JP5714311B2 - Method for imparting water solubility while maintaining pharmacological activity of poorly water-soluble pharmacologically active substance - Google Patents

Description

  The present invention relates to a water-soluble complex containing a poorly water-soluble pharmacologically active substance, a method for producing the same, and a method for imparting water solubility while maintaining the pharmacological activity of the poorly water-soluble pharmacologically active substance.

Even though it has excellent pharmacological activity, it is hardly soluble in water, and therefore there are many substances that are difficult to form into pharmaceutical preparations.
For example, 1'-acetoxychavicol acetate (ACA), which is one of the chevicol compounds, is a rhizome of the ginger family such as Languas galanga and Alpinia galanga, which is used as a substitute for stomach medicine, analgesics and ginger in southern China and Southeast Asia. It is a physiologically active substance having various effects, such as antitumor (Non-patent Document 1), anti-inflammatory (Non-patent Document 2), and collagen production promotion (Patent Document 1). In previous studies, ACA not only activates two apoptosis-inducing pathways via Fas and mitochondria (Non-Patent Document 3), but also has an inhibitory effect on the activation of NF-κB, a nuclear translocation transcription factor. It has been reported (Non-Patent Document 4).
However, ACA is poorly water-soluble and is usually used as a DMSO or EtOH solution in in vitro experiments. When a poorly water-soluble compound is used in an in vivo experiment, it is used with castor oil or a derivative thereof. However, it is necessary to consider toxicity due to these solvents. For this reason, water solubilization is a problem for use as a drug, but there has been no report of successful solubilization of ACA in water while maintaining pharmacological activity.

Curcumin is a curcuminoid yellow pigment component that is a kind of polyphenol contained in turmeric and turmeric, and has an antioxidant action and an anti-inflammatory action, and thus has been used in foods and drinks, health foods and the like. In addition, recent studies have revealed that curcumin has a protective ability (amyloid formation inhibitory ability) of nervous system damage in Alzheimer's disease (Non-patent Document 5). Furthermore, curcumin has been revealed to have specific physiological activities such as the ability to induce apoptosis of cancer cells, and has attracted attention for its antitumor action and anticancer activity (Patent Document 2, Non-Patent Document 6). Thus, the physiological activity and medical usefulness of curcumin have been actively studied.
However, since curcumin is sparingly water-soluble, it is extracted mainly from raw material turmeric and the like with an organic solvent. However, curcumin obtained by this method requires removal of an organic solvent and the like, and cannot be dissolved in water, so that it is difficult to use. In addition, curcumin does not dissolve in water, so even if it is taken as it is, it is not absorbed much in the intestinal tract and is excreted outside the body.

Ubiquinone such as coenzyme Q10 is one of the electron mediators present in the inner mitochondrial membrane and prokaryotic cell membrane, and is a benzoquinone derivative that mediates the electrons of respiratory chain complexes I and III in the electron transport system. is there. Ubiquinone has a relatively long isoprene side chain and is retained in the membrane due to its hydrophobicity. It is known that the antioxidant action of ubiquinone is effective in preventing senile deafness in mice (Non-patent Document 7). Thus, ubiquinone has been used in foods and drinks, health foods and the like because it has an antioxidant effect.
However, ubiquinone such as coenzyme Q10 is poorly water-soluble and is produced by fermentation. However, coenzyme Q10 obtained by this method cannot be dissolved in water and has a problem that it is difficult to use. In addition, since coenzyme Q10 does not dissolve in water, even if it is ingested as it is, it is not absorbed much in the intestinal tract and is excreted outside the body. Therefore, a method for increasing the absorption level and bioavailability level of coenzyme Q10 in plasma by using coenzyme Q10 as a soft gel capsule containing a solvent and carrier oil is described (Patent Document 3). However, since the method described above uses fats and oils, it is necessary to consider the toxicity and the like due to these fats and oils.

Vitamin D (vitamin D) is a kind of vitamin and is classified as a fat-soluble vitamin. Vitamin D is divided into vitamin D2 (ergocalciferol, Ergocalciferol) and vitamin D3 (cholecalciferol, Cholecalciferol). Vitamin D2 is abundant in plants and vitamin D3 is abundant in animals, and vitamin D3 plays an important role in humans. Vitamin D acts as an active vitamin D (calcitriol or 1,25-dihydroxycholecalciferol) to increase the calcium (Ca ²⁺ ) concentration in blood (Non-patent Document 8). Vitamin D has also been suggested to be involved in immune reactions (Non-Patent Document 9).
However, vitamin D has poor water solubility and is difficult to use. In addition, since vitamin D3 does not dissolve in water, even if it is taken as it is, it is not absorbed much in the intestinal tract and is excreted outside the body. Therefore, a method of solubilizing a composition containing a lipophilic drug (vitamin D), a nonionic solubilizer, and a lipophilic antioxidant in an aqueous solvent is described (Patent Document 4). However, in the above-described method, a nonionic solubilizer or a lipophilic antioxidant is used, so that there is a risk of inhibiting the original pharmacological activity of vitamin D.

Paclitaxel (Taxol) is a kind of anticancer agent and is a substance isolated from the bark of Pacific yew (Taxus brevifolia). By binding to microtubules and stabilizing to inhibit depolymerization, tumor cell division is inhibited (Non-patent Document 10).
However, paclitaxel (taxol) is sparingly water-soluble, and is currently used as an anticancer agent by an ethanol solution or a water-solubilizing technique such as a liposomal agent, and there are limitations on the administration method.

JP 2009-079004 JP 2009-195198 JP 2007-297395 A Special Table 2006-502185

Cancer Res. 2005, 65, 4417. Bioorg. Med. Chem. Lett. 2003, 13, 3197. Clin. Cancer Res. 2004, 10, 2120. J. Immunol. 2005, 174, 7383. J. Biol. Chem. 2005, 280, 5892. Bioorg. Med. Chem. Lett. 2003, 13, 3197. Proc. Natl. Acad. Sci. USA. 2009, 106, 19432. Voet, Donald; Voet, Judith G. (2004). Biochemistry. Volume one. Biomolecules, mechanisms of enzyme action, and metabolism, 3rd edition, pp. 663-664. New York: John Wiley & Sons. Am. J. Clin. Nutr. 2004, 80, 1717S. J. Natl. Med. Assoc. 1993, 85, 427.

