Method for solubilizing poorly water-soluble substances and solubilized composition for poorly water-soluble substances

Abstract

To provide a method for solubilizing a water-insoluble substance that uses a solubilizer having high biocompatibility and low environmental load and is applicable to a wide range of water-insoluble substances by a simple process, offering high versatility, and a composition comprising a solubilized water-insoluble substance.SOLUTION: A water-insoluble substance is solubilized by a method including the steps of: grinding a water-insoluble substance and a solubilizer, which is at least one selected from gelatin, polyglutamic acid and polylysine, using a ball mill, or a mortar and a pestle, to obtain a ground mixture; and adding water to the ground mixture, to obtain aqueous solution of the ground mixture.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for solubilizing a poorly water-soluble substance and a solubilized composition for a poorly water-soluble substance. [Background technology]

[0002] Many of the pharmacologically active substances and functional substances used in the fields of pharmaceuticals, food, chemistry, etc. are poorly water-soluble substances. If such poorly water-soluble substances could be solubilized, the versatility of these pharmacologically active substances and functional substances could be enhanced.

[0003] Conventional solubilizers for poorly water-soluble substances include sugars such as cyclodextrin and pullulan, as well as artificial cell membranes such as liposomes and polymeric micelles. Because sugars and liposomes are naturally occurring substances, they are highly biocompatible and have a low environmental impact, but because polymeric micelles are synthetic, their biocompatibility and environmental impact require long-term confirmation.

[0004] As an example of using sugars as a solubilizer, Non-Patent Document 1 describes the use of C 60 C was obtained by mixing fullerene with a γ-cyclodextrin solution and heating and stirring. 60 It is stated that a solubilized complex of fullerenes was obtained. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] T. Andersson, K. Nilsson, M. Sundahl, G. Westman, O. Wennerstrom, C60 embedded in γ-cyclodextrin: a water-soluble fullerene, J.Chem.Soc.,Chem.Commun.1992,604-606. Summary of the Invention [Problem to be solved by the invention]

[0006] Although heating and stirring is a common method for solubilizing poorly water-soluble substances, this method is not effective for poorly water-soluble substances with low thermal stability. Furthermore, selecting a solubilizer and setting a solubilization method depending on the type of poorly water-soluble substance, as in the past, is inefficient.

[0007] Therefore, the present invention has been made in consideration of these points, and its object is to provide a highly versatile method for solubilizing poorly water-soluble substances that can be applied to a wide variety of poorly water-soluble substances, and a solubilized composition for poorly water-soluble substances. [Means for solving the problem]

[0008] To achieve the above object, the present invention provides a method for solubilizing a poorly water-soluble substance, comprising the steps of: grinding and mixing the poorly water-soluble substance with a solubilizing agent selected from gelatin, polyglutamic acid, and polylysine using a ball mill or a mortar and pestle to obtain a ground mixture; and adding water to the ground mixture to obtain an aqueous solution of the ground mixture. The present invention also provides a solubilized composition for a poorly water-soluble substance, comprising the poorly water-soluble substance, a solubilizing agent, and water, wherein the solubilizing agent is at least one selected from gelatin, polyglutamic acid, and polylysine. [Effects of the Invention]

[0009] According to the method for solubilizing a poorly water-soluble substance and the solubilized composition for a poorly water-soluble substance of the present invention, it is possible to solubilize a wide variety of poorly water-soluble substances in a simple manner using a solubilizing agent that is highly biocompatible and has a low environmental impact. [Brief explanation of the drawings]

[0010] [Figure 1] 1 shows visible-ultraviolet absorption spectra of solubilized fullerene compositions obtained by different solubilization methods. [Figure 2]1 shows visible-ultraviolet absorption spectra of solubilized compositions of tetraphenylporphyrin obtained by different solubilization methods. [Figure 3] 1 is a visible-ultraviolet absorption spectrum of a solubilized fullerene composition. [Figure 4] 1 is a visible-ultraviolet absorption spectrum of a solubilized composition of tetraphenylporphyrin. [Figure 5] 1 is a graph showing the long-term stability of a solubilized fullerene composition. [Figure 6] 1 is a graph showing the long-term stability of a solubilized composition of tetraphenylporphyrin. [Figure 7] 1 is a graph showing the thermal stability of a solubilized composition of fullerene and tetraphenylporphyrin. [Figure 8] 1 shows a visible-ultraviolet absorption spectrum of a solubilized composition of a fullerene derivative. [Figure 9] 1 is a visible-ultraviolet absorption spectrum of a solubilized composition of phthalocyanine. [Figure 10] 1 is a visible-ultraviolet absorption spectrum of a solubilized composition of quinquithiophene. [Figure 11] 1 is a visible-ultraviolet absorption spectrum of a solubilized composition of β-carotene. [Figure 12] 1 is a visible-ultraviolet absorption spectrum of a solubilized composition of carbamazepine. [Figure 13] 1 shows the visible-ultraviolet absorption spectrum of a solubilized composition of paclitaxel. [Figure 14] 1 is a visible-ultraviolet absorption spectrum of a solubilized composition of curcumin. [Figure 15] 1 is a visible-ultraviolet absorption spectrum of a solubilized resveratrol composition. DETAILED DESCRIPTION OF THE INVENTION

