Emulsifying Solution Prepared by an AI-based Drug Delivery System (AIDDS™) Enhances Bioavailability of Silymarin

Materials

Silymarin was obtained from Naturesense Co. Ltd., medium chain triglyceride (MCT) from Wellga, Korea, polysorbate 80 from Kao Chemical, Japan, polyglyceryl-3 dioleate (Plurol^® Oleique CC 497) and propylene glycol monocaprylate (Type I, Capryol PGMC) from Gattefosse Co., France and propylene glycol from Samchun Pure Chemical Co., Korea.

Assay for silymarin

To determine the solubility of silymarin in various solvents, an excess amount of silymarin was added into each solvent and was continuously stirred for 72 hours at 37°C. At equilibrium, the mixture was centrifuged at 12,000 rpm for 10 min, and the supernatant was filtered through a membrane filter (0.45 μm, Advantec, Korea). The concentration of silymarin in the supernatant was determined by HPLC (Agilent 1260 infinity II; Agilent Technologies, Inc.) using a Capcellpak UG120 C18 column (diameter, 4.6 mm; length, 250 mm) filled with octadecylsilyl silica gel (diameter, 5 μm) at 1.0 ml/min of flow rate. Oven temperature was 40\\xcb\\x9aC and UV wavelength of detector was 280 nm. The mobile phases were set as A (20% aqueous methanol) and B (80% aqueous methanol) under following conditions: 0-5 min (15% B), 5-20 min (15-45% B), 20-40 min (45% B), 40-41 min (45-15% B), 41-55 min (15% B).

Construction of pseudo-ternary phase diagrams for selecting emulsifying solution for silymarin and provision of the data for establishing AIDDS™ (AI-based Drug Delivery System)

To increase the bioavailability of a lipophilic compound such as silymarin, it is important to find good emulsifying solutions (emulsifiers) to dissolve the substance. For this purpose, MCT was selected as a solvent, polysorbate 80 as a surfactant and various compounds as co-surfactants such as Plurol^® Oleique CC 497, Type I, Capryol PGMC and propylene glycol. And then MCT (solvent), polysorbate 80 (surfactant) and one of the above co-surfactants were mixed in various ratios. Each of the resulting mixed solutions was tested for its ability to dissolve silymarin and if it dissolved silymarin, it was selected as an emulsifier. After a series of the tests, the group of emulsifiers was obtained and also a group of the ratios of solvent, surfactant and co-surfactant corresponding to the individual emulsifiers obtained. Each of these ratios was plotted as a dot in the triangle whose 3 sides represent the amount of solvent, surfactant and co-surfactant, respectively. These dots formed the areas in the triangle as shown Fig. 1. Then, if any dot in the area is chosen and the solvent, surfactant and co-surfactant are mixed by the ratios indicated by the dot, a good emulsifier will be prepared. All of these procedures are very complicated and thus should be operated by a computer program in the future. This program is called AI-based Drug Delivery System (AIDDS™) and the data obtained in the present study will be used for establishing AIDDS™ program.

Fig. 1
Summary of our method to find emulsifying solution against silymarin. This method is plotting of the ratios in the triangle with which MCT as a solvent, Polysorbate 80 as a surfactant and a mixture of Plurol Oleique^® and Capryol PGMC (1:1) as a co-surfactant were mixed and then, the mixtures dissolve and emulsify silymarin. The plotting gives areas and any mixtures made by the ratios indicated by any points in the area will be emulsifiers that can dissolve silymarin. Details are described in METHODS.
MCT = medium chain triglyceride.

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Determination of droplet size of the emulsifiers prepared by the diagram

One of the criteria to assess an emulsifier is the size of the microemulsion droplet of the emulsion formed by the emulsifier and the solute when the emulsion is diluted with water. The smaller its size the better. The emulsifier (500 mg) and silymarin (65.0 mg) were mixed and emulsion was prepared. This emulsion were diluted 1000 times with water and the sizes of droplets distributed in the oil phase were determined by a analyzer (ZETASIZER Nano-ZS, Malvern, UK) according the procedure provided by the manufacturer.

Stability test of the silymarin-containing emulsifier

Another criterion to evaluate an emulsifier is the stability when it is in the state of the solution containing the solute. In order to evaluate their stability, the emulsifiers prepared were mixed silymarin and aliquots of the resulting solutions were added to glass vial and sealed. The sealed vials were then stored at either 25°C, relative humidity (RH) 60% or 40°C, relative humidity 75% for 3 months. The clarity, phase separation, concentration of silymarin, and droplet size in water (1:200) were investigated at intervals.