  The present invention provides a poorly water-soluble pharmacologically active substance complex that has practically sufficient water solubility and maintains pharmacological activity, and has practically sufficient water solubility without impairing the pharmacological activity of the poorly water soluble pharmacologically active substance. It is an object to provide a method that can be applied to an active substance.

The inventors of the present invention have made extensive studies to solve the above problems, and among β-1,3-1,6-D-glucans, the β-1 of the side chain with respect to the β-1,3 bond of the main chain is particularly important. , 6 bond ratio (branching degree) is 50 to 100%, and the complex with β-1,3-1,6-D-glucan significantly improves the water solubility of poorly water-soluble pharmacologically active substances. I found that I can do it. Surprisingly, the present inventors have found that the pharmacological activity of the poorly water-soluble pharmacologically active substance is maintained in spite of being a complex.
The present invention has been completed based on these findings, and provides the following poorly water-soluble pharmacologically active substance complex, its production method, and a method for imparting water solubility while maintaining the pharmacological activity of the poorly water-soluble pharmacologically active substance. To do.

Item 1. A complex of a poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan having a degree of branching of β-1,6 bonds with respect to β-1,3 bonds of 50 to 100%.
Item 2. Item 2. The complex according to Item 1, wherein the poorly water-soluble pharmacologically active substance is a substance having a molecular weight of 100 to 3,000.
Item 3. A poorly water-soluble pharmacologically active substance is represented by the following general formula (1)
(In the formula, the 2′-3 ′ bond is a single bond or a double bond. R ¹ , R ² , R ³ and R ⁴ may be the same or different and each represents a hydrogen atom, a methoxy group, or an RCOO— group (R is An alkyl group having 1 to 4 carbon atoms).
Item 2. The complex according to Item 1, which is a compound represented by:
Item 4. Item 2. The complex according to Item 1, wherein the poorly water-soluble pharmacologically active substance is curcumin.
Item 5. The poorly water-soluble pharmacologically active substance is represented by the following general formula (2)
[Wherein n represents an integer of 6 to 10. ]
Item 2. The complex according to Item 1, which is a ubiquinone represented by:
Item 6. Item 2. The complex according to Item 1, wherein the poorly water-soluble pharmacologically active substance is vitamin D.
Item 7. Item 2. The complex according to Item 1, wherein the poorly water-soluble pharmacologically active substance is paclitaxel (taxol).
Item 8. A poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan having a degree of branching of β-1,6 bonds to β-1,3 bonds of 50 to 100% remain in a solid state. A method of imparting water solubility while maintaining the pharmacological activity of a poorly water-soluble pharmacologically active substance, comprising a step of stirring and a step of adding water to the mixture and stirring.
Item 9. A poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan having a degree of branching of β-1,6 bonds to β-1,3 bonds of 50 to 100% are mixed in a polar solvent. A method of imparting water solubility while maintaining the pharmacological activity of a poorly water-soluble pharmacologically active substance, comprising a step of adding water to the resulting mixture and aging.
Item 10. A poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan having a degree of branching of β-1,6 bonds to β-1,3 bonds of 50 to 100% remain in a solid state. A method for producing a complex of a poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan, comprising a stirring step and a step of adding water to the mixture and stirring the mixture.
Item 11. A poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan having a degree of branching of β-1,6 bonds to β-1,3 bonds of 50 to 100% are mixed in a polar solvent. And a method of producing a complex of a poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan, which comprises a step of adding water to the resulting mixture and aging.

  According to the present invention, water solubility can be imparted to this substance without impairing the pharmacological activity of the poorly water-soluble pharmacologically active substance. This made it possible to formulate a poorly water-soluble pharmacologically active substance and absorb it in the living body. In addition, since the poorly water-soluble pharmacologically active substance is water-solubilized using β-1,3-1,6-D-glucan, which is also used as a food ingredient, the complex of the present invention has high safety, It can be used as health food, functional food, cosmetics, medicine, and the like.

It is a ¹ H NMR spectrum of glucan derived from Aureobasidium pullulans obtained in an experimental example. It is a figure which shows the particle size distribution of a culture solution when ultrasonically irradiating the glucan derived from an aureobasidium pullulans obtained in the experiment example. It is a figure which shows (beta) -1,3-1,6-D-glucan / ACA complex composition ratio computed from < ¹ > H NMR. It is a figure which shows the cytotoxicity with respect to RAW264.7 cell of (beta) -1,3-1,6-D-glucan and its ACA complex. The vertical axis of the graph represents the cell viability, and the concentration in the figure represents the ACA concentration. It is a figure which shows the inhibitory effect of the ACA complex with respect to IκBα degradation after LPS stimulation in macrophage cells. It is the microscope observation figure of a human origin fibroblast, and a type I collagen dyeing | staining image figure. It is an absorption spectrum of curcumin water-solubilized with β-1,3-1,6-D-glucan (aqua β). It is an absorption spectrum of coenzyme Q10 water-solubilized with β-1,3-1,6-D-glucan (aqua β). It is an absorption spectrum of vitamin D3 water-solubilized with β-1,3-1,6-D-glucan (aqua β). It is an absorption spectrum of paclitaxel (taxol) solubilized in water by β-1,3-1,6-D-glucan (aqua β).

Hereinafter, the present invention will be described in detail.
(1) Complex The complex of the present invention has a poorly water-soluble pharmacologically active substance and the ratio (branch degree) of β-1,6 bonds in the side chain to β-1,3 bonds in the main chain is 50 to 100%. It is a complex with a certain β-1,3-1,6-D-glucan.

(1-1) A poorly water-soluble pharmacologically active substance The poorly water-soluble pharmacologically active substance means a pharmacologically active substance having a solubility in water of 10 mg / ml or less. For example, a drug whose solubility at room temperature (15 to 25 ° C.) specified in the 15th revised Japanese Pharmacopeia, general rules is “very insoluble” and “almost insoluble” corresponds to a poorly water-soluble pharmacologically active substance To do.
In the present invention, the kind of poorly water-soluble pharmacologically active substance is not particularly limited.
Typically, the following general formula (1)
(In the formula, the 2′-3 ′ bond is a single bond or a double bond. R ¹ , R ² , R ³ and R ⁴ may be the same or different and each represents a hydrogen atom, a methoxy group, or an RCOO— group (R is An alkyl group having 1 to 4 carbon atoms).
The compound represented by these is mentioned.