[0011] (Solubilized composition of poorly water-soluble substance) The solubilized composition of a poorly water-soluble substance of the present invention comprises a poorly water-soluble substance and a solubilizing agent which is at least one selected from gelatin, polyglutamic acid, and polylysine, and is obtained by grinding and mixing the poorly water-soluble substance and the solubilizing agent using a ball mill or a mortar and pestle, and then adding water.

[0012] In the present invention, "poorly water-soluble" means that the solubility in water at 25°C is 0.1 g / L or less, and includes substances that are completely insoluble in water at 25°C.

[0013] The poorly water-soluble substance used in the present invention is not particularly limited, and examples thereof include various poorly water-soluble substances such as poorly water-soluble pharmacologically active substances, poorly water-soluble functional substances for use in the food industry such as beverages and supplements, and poorly water-soluble compounds in the chemical materials industry that have traditionally required dissolution in organic solvents.

[0014] Poorly water-soluble pharmacologically active substances include those classified as Class 2 (low solubility, good membrane permeability) or Class 4 (low solubility, poor membrane permeability) in the Biopharmaceutical Classification System established by the FDA. Specific examples include paclitaxel (taxol) and carbamazepine.

[0015] Examples of poorly water-soluble functional substances in the food and chemical material fields include porphyrin derivatives, phthalocyanine derivatives, fullerene derivatives, polyheterocyclic compounds such as oligothiophenes and polypyrroles, carotenoids such as α-carotene, β-carotene, γ-carotene, lycopene and cryptoxanthin, and polyphenols such as curcumin, resveratrol and astaxanthin.

[0016] These poorly water-soluble substances may be used alone or in combination of two or more.

[0017] The poorly water-soluble substance, when pulverized and mixed with a solubilizer using a ball mill or a mortar and pestle, has an average particle size of 50 to 1500 nm, preferably 80 to 600 nm, of dispersed particles. In the present invention, the average particle size is the average particle size obtained by dynamic light scattering. The measurement device used was a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK).

[0018] The solubilizing agent used in the present invention is a polypeptide, which is a naturally occurring substance. Specific examples include gelatin, collagen, polyglutamic acid, polylysine, etc., preferably gelatin, polyglutamic acid, and polylysine, and more preferably gelatin and polylysine. Collagen exhibits high solubility in the solubilized composition when mixed with carbamazepine. Gelatin, polyglutamic acid, and polylysine can be used as solubilizing agents regardless of the type of poorly water-soluble substance, making them highly versatile.

[0019] The solubilized composition of the present invention contains water as a solvent.

[0020] Furthermore, the solubilized composition of the present invention may contain other components as needed, as long as the effects of the present invention are not impaired.

[0021] (Method for solubilizing poorly water-soluble substances) The method of the present invention for solubilizing a poorly water-soluble substance includes the steps of: grinding and mixing the poorly water-soluble substance and at least one solubilizing agent selected from gelatin, polyglutamic acid, and polylysine using a ball mill or a mortar and pestle to obtain a ground mixture; and adding water to the ground mixture to obtain an aqueous solution of the ground mixture.

[0022] In the present invention, a grinding and mixing method using a high-speed vibration grinder as a type of ball mill is used. In the high-speed vibration grinder, hard balls reciprocate at high speed within the hollow portion of a container containing the poorly water-soluble substance and the solubilizer, thereby grinding and mixing the poorly water-soluble substance and the solubilizer. Specifically, this ball mill can grind particles with particle sizes ranging from several nanometers to several hundred micrometers by vibrating and rotating at a speed of 180 to 2100 rpm (3 to 35 Hz), for example.

