retained the initial level of CT only for ca. 21 days.
(a) (b) Figure 11. Influence of type of the alcoholamine on the molar ratio alcoholamine/CT in solutions prepared in varied temperature (a) and the influence of increasing concentrations of NaOH and of 96% ethanol on the molar ratio cosolvent/CT (b).
The molecular peculiarity of AMPD in comparison to TEOA, TIPA and TRIS is based on the number of OH groups in the particle, which are attached to the nonlinear carbon chain with the primary amine group. Both the OH groups' number and the number of carbon chains attached to the nitrogen atom may influence the dissolving potential of alcoholamines against CT ( Figure 10). The number of NaOH molecules interacting with CT increases with the increase in NaOH concentration, and the pattern is close to the linear dependence. The ethanol solution fits the pattern observed for NaOH solutions ( Figure  11b). The stability of CT solutions in alkaline solutions depended on the type of alkaline component. In the NaOH solutions, regardless of the NaOH concentrations, the level of CT decreased after 21 days, whereas in the case of alcoholamines, the CT level decreased after 42 days (Figure 7a-d). The short shelf time of CT may depend on the storage conditions of the solutions at 25 °C in the absence of direct light. CT solutions were also heated The molecular peculiarity of AMPD in comparison to TEOA, TIPA and TRIS is based on the number of OH groups in the particle, which are attached to the nonlinear carbon chain with the primary amine group. Both the OH groups' number and the number of carbon chains attached to the nitrogen atom may influence the dissolving potential of alcoholamines against CT ( Figure 10). The number of NaOH molecules interacting with CT increases with the increase in NaOH concentration, and the pattern is close to the linear dependence. The ethanol solution fits the pattern observed for NaOH solutions (Figure 11b). The stability of CT solutions in alkaline solutions depended on the type of alkaline component. In the NaOH solutions, regardless of the NaOH concentrations, the level of CT decreased after 21 days, whereas in the case of alcoholamines, the CT level decreased after 42 days (Figure 7a-d). The short shelf time of CT may depend on the storage conditions of the solutions at 25 • C in the absence of direct light. CT solutions were also heated up to 40-60 • C, as temperature may affect the solubility of CT. CT is temperature sensitive, so the producer's specification recommends storage of the powder at -20 • C and CT in solvent at −80 • C [50]. Song and coworkers found the instability of CT in methanol at room temperature and after exposure to light [51]. Our research was preparatory to the final hydrogel's formulation of CT. Further steps will include stabilization of the samples via proper storage of the mixtures, e.g., in low temperatures, in the absence of direct light, or via inhibition of the degradation processes with the use of selected substances, which will be developed in the future. However, the most interesting option includes the ex tempore preparation of the mixtures prescribed for an individual patient. Some preparations against acne have been developed in the ex tempore form, i.e., due to the instability of erythromycin in suspension with zinc salt, clindamycin hydrochloride in lotion [52], or azelaic acid in a liposomal formulation [53].

Conclusions
Alcohols, including methanol, ethanol, and isopropanol, are good solvents for CT. The alkalic environment of NaOH solutions, as well as the alcoholamine solutions, favor the dissolution of CT. CT dissolution is mainly dependent on the pH, and the presence of ethanol is a conducive factor for CT aqueous solubility, similar to the increased temperature and sonification applied during the dissolution procedure. There are only slight differences between TIPA, TEOA, and TRIS in the solubilizing potential. Between the analyzed alcoholamines, AMPD seems to be the most interesting alkalic component, which may be used to enhance the solubility of CT, as confirmed by the number of AMPD particles on the CT particle in the aqueous solution. Alcoholamines enable higher stability compared to NaOH in respective solutions, which is very important for the further development of CT in various pharmaceutical formulations.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/pharmaceutics13070992/s1, Table S1: Factors applied for dissolution of 1 mg of CT, evaluated in the terms of visual appearance, Table S2: The composition of samples applied in the titration studies, Table S3: The composition and preparation parameters of the HPLC samples.