Examples of the compound represented by the general formula (1) include 1′-acetoxychabicol acetate (2′-3 ′: C═C, R ¹ = R ⁴ = CH ₃ COO—, R ² = R ³ = H -, hereinafter referred to as ACA), dihydro-1'Acetoxychavicol acetate (2'-3 ': C- C, R 1 = R 4 = CH 3 COO-, R 2 = R 3 = H -), 1 '-Acetoxyeugenol acetate (2'-3': C = C, R ¹ = R ⁴ = CH ₃ COO-, R ² = CH ₃ O-, R ³ = H-), 1 ', 2-diacetoxy chabi Cole acetate (2′-3 ′: C═C, R ¹ = R ³ = R ⁴ = CH ₃ COO—, R ² = H—) and the like are included. Among them, ACA is preferable because it has strong pharmacological activity and is useful as a medicine. The molecular weight of ACA is 234.
Further, the compound represented by the general formula (1) includes optical isomers, for example, racemates, R isomers, and S isomers.

Curcumin is also a suitable poorly water-soluble pharmacologically active substance. The structural formula of curcumin is shown below.
Curcumin can be used from any organism. For example, in addition to turmeric (Curcuma longa L.), those derived from plants such as mango schiger, Indian arrow root, gadgets, yellow gadgets, black gadgets, galan girls and the like can be exemplified. The molecular weight of curcumin is 368.38.

Ubiquinone is also a suitable poorly water-soluble pharmacologically active substance. The structural formula of ubiquinone is shown below.
[Wherein n represents an integer of 6 to 10. ]

The molecular weight of the ubiquinone represented by the general formula (2) is 599 to 863. Among the ubiquinones represented by the general formula (2), those having n = 10 (coenzyme Q10) are preferable. Coenzyme Q10 used in the present invention may be extracted from the heart of an animal such as a cow, or may be obtained by a synthesis method or a fermentation method. Commercial products can also be purchased. Commercially available products include food ingredients Coenzyme Q10 (trade name, manufactured by Nisshin Pharma Co., Ltd.), Kaneka Coenzyme Q10 (trade name, manufactured by Kaneka Chemical Co., Ltd.), CoEnzyme Q10 (trade name, manufactured by Asahi Kasei Co., Ltd.) BioQ ₁₀ (manufactured by Mitsubishi Gas Chemical Company), coenzyme Q10 manufactured by Wako Pure Chemical Industries, Ltd., and the like. Although the purity of ubiquinone is not limited, since the complex of the present invention is applied to medicines, supplements, beverages, foods, and cosmetics, it is desirable to use a compound having high purity according to the use.

Vitamin D is also a suitable poorly water-soluble pharmacologically active substance. Examples of vitamin D include ergocassipherol (vitamin D2) and cholecalciferol (vitamin D3). Ergociferol has a molecular weight of 396.65 and cholecalciferol has a molecular weight of 384.64. Among vitamin D, vitamin D3 represented by the following formula (3) is preferable.

Paclitaxel (Taxol) is also a suitable poorly water-soluble pharmacologically active substance. The structure of paclitaxel is shown in the following formula (4). The molecular weight of paclitaxel is 853.9.

Other poorly water-soluble pharmacologically active substances include fat-soluble vitamins and coenzymes, chemotherapeutic drugs; antifungal drugs; oral contraceptives, nicotine or nicotine replacement drugs, minerals, analgesics, antacids, muscle relaxants, antihistamines , Decongestants, anesthetics, antitussives, diuretics, anti-inflammatory drugs, antibiotics, antiviral drugs, psychotherapeutic drugs, antidiabetic and cardiovascular drugs, dietary supplements and nutritional supplements, etc. it can.
The fat-soluble vitamin and coenzyme are not particularly limited, and examples thereof include nicotinic acid, pyridoxine, choline, paraaminobenzoic acid, vitamin A and carotenoid, retinoic acid, vitamin E, vitamin K and the like. Examples of carotenoids include carotenes such as α-carotene, β-carotene, γ-carotene, δ-carotene and lycopene; xanthophylls such as lutein, zeaxanthin, canthaxanthin, fucoxanthin, anthraxanthin and violaxanthin.
Of the poorly water-soluble pharmacologically active substances exemplified above, plant-based substances are preferable. For example, polyphenols (such as grape seed extraction polyphenols), isoflavones (such as soybean isoflavones), resveratrol, epigenin, aloe vera, echinacea, chamomile, Hammelis Extracts, anti-inflammatory plant extracts such as Fever Hupartenolide; anti-psoriatic drugs such as Chinese zizpus jujuba; astringents such as Hammelis; antibacterial drugs such as Artemisia, Chamomile and Golden Seal; such as Echinacea Examples include immunomodulators; anti-aging drugs; anticancer drugs such as docetaxel and camptothecin; anti-photopathic drugs;
Cilostazol, rebamipide, aripiprazole, cyclosporine, nifedipine, ceramide and the like can also be mentioned.

  In a complex with β-1,3-1,6-D-glucan in which the ratio (branch degree) of β-1,6 bonds in the side chain to β-1,3 bonds in the main chain is 50 to 100% The size of the poorly water-soluble pharmacologically active substance may influence whether or not the water-solubilization can be achieved. Molecular weight is one indicator of the size of a substance. In the present invention, the molecular weight of the poorly water-soluble pharmacologically active substance is preferably about 100 to 3,000, more preferably about 150 to 2,000, and even more preferably about 200 to 1,000.

(1-2) β-1,3-1,6-D-glucan In β-1,3-1,6-D-glucan used in the present invention, the side chain for β-1,3 bonds of the main chain The degree of branching, which is the ratio of β-1,6 bonds, is usually about 50 to 100%, preferably about 75 to 100%, more preferably about 85 to 100%.
β-1,3-1,6-D-glucan has the above-mentioned degree of branching because β-1,3-1,6-D-glucan is converted into exo-type β-1,3-glucanase (Cytalase M, It can be confirmed from the release of glucose and gentiobiose as degradation products when hydrolyzed by KAI Kasei Co., Ltd., and the integration ratio of NMR (supervised by Tadayuki Imanaka, development of the use of microorganisms, 1012-1015, NT)・ S (2002).