[0023] In the grinding and mixing using a mortar and pestle, the material of the mortar and pestle is not limited, and for example, porcelain, glass, or agate mortars and pestles can be used, but porcelain mortars and pestles are preferred. [Example]

[0024] Examples of the present invention will be described below. However, the present invention is not limited to these examples, and these examples can be modified or changed based on the spirit of the present invention, and such modifications are not excluded from the scope of the present invention.

[0025] (Synthesis of solubilized compositions of poorly water-soluble substances using a high-speed vibrating mill or a mortar and pestle) Example 1 As a poorly water-soluble substance, fullerene (C in the figure and table) 60 A solubilized composition was synthesized by high-speed vibration milling using a soluble fiber (referred to as "A" in the figures and tables) and gelatin (referred to as "GEL" in the figures and tables) as a solubilizing agent.

[0026] Specifically, 1.4 mg (2.0 μmol) of fullerene and 10.0 mg of gelatin were placed in a cylindrical agate container together with an agate ball, and then subjected to high-speed vibration milling at 30 Hz for 20 minutes using a high-speed vibration mill (Retsch, MM200 model) to obtain a milled mixture. 2.0 mL of ultrapure water was then added to the milled mixture for extraction. The resulting extract was subjected to ultrasonic irradiation for 60 minutes, after which the insoluble fullerene was precipitated using a centrifuge (4500 rpm, 20 minutes), and the supernatant was separated. This supernatant is a solubilized composition of fullerene and gelatin.

[0027] Example 2 As a poorly water-soluble substance, fullerene (C in the figure and table) 60 The solubilized composition was synthesized by a kneading method using a mortar and pestle, using cellulose acetate (referred to as "C1" in the figures and tables) as a solubilizer and gelatin (referred to as "GEL" in the figures and tables).

[0028] Specifically, 1.4 mg (2.0 μmol) of fullerene and 10.0 mg of gelatin were first placed in a porcelain mortar and ground for 20 minutes using a porcelain pestle to obtain a ground mixture. 2.0 mL of ultrapure water was then added to the ground mixture for extraction. The resulting extract was subjected to ultrasonic irradiation for 60 minutes, after which the insoluble fullerene was precipitated using a centrifuge (4500 rpm, 20 minutes), and the supernatant was separated. This supernatant is a solubilized composition of fullerene and gelatin.

[0029] Example 3 A solubilized composition was synthesized by the high-speed vibration milling method in the same manner as in Example 1, using 1.1 mg (2.0 μmol) of tetraphenylporphyrin (referred to as TPP in the figures and tables) as the poorly water-soluble substance and gelatin as the solubilizing agent.

[0030] Example 4 A solubilized composition was synthesized using 1.1 mg (2.0 μmol) of tetraphenylporphyrin (referred to as TPP in the figures and tables) as the poorly water-soluble substance and gelatin as the solubilizing agent by the kneading method using a mortar and pestle, as in Example 2.

[0031] (Synthesis of solubilized composition of poorly water-soluble substance by heating and stirring) -Comparative Example 1- Next, a solubilized composition of the poorly water-soluble substance was synthesized by heating and stirring using fullerene as the poorly water-soluble substance and gelatin as the solubilizing agent.

[0032] Specifically, 1.4 mg (2.0 μmol) of fullerene, 10.0 mg of gelatin, and 2.0 mL of ultrapure water were placed in a recovery flask and heated and stirred at 80° C. for 24 hours. After that, the insoluble fullerene was precipitated using a centrifuge (4500 rpm, 20 minutes), and the supernatant was separated.

[0033] (Synthesis of solubilized compositions of poorly water-soluble substances by ultrasonic irradiation) -Comparative Example 2- Next, a solubilized composition of the poorly water-soluble substance was synthesized by using fullerene as the poorly water-soluble substance and gelatin as the solubilizing agent through ultrasonic irradiation.

[0034] Specifically, 1.4 mg (2.0 μmol) of fullerene, 10.0 mg of gelatin, and 2.0 mL of ultrapure water were placed in a recovery flask and subjected to ultrasonic irradiation for 2 hours. After that, the insoluble fullerene was precipitated by centrifugation (4500 rpm, 20 minutes), and the supernatant was separated.

[0035] -Comparative Example 3- A solubilized composition was synthesized by heating and stirring in the same manner as in Comparative Example 1, using 1.1 mg (2.0 μmol) of tetraphenylporphyrin as the poorly water-soluble substance and gelatin as the solubilizing agent.