Dissolution of silymarin into surrounding medium

The dissolution of silymarin into the surrounding media can be a factor to determine its absorption in vivo. Thus, dissolution of silymarin in the emulsifiers into the surrounding media was measured and compared to that of its solid state. Silymarin in the emulsifiers or in HPMC (hydroxy propyl methyl cellulose) capsules was placed in 900 mL of distilled water or 2 phosphate-buffered solutions (PBSs) of pH 1.2 or pH 6.8 and maintained at 37^oC with stirring by revolution at 100 rpm. Aliquots (5 mL) of water or 2 buffer solutions were taken at 0, 30, 60, and 120 min and filtered through a 0.45-µm membrane filter. The concentrations of silymarin in the filtrates were assessed by measuring silybin using the HPLC as described above. Silybin is a major component of silymarin.

Pharmacokinetics study

The benefit of the emulsifiers prepared in the present study was evaluated by pharmacokinetic study in rats. Male Sprague–Dawley rats (200–300 g) were supplied by ORIENTBIO Inc., Korea. The rats had free access to water and were fasted for 16 hours prior to experiments. Eighteen rats were randomly assigned to 3 groups (n = 6 in each group). One of the 3 silymarin preparations was administered orally to each group; silymarin in 10 mL of the emulsifier or saline and a silymarin product (selected from the market) in 10 mL of saline. The dose of silymarin administered was 66.7 mg/kg. Following administration of silymarin preparations, rats were given 1 mL of water. And then, blood samples (0.5 mL) were collected from the jugular vein at 0, 0.25, 0.5, 1, 2, 3, 4, 6, 8 and 12 hours, immediately placed into heparinized tubes, centrifuged at 12,000 rpm for 3 minutes at 4°C. The plasma obtained was stored at −80°C until analyzed. Each plasma sample (20 µL) was mixed with 10 µL of naringin in acetonitrile (50 µg/mL) as an internal standard and diluted with 300 µL of acetonitrile. The solution was further mixed for 3 minutes and centrifuged at 12,000 rpm for 3 minutes at 4°C. Aliquots (2 µL) of the supernatant was used for analysis.

The concentrations of silymarin in the aliquots were assessed by measuring silybin, a major component of silymarin, using the liquid chromatography-mass spectrometry (LC/MS) [Prominence (main body; SHIMADZU Co. Ltd.), API4000 (detector; AB Sciex, Pte. Ltd.), and an ESI (ionizer)source in negative ion mode]. For the LC analysis, the mobile phase consisted of 50:50 (v/v) mixture of 0.02% formic acid in water and acetonitrile. The flow rate was 0.2 mL/min. The spray voltage of the MS was set at 4.2 kV. The temperature of the drying gas (N₂) was kept at 400°C. Concentrations of silybin were calculated based on the area ratios (A_silybin/A_naringin), and the areas of the silybin were linear with respect to silymarin concentrations. Thus, a straight regression line with a correlation coefficient above 0.99 was obtained over the concentration range of 50–37,500 ng/mL. The lower limit of detection was 50 ng/mL. This analysis technique provided acceptable results with respect to precision and accuracy (r > 0.99).

From this analysis, maximum plasma concentration of silybin (C_max) and the time to reach maximum plasma concentration (T_max) were obtained. The area under the silybin concentration-time curve was calculated from 0 to 12 hours (AUC_0-12h) according to the interpolation method.6 Pheonix WinNolin (ver.6.2, Pharsight-A Certara Company, USA) was used for PK calculation.

Statistical analysis

All experimental results were expressed as mean ± standard error. The statistical significance between groups was tested by analysis of variance using IBM SPSS software version 24.0 (IBM Analytics, Armonk, NY, USA). Statistical significance was defined using two-tailed tests, and values less than 0.05 were considered statistically significant.

Searching for efficient emulsifying solutions (emulsifiers) for silymarin by the technique of pseudo-ternary phase diagrams

In order to increase the bioavailability of lipophilic drugs, they should be well emulsified in the suitable emulsifying solutions (emulsifiers) since these drugs in the state of microemulsion are well dissolved out into the surrounding medium in the gut, and thus well absorbed. So it is important to find a good emulsifying solution for a certain lipophilic compound but not easy. In the present study, we tried to find good emulsifiers targeting silymarin, a wide used health supplement by constructing the pseudo-ternary phase diagrams.