(a) β-1,3-1,6-D-glucan produced by microorganisms belonging to the genus Aureobasidium β-1,3-1,6-D-glucan having the above - mentioned degree of branching is derived from the genus Aureobasidium ( It can be obtained from microorganisms belonging to Aureobasidium sp.).
Β-1,3-1,6-D-glucan derived from microorganisms belonging to the genus Aureobasidium sp. Has a ¹ H NMR spectrum of about 4.7 ppm in a solution containing 1N sodium hydroxide heavy aqueous solution as a solvent. And two signals of about 4.5 ppm. Since it is well known that NMR measurement values change due to subtle changes in conditions and are accompanied by errors, “about 4.7 ppm” and “about 4.5 ppm” are measured values within the normally expected range. A numerical value including a fluctuation range (for example, ± 0.2).
The β-1,3-1,6-D-glucan having the above-mentioned degree of branching preferably has a viscosity of an aqueous solution at 30 ° C., pH 5.0, and concentration 0.5 (w / v%), preferably 200 cP (mPa · m). s) or less, more preferably 100 cP (mPa · s) or less, and still more preferably 50 cP (mPa · s) or less. The lower limit of the viscosity is usually about 10 cP (mPa · s).
In the present invention, the viscosity is a value measured using a BM type rotational viscometer.
Since β-1,3-1,6-D-glucan produced by microorganisms of the genus Aureobasidium is secreted outside the fungus body, compared to β-glucan contained in the cell walls of mushrooms and baker's yeast, It is preferable in that it can be easily recovered and is water-soluble. Aureobasidium microorganisms can produce high-molecular-weight glucans having a molecular weight of 1 million or more to low-molecular-weight glucans having a molecular weight of about tens of thousands according to the culture conditions.

Among them, those produced by Aureobasidium pullulans are preferable, and Aureobasidium pullulans GM-NH-1A1 strain or GM-NH-1A2 strain (incorporated administrative agency National Institute of Industrial Science and Technology patent biological deposit center) Those produced by FERM P-19285 and FERM P-19286, respectively) are preferred. The GM-NH-1A1 and GM-NH-1A2 strains are mutants of the Aureobasidium sp. K-1 strain. The Aureobasidium genus K-1 strain is known to produce two types of β-1,3-1,6-D-glucan having a molecular weight of 2 million or more and about 1 million.
In addition, β-1,3-1,6-D-glucan produced by an Aureobasidium microorganism usually has a sulfur-containing group, whereas β-glucan produced by the K-1 strain has a sulfoacetic acid group. (Arg. Biol. Chem., 47, 1167-1172 (1983)), science and industry, 64, 131-135 (1990)). It is considered that β-1,3-1,6-D-glucan produced by the GM-NH-1A1 strain and the GM-NH-1A2 strain also has a sulfoacetic acid group. Among microorganisms belonging to the genus Aureobasidium, there are also species and strains that produce β-1,3-1,6-D-glucan containing a phosphorus-containing group such as a phosphate group, a malate group, and the like.

The GM-NH-1A1 strain and the GM-NH-1A2 strain have a high molecular weight β-glucan (fine particle glucan) having an apparent main peak of 500 to 2.5 million and an apparent peak of 2 to 2, as will be described later in Examples. It is a strain that produces both 300,000 low molecular weight β-glucan. The fine particle glucan has a primary particle diameter of about 0.05 to 2 μm.
The solubility of β-1,3-1,6-D-glucan depends on pH and temperature. This β-1,3-1,6-D-glucan has a primary particle diameter of 0.05 to 2 μm when it is intended to prepare a 2 mg / ml aqueous solution at pH 3.5 and a temperature of 25 ° C. Fine particles are formed, and the remainder is dissolved in water. In the present invention, the particle diameter is a value measured by a laser diffraction scattering method.

β-1,3-1,6-D-glucan has a viscosity lower than that of natural β-1,3-1,6-D-glucan produced by microorganisms belonging to the genus Aureobasidium preferable. This low-viscosity β-1,3-1,6-D-glucan has an aqueous solution with a viscosity of usually 200 cP (mPa · s) or less at 30 ° C., pH 5.0 and a concentration of 0.5 (w / v%). More preferably 100 cP (mPa · s) or less, still more preferably 50 cP (mPa · s) or less, and even more preferably 10 cP or less.
This low-viscosity glucan may have the same primary structure as natural β-1,3-1,6-D-glucan produced by an Aureobasidium microorganism. Specifically, the ¹ HNMR spectrum of a solution using 1N sodium hydroxide heavy aqueous solution as a solvent has two signals of about 4.7 ppm and about 4.5 ppm. Since it is well known that NMR measurement values change due to subtle changes in conditions and are accompanied by errors, “about 4.7 ppm” and “about 4.5 ppm” are measured values within the normally expected range. A numerical value including a fluctuation range (for example, ± 0.2).

β-1,3-1,6-D-glucan preferably has a metal ion concentration of 0.4 g or less per gram of solid content of β-1,3-1,6-D-glucan. It is more preferably 2 g or less, and even more preferably 0.1 g or less. When the raw material β-1,3-1,6-D-glucan is in an aqueous solution state, the metal ion concentration is preferably 120 mg or less per 100 ml of the aqueous solution, more preferably 50 mg or less, Even more preferably, it is 20 mg or less.
The metal ions referred to here include alkali metal ions, alkaline earth metal ions, Group 3 to Group 5 metal ions, transition metal ions, and the like. Include alkali-derived potassium ions, sodium ions and the like used in the production of low viscosity β-1,3-1,6-D-glucan. The metal ion concentration can be adjusted by ultrafiltration or dialysis.
When the metal ion concentration is in the above range, β-1,3-1,6-D-glucan gelation, aggregation, and precipitation are unlikely to occur when stored in an aqueous solution or when sterilized by heating in an aqueous solution. . Further, when used in solid form, aggregation or the like hardly occurs when re-dissolving.