[0036] -Comparative Example 4- A solubilized composition was synthesized by ultrasonic irradiation in the same manner as in Comparative Example 2, using 1.1 mg (2.0 μmol) of tetraphenylporphyrin as the poorly water-soluble substance and gelatin as the solubilizing agent.

[0037] (Measurement of ultraviolet-visible absorption spectrum) The UV-visible absorption spectra of the solubilized compositions obtained by the above methods were measured to confirm whether the poorly water-soluble substance was dissolved in water. Specifically, the solubilized compositions obtained by the above methods were placed in 1 mm cells and measured using a UV-visible spectrophotometer (Shimadzu, UV-3600). The UV-visible absorption spectra of the solubilized compositions of fullerene and gelatin obtained in Example 1, Example 2, Comparative Example 1, and Comparative Example 2 are shown in Figure 1, and the UV-visible absorption spectra of the solubilized compositions of tetraphenylporphyrin and gelatin obtained in Example 3, Example 4, Comparative Example 3, and Comparative Example 4 are shown in Figure 2.

[0038] 1 and 2, in both cases where fullerene or tetraphenylporphyrin was used as the poorly water-soluble substance, almost no absorption was observed in the solubilized composition synthesized by heating and stirring and ultrasonic irradiation, while absorption due to fullerene and tetraphenylporphyrin was observed in Examples 1 to 4, which used the high-speed vibration grinding method and mortar and pestle. These results demonstrate that it is difficult to solubilize poorly water-soluble substances using heating and stirring and ultrasonic irradiation, and that the high-speed vibration grinding method or the method using a mortar and pestle, preferably the high-speed vibration grinding method, is appropriate.

[0039] Next, using a high-speed vibration milling method, we compared the solubility of various poorly water-soluble substances when polypeptides were used as solubilizers with that when conventionally used polysaccharides were used.

[0040] (Synthesis of fullerene-solubilized composition) Examples 5 and 6 and Comparative Examples 5 to 7 1.4 mg (2.0 μmol) of fullerene, a poorly water-soluble substance, was pulverized and mixed with each solubilizing agent by high-speed vibration pulverization, and solubilized compositions were synthesized in the same manner as in Example 1. The solubilizing agents used were 10.0 mg of poly-γ-glutamic acid (referred to as γ-PGA in the figures and tables) in Example 5, 10.0 mg of α-poly-L-lysine (referred to as α-PL in the figures and tables) in Example 6, 10.0 mg of γ-cyclodextrin (referred to as γ-CDx in the figures and tables) in Comparative Example 5, 10.0 mg of pullulan (referred to as PL in the figures and tables) in Comparative Example 6, and collagen (referred to as COL in the figures and tables) in Comparative Example 7.

[0041] The solubilized compositions obtained as the supernatant in Examples 1, 5, and 6 and Comparative Examples 5 to 7 were each diluted 10-fold and placed in a 1 mm cell, and measured using an ultraviolet-visible spectrophotometer (Shimadzu, UV-3600) in the same manner as above. The measurement results are shown in Figure 3. As shown in Figure 3, Examples 1, 5, and Comparative Example 5 showed large absorption, indicating that fullerene was well dissolved.

[0042] (Synthesis of solubilized tetraphenylporphyrin composition) Examples 7 and 8 and Comparative Examples 8 to 10 1.1 mg (2.0 μmol) of tetraphenylporphyrin, a poorly water-soluble substance, was ground and mixed with each solubilizing agent by high-speed vibration grinding, and solubilized compositions were synthesized using the same procedure as in Example 1. As solubilizing agents, 10.0 mg of poly-γ-glutamic acid was used in Example 7, 10.0 mg of α-poly-L-lysine in Example 8, 10.0 mg of trimethyl-β-cyclodextrin (referred to as TMe-β-CDx in the figures and tables) in Comparative Example 8, 10.0 mg of pullulan in Comparative Example 9, and collagen in Comparative Example 10 were used.

[0043] The solubilized compositions obtained as supernatants in Examples 3, 7, and 8 and Comparative Examples 9 and 10 were diluted 10-fold, and the supernatant obtained in Comparative Example 8 was diluted 100-fold, placed in 1 mm cells, and measured using an ultraviolet-visible spectrophotometer (Shimadzu, UV-3600) in the same manner as above. The measurement results are shown in Figure 4. As shown in Figure 4, Examples 3, 7, and 8 and Comparative Example 9 showed large absorption, indicating that tetraphenylporphyrin was well dissolved.