As described in the METHOD, MCT as a solvent, polysorbate 80 as a surfactant and one or 2 of the compounds (Plurol^® Oleique CC 497, Capryol PGMC and propylene glycol) as co-surfactants were mixed in various ratios. Each of the resulting mixed solutions was tested for its ability to dissolve silymarin. Of total mixed 36 solutions, 1236 solutions were shown to dissolve silymarin, i.e. identified as silymarin-emulsifiers. From the 36 solution, 1236 ratios of MTC, polysorbate 80 and a 1: 1 mixture of Plurol Oleique CC 497 and Capryol PGMC were plotted as dots in the triangle, which formed the areas shown in Fig. 1. One thing to be noted is that co-surfactants showed an increased emulsifying activity when used in combination, therefore, a 1:1 mixture of Plurol Oleique CC 497 and Capryol PGMC was used as a co-surfactant. Among 36 emulsifying solutions, those mixed with the ratios of 1:1:7, 1:2:6, 1:3:5, 6:1:2, 6:2:1, and 7:1:1 were shown to emulsify silymarin very clearly. Thus, one of these rations was chosen and the emulsifying solution with this ratio was prepared and evaluated for its quality. This solution is called the EMULSIFIER^AIDDS™.

Size of emulsion droplet of the EMULSIFIER^AIDDS™

The droplet size of micro-emulsions is the most important characteristic of an emulsifying solution when it contains a solute. The droplet size is closely related to stability and in vivo fate.7, 8, 9 Therefore, droplet size of the micro-emulsions of the mixture of EMULSIFIER^AIDDS™ and silymarin in various media was measured. As shown in Table 1, the droplet size of the micro-emulsions were increased when the solution contained silymarin but around 100 nm which is the range of good and fine microemulsion.10

Table 1
The mean particle size of emulsion made with of EMUSIFIER^a and silymarin in various media

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Stability of the silymarin-containing TPD-emulsifying solution -

Another criterion to evaluate an emulsifying solution is the stability when it is in the state of the solution containing the solute. After storage at 25°C/relative humidity 60% or 40°C/relative humidity 75% for 3 months, the emulsion made with EMULSIFIER^AIDDS™ and silymarin was shown to be clear and transparent without any phase separation. Furthermore, the concentration of silymarin and the droplet size of the microemulsion did not change significantly as compared to the initial state (data not shown).

These findings suggest that EMULSIFIER^AIDDS™ can form a very stable emulsion with silymarin.

Comparison of dissolution of silymarin into surrounding medium between the emulsified and solid states

The dissolution of silymarin into the surrounding media can be a factor to determine its absorption in vivo. Thus, dissolution of silymarin in the emulsifiers into surrounding media (water and PBS pH 1.2, 6.8) was compared to that of its solid state as described in the METHOD. As shown in Fig. 2, crude silymarin showed negligible release (< 25%), even after 120 minutes in distilled water. In contrast, the emulsion made with EMULSIFIER^AIDDS™ showed the rapid release in the 3 different media. Strikingly, at 60 minutes, approximately 90% of the silymarin was dissolved into media. Thus, it was confirmed that the emulsion made with EMULSIFIER^AIDDS™ and silymarin was stable and releasing silymarin into the surrounding media rapidly.

Fig. 2
Comparison of release of silymarin emulsified in EMUSIFIER into surrounding media to that of silymarin in solid state. Emulsion of silymarin in EMULSIFIER^a or silymarin in HPMC capsules was placed in 900 mL of distilled water or 2 phosphate-buffered solutions of pH 1.2 or pH 6.8 and maintained at 37^oC with stirring by revolution at 100 rpm. Aliquots (5 mL) of surrounding median (water or 2 buffer solutions) were taken at 0, 30, 60, and 120 minutes and assessed for silybin using the HPLC. Silybin is a major component of silymarin. For details, see METHOD.
^aAn emulsifying solution prepared by our method as described in METHODS.

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Pharmacokinetic studies

The final benefit of silymarin emulsion made with the EMULSIFIER^AIDDS™ is the rapid increase of silymarin concentration in the blood when given orally. Thus, pharmacokinetic study was performed in rats with 3 silymarin preparations, which were 1) silymarin dissolved in 10 mL EMULSIFIER^AIDDS™, 2) silymarin in 10 mL saline and 3) a silymarin product from the market in 10 mL saline. The oral dose of each preparation was 66.7 mg/kg. Silymarin concentration was assessed by analysis of silybin, a major component in silymarin. The results are shown in Fig. 3 and Table 2. AUC_0-12h, C_max and T_max were outstandingly better in silymarin dissolved in 10 mL EMULSIFIER^AIDDS™ than in saline.

Fig. 3
Pharmacokinetics of silymarin emulsified in EMUSIFIER in rats. Three silymarin in different states were prepared; 1) in 10 mL of EMUSIFIER, 2) in 10 mL saline and 3) a silymarin product from the market in 10 mL saline. The oral dose of silymarin in each preparation was 66.7 mg/kg. Silymarin concentration in blood was assessed by analyzing silybin, a major component of silymarin. Details are described in METHODS.
^*P < 0.05 compared with the control by ANOVA. Data are shown as means ± standard deviation (n = 6 mice/group).

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Table 2
Pharmacokinetic parameters of silymarin in three different states in rats

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