(b) Production method of β-1,3-1,6-D-glucan by microorganisms belonging to the genus Aureobasidium β-1,3-1,6-D-glucan having a degree of branching of 50 to 100% is, for example, It can be obtained as a precipitate by adding an organic solvent to the culture supernatant of a microorganism that produces sucrose.
Various methods for producing β-1,3-1,6-D-glucan by culturing microorganisms belonging to the genus Aureobasidium have been reported. Examples of the carbon source that can be used in the culture medium include carbohydrates such as sucrose, glucose, and fructose, and organic nutrient sources such as peptone and yeast extract. Examples of the nitrogen source include inorganic nitrogen sources such as ammonium sulfate, sodium nitrate, and potassium nitrate. In some cases, inorganic salts such as sodium chloride, potassium chloride, phosphate, magnesium salt, calcium salt, trace metal salts such as iron, copper, manganese and vitamins are used as appropriate in order to increase the production amount of β-glucan. It is also an effective method to add a kind or the like.

Aureobasidium microorganisms are reported to produce high concentrations of β-1,3-1,6-D-glucan when cultured in a medium containing ascorbic acid in a Zapec medium containing sucrose as a carbon source (Arg. Biol. Chem., 47, 1167-1172 (1983)); Science and Industry, 64, 131-135 (1990); JP-A-7-51082). However, the medium is not particularly limited as long as microorganisms grow and produce β-1,3-1,6-D-glucan. If necessary, organic nutrient sources such as yeast extract and peptone may be added.
Conditions for aerobic culture of microorganisms belonging to the genus Aureobasidium in the above medium are about 10 to 45 ° C, preferably about 20 to 35 ° C, about 3 to 7, preferably about 3.5 to 5. PH conditions and the like.
In order to effectively control the culture pH, it is also possible to control the pH of the culture solution with an alkali or an acid. Furthermore, you may add an antifoamer suitably for the defoaming of a culture solution. The culture time is usually about 1 to 10 days, preferably about 1 to 4 days, whereby β-glucan can be produced. The culture time may be determined while measuring the production amount of β-glucan.
When microorganisms belonging to the genus Aureobasidium are cultured under aeration and agitation for about 4 to 6 days under the above conditions, 0.1-β-glucan polysaccharide containing β-1,3-1,6-D-glucan as a main component is contained in the culture solution. % (W / v) to several% (w / v), and the viscosity of the culture solution is several hundred cP ([mPa · s]) at 30 ° C. using a BM type rotational viscometer (manufactured by Toki Sangyo Co., Ltd.). ) To several thousand cP ([mPa · s]). By adding, for example, an organic solvent to the supernatant obtained by centrifuging this culture, β-1,3-1,6-D-glucan can be obtained as a precipitate.

<Method for producing low viscosity β-1,3-1,6-D-glucan>
When an alkali is added to the above-described culture solution containing β-1,3-1,6-D-glucan having high viscosity while stirring at room temperature, the viscosity is drastically decreased.
The alkali is not particularly limited as long as it is water-soluble and can be used as a pharmaceutical or food additive. For example, an alkali carbonate aqueous solution such as a calcium carbonate aqueous solution, a sodium carbonate aqueous solution, a potassium carbonate aqueous solution, or an ammonium carbonate aqueous solution; an alkali hydroxide aqueous solution such as a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, or a calcium hydroxide aqueous solution; or an ammonia aqueous solution is used. it can. The alkali may be added so that the pH of the culture solution is 12 or more, preferably 13 or more. For example, when using sodium hydroxide to increase the pH of the culture solution, the final concentration of sodium hydroxide is preferably 0.5% (w / v) or higher, more preferably 1.25% (w / v) or higher. Add so that. When alkali is added to the culture solution and stirred well, the viscosity of the culture solution decreases instantaneously.
Next, insoluble substances such as bacterial cells are separated from the culture solution after the alkali treatment. Since the viscosity of the culture solution is low, a method of allowing the cells to settle naturally (decant method), centrifuging, filtering the whole volume using filter paper or filter cloth, filter press, and membrane filtration (limitation of MF membrane, etc.) Insoluble substances and glucan can be easily separated by a method such as external filtration. In the case of total filtration with filter paper or filter cloth, it is one means to use a filter aid such as celite. Industrially, removal of bacterial cells by a filter press is preferred. Further, the β-D-glucan solution before removal of the insoluble substance may be diluted with water as necessary. If the concentration is too high, it is difficult to remove insoluble substances, and if it is too low, it is not efficient. The β-D-glucan concentration is about 0.1 mg / ml to 20 mg / ml, preferably about 0.5 mg / ml to 10 mg / ml, more preferably about 1 mg / ml to 5 mg / ml.
Next, the solution containing glucan is neutralized by adding an acid. Neutralization may be performed before removal of insoluble matter. The acid is not particularly limited as long as it can be used as a pharmaceutical or food additive. For example, hydrochloric acid, phosphoric acid, sulfuric acid, citric acid, malic acid and the like can be used. The amount of acid used may be such that the solution or culture solution becomes neutral (pH 5-8). That is, neutralization does not necessarily need to be adjusted to pH 7.