[0044] (Evaluation of long-term stability) The solubilized compositions thus obtained were evaluated for long-term stability. The solubilized compositions obtained as supernatants in Examples 1, 3, 5 to 8 and Comparative Examples 5, 6, 8, and 9 were diluted 10-fold, placed in 1 mm cells, and left at room temperature (25°C). After 1 day, 3 days, and 7 days, the UV-visible absorption spectra were measured. The long-term stability was evaluated from the change in absorbance at each absorption maximum (Abs / Abs0, where Abs0 is the initial value).

[0045] The absorption maxima were 260 nm for the fullerene-cyclodextrin composition (Comparative Example 5), 264 nm for the fullerene-pullulan composition (Comparative Example 6), 262 nm for the fullerene-gelatin composition (Example 1), 261 nm for the fullerene-poly-γ-glutamic acid composition (Example 5), 262 nm for the fullerene-α-poly-L-lysine composition (Example 6), 416 nm for the tetraphenylporphyrin-cyclodextrin composition (Comparative Example 8), 431 nm for the tetraphenylporphyrin-pullulan composition (Comparative Example 9), 423 nm for the tetraphenylporphyrin-gelatin composition (Example 3), 429 nm for the tetraphenylporphyrin-poly-γ-glutamic acid composition (Example 7), and 420 nm for the tetraphenylporphyrin-α-poly-L-lysine composition (Example 8). The results of Examples 1, 5, and 6 and Comparative Examples 5 and 6 are shown in FIG. 5, and the results of Examples 3, 7, and 8 and Comparative Examples 8 and 9 are shown in FIG.

[0046] As shown in Figure 5, none of the fullerene solubilized compositions showed a significant loss of stability. However, as shown in Figure 6, in the tetraphenylporphyrin solubilized compositions, the absorbance of the tetraphenylporphyrin-cyclodextrin composition (Comparative Example 8) fell to less than half of the initial value after one day, and visually visible precipitation was observed.

[0047] (Evaluation of thermal stability) Next, thermal stability was evaluated. The solubilized compositions obtained as supernatants in Examples 1, 3, 5 to 8 and Comparative Examples 5, 6, 8, and 9 were diluted 10-fold and placed in 1 mm cells. After heating at 80°C for 2 hours, the UV-visible absorption spectrum of each solubilized composition was measured at room temperature. Long-term stability was evaluated from the absorbance change at each absorption maximum (Abs / Abs0, Abs0 is the initial value). Note that each absorption maximum from which the absorbance change was determined was the same as that observed in the long-term stability evaluation described above. The results of the thermal stability evaluation are shown in Figure 7.

[0048] As shown in FIG. 7, in Comparative Examples 5 and 8, in which cyclodextrin was used as a solubilizer, the absorbance was significantly reduced, indicating that the thermal stability was low.

[0049] The results of the solubility evaluation, long-term stability evaluation, and thermal stability evaluation for Examples 1, 3, and 5 to 8 and Comparative Examples 5 to 10 are shown in Table 1.

[0050] [Table 1]

[0051] In Table 1, the solubility was evaluated based on the absorbance of the UV-visible absorption spectrum: ◎ indicates very good solubility, ◯ indicates some solubility, △ indicates slight solubility, and × indicates no solubility. Those marked with - had low solubility, and therefore were not evaluated for long-term stability or thermal stability. Note that the UV-visible absorption spectrum showed broadening of peaks due to association of poorly water-soluble substances caused by the solubilizer, so the solubility was determined by the peak area.

[0052] In addition, in Table 1, the evaluation of long-term stability and thermal stability was carried out as follows: ◎ if it could be said that 90% or more of the poorly water-soluble substance remained in the aqueous solution based on the peak intensity; 〇 if it could be said that 80-89% remained; △ if it could be said that 50-79% remained; and × if it could be said that less than 50% remained.

[0053] According to Table 1, Examples 1, 3, and 5 to 8, which used gelatin, poly-γ-glutamic acid, and α-poly-L-lysine as solubilizers, showed good solubility, long-term stability, and thermal stability, and relatively better results than Comparative Examples 5 to 10.

[0054] Next, the high-speed vibration grinding method was used to prepare solubilized compositions of various other poorly water-soluble substances, and the solubility was compared between cases where polypeptides were used as solubilizing agents and cases where conventionally used polysaccharides were used.