β-1,3-1,6-D-glucan obtained by neutralization after alkaline treatment at pH 12 or higher has a viscosity of usually 200 cP at 30 ° C., pH 5.0, and concentration 0.5 (w / v%). Hereinafter, depending on the case, it is 100 cP or less, 50 cP or less, or 10 cP or less. The viscosity varies depending on the production method or purification method.
The alkali-treated low viscosity β-1,3-1,6-D-glucan does not increase in viscosity even when neutralized. Furthermore, at room temperature (15 to 35 ° C.), even if the liquid is acidified so that the pH is less than 4, the viscosity does not increase.
In addition, instead of removing the cells after neutralizing and neutralizing the culture supernatant, alkali treatment and neutralization can be performed after removing the cells from the culture supernatant.
In order to remove soluble contaminants (for example, salts) having a lower molecular weight than glucan from the resulting aqueous solution of glucan, ultrafiltration may be performed, for example.
Moreover, after carrying out alkali treatment and sterilization, it is possible to perform ultrafiltration under alkaline conditions without neutralization, and thereby purified β-1,3-excellent in transparency, thermal stability and long-term storage stability. 1,6-D-glucan is obtained. The alkaline condition is pH 10 or more, preferably 12 or more, and the upper limit of pH is usually about 13.5.
The β-1,3-1,6-D-glucan contained in the aqueous solution thus obtained is temporarily removed from the aqueous solution, whether it is dried to form a solid formulation or used as a formulation as an aqueous solution. It can be deposited. The precipitation method of β-1,3-1,6-D-glucan is not particularly limited. For example, an aqueous solution in which the concentration of glucan is increased to 1 w / w% or more by ultrafiltration or the like is added to ethanol. By adding the alcohol to the aqueous solution at a volume ratio equal to or greater than, preferably equal to or greater than twice, β-1,3-1,6-D-glucan can be precipitated. In this case, the pH is adjusted to an acidic value with an organic acid such as citric acid, preferably less than pH 4, more preferably pH 3-3.7, and ethanol is added to obtain high purity β-1,3-1,6. -Glucan powder can be obtained.
By reducing the viscosity of β-1,3-1,6-D-glucan, concentration by ultrafiltration and the like can be easily performed, so that the amount of alcohol used for alcohol precipitation can be reduced.
When obtained as a solid, the low-viscosity β-1,3-1,6-D-glucan aqueous solution may be directly dried, or the precipitated β-1,3-1,6-D-glucan is dried. May be. Drying can be performed by a known method such as spray drying or freeze drying.

(1-3) A poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan in a ratio complex of a poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan The ratio (poorly water-soluble pharmacologically active substance: β-1,3-1,6-D-glucan) is preferably about 1: 0.01 to 1000, and preferably about 1: 0.1 to 100 in terms of dry weight. More preferably, about 1: 0.1-50 is still more preferable, and about 1: 0.1-20 is especially preferable. If it is the said range, sufficient water solubility will be acquired.

(2) Manufacturing method of composite
(2-1) High-speed vibration pulverization method The method for producing the composite of the present invention described above is not particularly limited.
For example, a poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan having a degree of branching of β-1,6 bonds with respect to β-1,3 bonds of 50 to 100% in a solid state. It can be produced by a method comprising a step of stirring as it is and a step of adding water to the mixture and stirring.
Mixing ratio of poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan (poorly water-soluble pharmacologically active substance: β-1,3-1,6-D-glucan) 1: 0.01-1000 is preferable, about 1: 0.1-100 is more preferable, about 1: 0.1-50 is still more preferable, About 1: 0.1-20 is especially preferable.
For stirring in the solid state, it is sufficient that the poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan are sufficiently pulverized and mixed. Can be used. For example, a pulverization / mixing method using a planetary or centrifugal ball mill; a high-speed vibration pulverization method using a high-speed vibration pulverization apparatus that vibrates the container at a high speed by placing the material to be mixed into a container together with a hard ball, and the like.
Among them, the high-speed vibration pulverization method is preferable, and the frequency thereof can be set to about 10 to 120 s ⁻¹ (about 10 to 120 Hz), for example.
Also, the vibration time can be, for example, about 5 to 60 minutes. The amplitude is, for example, when the bottom diameter of the container hollow part is 12 mm and the length of the container hollow part in the longitudinal direction is 50 mm, usually about 5 to 100 mm is based on the center point of vibration of the container subjected to vibration. In the case of maximum displacement, it means the length from the center point to the maximum displacement point. In addition, when the bottom diameter of the container hollow portion is 12 mm and the length of the container hollow portion in the longitudinal direction is 50 mm, the hard ball diameter may be about 1 to 10 mm. Examples of the hard ball material include agate, stainless steel, alumina, zirconia, tungsten carbide, chromium steel, and Teflon (registered trademark). The number of hard spheres provided in the container can be about 1-100.

Stirring in the solid state can be performed at room temperature.
Water is added to the mixture of the poorly water-soluble pharmacologically active substance thus obtained and β-1,3-1,6-D-glucan and stirred. The water may contain a buffering agent, a salt and the like as long as the formation of the complex is not inhibited. The amount of water can be about 10 to 10,000 parts by weight with respect to 1 part by weight of the mixture. Stirring in this step can be performed using, for example, a magnetic stirrer. This stirring step is preferably performed at room temperature for at least 2 days, for example, 4 to 7 days.
Thereby, a liquid in which a complex of a poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan is dissolved is obtained. If necessary, water-insoluble contaminants may be removed by centrifugation at about 5,000 to 20,000 g for about 5 to 60 minutes.

(2-2) Method of mixing and aging in a polar solvent The complex of the present invention described above has a poorly water-soluble pharmacologically active substance and a degree of branching of β-1,6 bonds to β-1,3 bonds of 50 to 50. It can also be produced by a method comprising a step of mixing 100% β-1,3-1,6-D-glucan in a polar solvent and a step of aging by adding water to the resulting mixture. .
Use ratio of poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan (poorly water-soluble pharmacologically active substance: β-1,3-1,6-D-glucan) in dry weight ratio 1: 0.01-1000 is preferable, about 1: 0.1-100 is more preferable, about 1: 0.1-50 is still more preferable, About 1: 0.1-20 is especially preferable.
The amount of the polar solvent used can be about 10 to 10,000 parts by weight with respect to 1 part by weight of β-1,3-1,6-D-glucan.
The polar solvent may be a protic polar solvent such as water, acetic acid, formic acid or lower alcohol, or an aprotic polar solvent such as dimethyl sulfoxide, acetone, acetonitrile, N, N-dimethylformamide, methylene chloride or tetrahydrofuran. However, an aprotic polar solvent is particularly preferable, and dimethyl sulfoxide is more preferable.

A poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan may be mixed with a polar solvent at the same time, one of which is first mixed with a polar solvent and then the other is added thereto. You may mix.
Mixing can be performed using, for example, a magnetic stirrer.
Subsequently, about 10 to 10,000 parts by weight of water is preferably added to 1 part by weight of the mixture, and the mixture is usually aged by allowing to stand at room temperature for 1 to 3 days. The water may contain a buffering agent, a salt and the like as long as the formation of the complex is not inhibited.
Thereby, a liquid in which a complex of a poorly water-soluble pharmacologically active substance and β-1,3-1,6-D-glucan is dissolved is obtained. If necessary, water-insoluble contaminants may be removed by centrifugation at about 5,000 to 20,000 g for about 5 to 60 minutes.
The production method of the complex of the present invention described above can also be regarded as a method of imparting water solubility while maintaining the pharmacological activity of the poorly water-soluble pharmacologically active substance.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.
Experimental example (production of purified β-1,3-1,6-D-glucan)
(1) Preparation of low viscosity β-1,3-1,6-D-glucan
(1-1) beta-glucan a liquid medium 100ml having the composition shown in Table 1 supra after culture production placed on shouldered flask 500ml volume, at 121 ° C., 15 min, after pressurized steam sterilization, Aureobasidium pullulans GM-NH-1A1 strain (FERM P-19285) was aseptically inoculated from a slant of the same medium composition with 1 platinum ear inoculum and stirred at a rate of 130 rpm at 24 ° C. for 24 hours at 30 ° C. A seed culture solution was prepared by culturing for a period of time.
Next, 200 L of medium having the same composition is placed in a 300 L culture apparatus (manufactured by Maruhishi Bioengineer), autoclaved at 121 ° C. for 15 minutes, and 2 L of the seed culture obtained as described above is aseptic. The cells were inoculated and aerated and stirred at 200 rpm, 27 ° C., 40 L / min. The pH of the medium was controlled within the range of pH 4.2 to 4.5 using sodium hydroxide and hydrochloric acid. The turbidity after 96 hours was 23 OD at OD 660 nm, the polysaccharide concentration was 0.5% (w / v), and the substituted sulfoacetic acid content calculated from the sulfur content was 0.09%.

<Measurement of polysaccharide concentration>
The polysaccharide concentration was collected by sampling several ml of the culture solution, centrifuging and removing the cells, and then adding ethanol to the supernatant so that the final concentration was 66% (v / v) to precipitate the polysaccharide. Then, it melt | dissolved in ion-exchange water and quantified with the phenol sulfuric acid method.

<Measurement of substituted sulfo content>
Similarly, ethanol was added to the culture supernatant from which the cells had been removed so that the final concentration was 66%, and β-glucan was collected by precipitation. Thereafter, the sample was dissolved again in ion-exchanged water, centrifuged again, sodium chloride was added to the supernatant so that the final concentration was 0.9%, and β-glucan was again collected with 66% ethanol. This β-glucan recovery and purification operation was further repeated twice, and the resulting β-glucan aqueous solution was dialyzed against ion-exchanged water and then freeze-dried to obtain a β-glucan powder.
This β-glucan powder was combusted and absorbed by the combustion tube and analyzed by ion chromatography. As a result, the S content was 239 mg / kg, and the substituted sulfoacetic acid content calculated from this value was 0.09%.

(1-2) Alkali treatment When the viscosity of the culture solution obtained as described above was measured at 30 ° C. and 12 rpm using a BM type rotational viscometer (manufactured by Tokyo Keiki), 1500 cP ((mPa · s) )Met. The rotor used for the measurement was selected appropriately according to the viscosity.
When 25% (w / w) sodium hydroxide was added to this culture solution so that the final concentration of sodium hydroxide was 2.4% (w / v) and stirred (pH 13.6), the viscosity decreased instantaneously. did. Subsequently, after neutralizing with a 50% (w / v) aqueous citric acid solution to pH 5.0, the viscosity at a concentration of 0.5 (w / v%) was measured. ) Was 20 cP ([mPa · s]).
Next, 1 wt% of KC Flock (manufactured by Nippon Paper Industries Co., Ltd.) was added to the culture broth as a filter aid, and the cells were removed using a Kamata filter press (manufactured by Kamata Kikai). About 230 L). The polysaccharide concentration was 0.5% (w / v), and the recovery rate was almost 100%.

(1-3) Desalination of β-glucan aqueous solution After dilution of the above β-glucan aqueous solution (culture filtrate) to 0.3%, ultrafiltration (UF) membrane (molecular weight cut 50,000, manufactured by Nitto Denko Corporation) Was used, and finally the sodium ion concentration was lowered to 20 mg / 100 ml, and then the pH was adjusted to 3.5 with a 50% (w / v) aqueous citric acid solution.
Subsequently, sterilization was performed by holding at 95 ° C. for 3 minutes using a hot filling heating unit (manufactured by Nisaka Manufacturing Co., Ltd.) to obtain a final β-glucan aqueous solution. The concentration of β-glucan at this time was 0.22% (w / v) as measured by the phenol sulfuric acid method. The total yield from the culture was about 73%.

<Measurement of sulfur content>
Further, the obtained β-glucan aqueous solution was dialyzed with ion-exchanged water and then freeze-dried to obtain β-glucan powder. From the compositional analysis result of this β-glucan, the S content was 330 mg / kg, and the substituted sulfoacetic acid content calculated from this was 0.12%.
<Confirmation of combined state>
Moreover, since the wavelength shift from 480 nm to about 525 nm could be confirmed by the Congo red method, the above-described culture filtrate that had been desalted contained glucan containing β-1,3 bonds. Proven (K. Ogawa, Carbohydrate Research, 67, 527-535 (1978), supervised by Tadayuki Imanaka, Development of the use of microorganisms, 1012-1015, NTS (2002)). The shift difference to the maximum value at that time was Δ0.48 / 500 μg polysaccharide.
15 ml of the culture filtrate was taken out, 30 ml of ethanol was added, and centrifuged at 4 ° C. and 1000 rpm for 10 minutes to collect the precipitated polysaccharide. Wash with 66% ethanol, centrifuge at 4 ° C, 1000 rpm for 10 minutes, add 2 ml of ion-exchanged water and 1 ml of 1N sodium hydroxide aqueous solution to the precipitated polysaccharide, stir, then heat at 60 ° C for 1 hour to precipitate. Dissolved. Next, after freezing at −80 ° C., vacuum lyophilization was performed overnight, and the dried powder was dissolved in 1 ml of 1N sodium hydroxide heavy aqueous solution and subjected to two-dimensional NMR.
FIG. 1 shows a ¹ H NMR spectrum having a correlation with 106 ppm of two-dimensional NMR ( ¹³ C- ¹ H COSY NMR). In this spectrum, two signals of 4.7 ppm and around 4.5 ppm were obtained.
As a result, it was proved that this β-glucan is β-1,3-1,6-D-glucan (supervised by Tadayuki Imanaka, development of the use of microorganisms, 1012-1015, NTS ( 2002)). From the integration ratio of each ¹ H NMR signal, the ratio of β-1,3 bond / β-1,6 bond was found to be 1.15. Therefore, the degree of branching of the β-1,6 bond in the side chain relative to the β-1,3 bond in the main chain is about 87%.