[0055] (Synthesis of solubilized fullerene derivative composition) Examples 9 to 11 and Comparative Examples 11 to 13 A poorly water-soluble fullerene derivative, C 60 -N,N-dimethylpyrrolidinium iodide (C in the figure and table) 601.8 mg (2.0 μmol) of PEG-1 (hereinafter referred to as "PEG-1") was ground and mixed with each solubilizing agent by high-speed vibration grinding, and solubilized compositions were synthesized using the same procedure as in Example 1. As solubilizing agents, 10.0 mg of gelatin was used in Example 9, 10.0 mg of poly-γ-glutamic acid in Example 10, 10.0 mg of α-poly-L-lysine in Example 11, 10.0 mg of γ-cyclodextrin in Comparative Example 11, 10.0 mg of pullulan in Comparative Example 12, and collagen in Comparative Example 13 were used.

[0056] The solubilized compositions obtained as supernatants in Examples 9 to 11 and Comparative Examples 11 to 13 were diluted 10-fold, placed in 1 mm cells, and measured using an ultraviolet-visible spectrophotometer (Shimadzu, UV-3600) in the same manner as above. The measurement results are shown in Figure 8.

[0057] (Synthesis of solubilized phthalocyanine composition) Examples 12 to 14 and Comparative Examples 14 to 16 1.0 mg (2.0 μmol) of phthalocyanine (referred to as Pc in the figures and tables), a poorly water-soluble substance, was ground and mixed with each solubilizing agent by high-speed vibration grinding, and solubilized compositions were synthesized using the same procedure as in Example 1. The solubilizing agents used were 10.0 mg of gelatin in Example 12, 10.0 mg of poly-γ-glutamic acid in Example 13, 10.0 mg of α-poly-L-lysine in Example 14, 10.0 mg of trimethyl-β-cyclodextrin in Comparative Example 14, 10.0 mg of pullulan in Comparative Example 15, and collagen in Comparative Example 16.

[0058] The solubilized compositions obtained as supernatants in Examples 12 to 14 and Comparative Examples 14 to 16 were diluted 10-fold, placed in 1 mm cells, and measured using an ultraviolet-visible spectrophotometer (Shimadzu, UV-3600) in the same manner as above. The measurement results are shown in Figure 9.

[0059] (Synthesis of Solubilized Oligothiophene Compositions) Examples 15 to 16 and Comparative Examples 17 to 19 2.1 mg (5.0 μmol) of quinchithiophene (referred to as 5T in the figures and tables), a type of oligothiophene that is a poorly water-soluble substance, was ground and mixed with each solubilizer by high-speed vibration grinding, and solubilized compositions were synthesized using the same procedure as in Example 1. The solubilizers used were 10.0 mg of gelatin in Example 15, 10.0 mg of poly-γ-glutamic acid in Example 16, 10.0 mg of α-poly-L-lysine in Example 17, 10.0 mg of trimethyl-β-cyclodextrin in Comparative Example 17, 10.0 mg of pullulan in Comparative Example 18, and collagen in Comparative Example 19.

[0060] The solubilized compositions obtained as the supernatants in Examples 15 to 17 and Comparative Examples 17 to 19 were placed in 1 mm cells and measured using an ultraviolet-visible spectrophotometer (Shimadzu, UV-3600) in the same manner as above. The measurement results are shown in Figure 10. Quinchithiophene was almost insoluble when cyclodextrin, pullulan, or collagen was used as the solubilizer, but showed good solubility when gelatin, polyglutamic acid, and polylysine were used as the solubilizer.

[0061] (Synthesis of carotenoid solubilized composition) Examples 18 to 20 and Comparative Examples 20 to 22 1.1 mg (2.0 μmol) of β-carotene (referred to as β-C in the figures and tables), a type of carotenoid that is a poorly water-soluble substance, was ground and mixed with each solubilizing agent by high-speed vibration grinding, and solubilized compositions were synthesized using the same procedure as in Example 1. The solubilizing agents used were 10.0 mg of gelatin in Example 18, 10.0 mg of poly-γ-glutamic acid in Example 19, 10.0 mg of α-poly-L-lysine in Example 20, 10.0 mg of γ-cyclodextrin in Comparative Example 20, 10.0 mg of pullulan in Comparative Example 21, and collagen in Comparative Example 22.

[0062] The solubilized compositions obtained as the supernatants in Examples 18 to 20 and Comparative Examples 20 to 22 were placed in 1 mm cells and measured using an ultraviolet-visible spectrophotometer (Shimadzu, UV-3600) in the same manner as above. The measurement results are shown in Figure 11. β-carotene was almost insoluble when cyclodextrin or collagen was used as the solubilizing agent, but showed good solubility when gelatin, polyglutamic acid, and polylysine were used as the solubilizing agent.

[0063] (Synthesis of solubilized carbamazepine composition) Examples 21 to 23 and Comparative Examples 23 to 25 1.2 mg (5.0 μmol) of carbamazepine (referred to as CBZ in the figures and tables), a poorly water-soluble substance, was ground and mixed with each solubilizing agent by high-speed vibration grinding, and solubilized compositions were synthesized using the same procedure as in Example 1. As solubilizing agents, 10.0 mg of gelatin was used in Example 21, 10.0 mg of poly-γ-glutamic acid in Example 22, 10.0 mg of α-poly-L-lysine in Example 23, 10.0 mg of trimethyl-β-cyclodextrin in Comparative Example 23, 10.0 mg of pullulan in Comparative Example 24, and collagen in Comparative Example 25 were used.

[0064] The solubilized compositions obtained as supernatants in Examples 21 to 23 and Comparative Examples 23 to 25 were diluted 10-fold, placed in 1 mm cells, and measured using an ultraviolet-visible spectrophotometer (Shimadzu, UV-3600) in the same manner as above. The measurement results are shown in Figure 12.

[0065] (Synthesis of solubilized paclitaxel composition) Examples 24 to 26 and Comparative Examples 26 to 28 1.7 mg (2.0 μmol) of the poorly water-soluble substance paclitaxel (referred to as PTX in the figures and tables) was ground and mixed with each solubilizing agent by high-speed vibration grinding, and solubilized compositions were synthesized using the same procedure as in Example 1. The solubilizing agents used were 10.0 mg of gelatin in Example 24, 10.0 mg of poly-γ-glutamic acid in Example 25, 10.0 mg of α-poly-L-lysine in Example 26, 10.0 mg of trimethyl-β-cyclodextrin in Comparative Example 26, 10.0 mg of pullulan in Comparative Example 27, and collagen in Comparative Example 28.

[0066] In Comparative Examples 26 and 27, the solubilized compositions obtained as supernatants were directly placed in 1 mm cells for measurement, but in Examples 24 to 26 and Comparative Example 28, the absorption spectra of paclitaxel and the solubilizer overlapped in the aqueous solution, making it difficult to confirm the results. Therefore, the aqueous solutions were lyophilized, and paclitaxel was extracted with ethanol. This was then placed in a 1 mm cell and measured using a UV-visible spectrophotometer (Shimadzu, UV-3600) in the same manner as above. The measurement results are shown in Figure 13.

[0067] (Synthesis of solubilized curcumin composition) Examples 27 to 29 and Comparative Examples 29 to 31 1.8 mg (5.0 μmol) of curcumin (referred to as Cur in the figures and tables), a poorly water-soluble substance, was ground and mixed with each solubilizing agent by high-speed vibration grinding, and solubilized compositions were synthesized using the same procedure as in Example 1. The solubilizing agents used were 10.0 mg of gelatin in Example 27, 10.0 mg of poly-γ-glutamic acid in Example 28, 10.0 mg of α-poly-L-lysine in Example 29, 10.0 mg of trimethyl-β-cyclodextrin in Comparative Example 29, 10.0 mg of pullulan in Comparative Example 30, and collagen in Comparative Example 31.

[0068] The solubilized compositions obtained as supernatants in Examples 27 to 29 and Comparative Examples 29 to 31 were diluted 10-fold, placed in 1 mm cells, and measured using an ultraviolet-visible spectrophotometer (Shimadzu, UV-3600) in the same manner as above. The measurement results are shown in Figure 14.

[0069] (Synthesis of solubilized resveratrol composition) Examples 30 to 32 and Comparative Examples 32 to 34 1.1 mg (5.0 μmol) of curcumin (referred to as Res in the figures and tables), a poorly water-soluble substance, was ground and mixed with each solubilizing agent by high-speed vibration grinding, and solubilized compositions were synthesized using the same procedure as in Example 1. The solubilizing agents used were 10.0 mg of gelatin in Example 30, 10.0 mg of poly-γ-glutamic acid in Example 31, 10.0 mg of α-poly-L-lysine in Example 32, 10.0 mg of trimethyl-β-cyclodextrin in Comparative Example 32, 10.0 mg of pullulan in Comparative Example 33, and collagen in Comparative Example 34.

[0070] The solubilized compositions obtained as supernatants in Examples 30 to 32 and Comparative Examples 32 to 34 were diluted 10-fold, placed in 1 mm cells, and measured using an ultraviolet-visible spectrophotometer (Shimadzu, UV-3600) in the same manner as above. The measurement results are shown in Figure 15.

[0071] Table 2 shows the evaluation results of solubility in Examples 2, 4, and 9 to 32 and Comparative Examples 1 to 4 and 11 to 34. In Table 2, as in Table 1, the absorbance of the ultraviolet-visible absorption spectrum indicates that the sample is very soluble, ◯ indicates that the sample is soluble to some extent, △ indicates that the sample is soluble to some extent, and × indicates that the sample is not soluble at all.

[0072] [Table 2]

[0073] As shown in Tables 1 and 2, when gelatin, poly-γ-glutamic acid, and α-poly-L-lysine were used as solubilizers for tetraphenylporphyrin, fullerene derivatives, carbamazepine, paclitaxel, curcumin, and resveratrol, and solubilized compositions were obtained by high-speed vibration milling. These compositions exhibited solubility equivalent to that of conventional solubilizers such as pullulan and cyclodextrin. Furthermore, when gelatin, poly-γ-glutamic acid, and α-poly-L-lysine were used as solubilizers, high solubility was observed for fullerene, phthalocyanine, quinquethiophene, β-carotene, and carbamazepine, which exhibited low solubility when pullulan or cyclodextrin was used.

[0074] Gelatin, poly-γ-glutamic acid, and α-poly-L-lysine can be milled and mixed with various types of poorly water-soluble substances using high-speed vibration milling to obtain solubilized compositions. These compositions are relatively more soluble and versatile than the sugars traditionally used as solubilizers. Furthermore, when solubilized compositions using gelatin, poly-γ-glutamic acid, and α-poly-L-lysine as solubilizers were obtained using high-speed vibration milling, they demonstrated superior long-term and thermal stability compared to compositions using cyclodextrin. Because gelatin, poly-γ-glutamic acid, and α-poly-L-lysine are naturally derived, they are highly biocompatible and have a low environmental impact. These results suggest that gelatin, poly-γ-glutamic acid, and α-poly-L-lysine are useful solubilizers.

[0075] Collagen, which is the same polypeptide as gelatin, poly-γ-glutamic acid, and α-poly-L-lysine, exhibited extremely high solubility as a solubilized composition with carbamazepine, and is therefore useful as a solubilizing agent for carbamazepine.

[0076] (Measurement of average particle size) Particle size was determined by dynamic light scattering. The measurement device used was a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). The hydrodynamic diameter (Dhy) and polydispersity (PDI) are summarized in Table 3. The results in Tables 1 to 3 suggest that the solubilization effect is high when the average particle size of dispersed particles of poorly water-soluble substances ground and mixed using high-speed vibration grinding and a mortar and pestle is 50 to 1500 nm, preferably 80 to 600 nm.

[0077] [Table 3]

Claims

1. a step of grinding and mixing a poorly water-soluble substance having a solubility in water at 25°C of 0.1 g / L or less and a solubilizer which is at least one selected from gelatin, polyglutamic acid, and polylysine using a ball mill or a mortar and pestle to obtain a ground mixture; and a step of adding water to the pulverized mixture to obtain an aqueous solution of the pulverized mixture.

2. 2. The method for solubilizing a poorly water-soluble substance according to claim 1, wherein the poorly water-soluble substance is at least one selected from the group consisting of porphyrin derivatives, phthalocyanine derivatives, fullerene derivatives, polyheterocyclic compounds, carotenoids, carbamazepine, paclitaxel, and polyphenols.

3. The composition contains a poorly water-soluble substance having a solubility in water of 0.1 g / L or less at 25°C, a solubilizing agent, and water, the solubilizing agent is at least one selected from gelatin, polyglutamic acid, and polylysine; A solubilized composition for a poorly water-soluble substance, characterized in that the dispersed particles of the poorly water-soluble substance have an average particle size of 50 to 1500 nm.

4. 4. The solubilized composition for a poorly water-soluble substance according to claim 3, wherein the dispersed particles of the poorly water-soluble substance have an average particle size of 80 to 600 nm.

5. The solubilized composition for a poorly water-soluble substance according to claim 3 or 4, characterized in that the poorly water-soluble substance is at least one selected from the group consisting of porphyrin derivatives, phthalocyanine derivatives, fullerene derivatives, polyheterocyclic compounds, carotenoids, carbamazepine, paclitaxel, and polyphenols.

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