<Particle size measurement>
Next, when the particle size of the culture solution was measured using a laser diffraction / scattering type particle size distribution measuring apparatus (LA-920 manufactured by HORIBA), the particles were about 0.3 μm and 100 μm in size. I was seen. Subsequently, when particle size measurement was performed while irradiating with ultrasonic waves, the 100 μm peak disappeared as soon as it was seen, the 0.3 μm peak increased, and finally only 0.3 μm. The particle size distribution of the culture solution when irradiated with ultrasonic waves is shown in FIG.
The peak of 0.3 μm is the peak due to the primary particles of β-1,3-1,6-D-glucan, and the peak of 100 to 200 μm is the primary particles of β-1,3-1,6-D-glucan. This is considered to be a peak due to aggregated secondary particles.
Further, it was confirmed that the secondary particles disappeared in the same manner even when stirred with a magnetic stirrer and lightly shaken, and easily broken into primary particles. Therefore, the secondary particles are considered to be very loosely aggregated (slowly aggregated state).

<Molecular weight measurement>
Further, Toyopearl HW65 manufactured by Tosoh Corporation (column size 75 cm × φ1 cm, excluded molecular weight 2.5 million (dextran)) was subjected to gel filtration chromatography using 0.1 M sodium hydroxide aqueous solution as an eluent, and dissolved β- When the molecular weight of a solution containing 1,3-1,6-D-glucan and β-1,3-1,6-D glucan primary particles was measured, the dissolved β-1,3-1,6- Two types of low molecular fractions having peaks of 2 to 300,000 derived from D-glucan and apparently high molecular fractions of 500 to 2.5 million derived from primary particles were detected. Shodex pullulan was used as a molecular weight marker.
In order to separate the water-soluble β-1,3-1,6-D-glucan and the fine particles, a β-1,3-1,6-D-glucan solution containing the fine particle fraction and the soluble fraction is used. When filtration was performed with a filter (0.2 μm) manufactured by Advantech, 500 to 2.5 million polymer fractions disappeared. From this, it was found that the polymer fraction corresponds to primary particles of β-1,3-1,6-D-glucan and secondary particles in which primary particles are aggregated. Therefore, the molecular weight of water-soluble β-1,3-1,6-D-glucan is considered to be 2 to 300,000.

(2) Preparation of powdered β-glucan In (1-2), β-1,3-1,6-D-glucan containing fine particles β-1,3-1,6-D-glucan prepared by alkali treatment and fungus body removal treatment Ethanol was added to the 1,6-D-glucan aqueous solution so that the final concentration was 66% (v / v) to precipitate the polysaccharide glucan and recovered by centrifugation. Next, ethanol and water were removed by a freeze-drying method to obtain dry β-1,3-1,6-D-glucan. The yield at that time was 95% or more compared to the total sugar concentration before ethanol precipitation.
Subsequently, the obtained dried β-1,3-1,6-D-glucan was dissolved and dispersed in water so that the final concentration was 0.3% (w / v), and then the same as described above. Gel chromatography was performed using Toyopearl HW65 (column size: 75 cm × φ1 cm, excluded molecular weight: 2.5 million (dextran)) manufactured by Toyopearl, and the molecular weight was measured. It was found that the molecular weight was composed of two types, a low molecular fraction having a peak of 2 to 300,000 and a high molecular fraction having an apparent appearance of 500 to 2.5 million. Here, a pullulan manufactured by Shodex was used as a molecular weight marker.
On the other hand, in order to separate water-soluble β-1,3-1,6-D-glucan and fine particles, an aqueous solution of β-1,3-1,6-D-glucan prepared by this method (fine particles and solubilized glucan are mixed). When the product was filtered with a filter (0.2 μm) manufactured by Advantech, 500 to 2.5 million polymer fractions disappeared. Therefore, even if the β-1,3-1,6-D-glucan obtained by this method is dried, it is the same as β-1,3-1,6-D-glucan before drying if it is redissolved. It was demonstrated to reproduce the physical behavior of

(3) Production of high-purity β-1,3-1,6-D-glucan powder (1) 90 L of a culture solution (polysaccharide concentration 0.5% (5 mg / ml)) reduced in viscosity by alkali treatment After neutralization with 9 kg of 50% citric acid aqueous solution, the cells were removed through a Kamata filter press 40D-4 pre-coated with 1.8 kg of filter aid (Nippon Paper Chemicals powder cellulose KC floc). The filtrate was concentrated to 9 L with an ultrafiltration spiral element (NTU3150-S4 manufactured by Nitto Denko). While stirring the concentrate, the pH was adjusted to 3.0-3.5 with citric acid, and 18 L of ethanol was added to obtain a glucan / ethanol / water slurry. The viscosity of the slurry was 22 mPa · s (30 ° C.) with a BM viscometer. The mixture was allowed to stand at room temperature for 3 hours, and about 17 L of the supernatant (ethanol / water) was removed. The viscosity of the remaining slurry was 45 mPa · s (30 ° C.). 10 L of this concentrated slurry was spray-dried using a spray dryer R-3 of Sakamoto Giken Co., Ltd. to obtain 360 g of β-1,3-1,6-D-glucan powder (recovery rate 80%). The purity of the obtained β-1,3-1,6-D-glucan was 90% or more as a result of NMR spectrum analysis.
When the obtained β-1,3-1,6-D-glucan powder was dissolved in 1N sodium hydroxide heavy aqueous solution and the NMR spectrum was measured, the ¹ H NMR spectrum was about 4.7 ppm and about 4. Two signals of 5 ppm were obtained. Further, the viscosity of the obtained β-1,3-1,6-D-glucan powder aqueous solution having a concentration of 0.5 (w / v%) was 200 cP or less (pH 5.0, 30 ° C.). The purified β-glucan obtained by the method described above was subjected to the following test.

Examples 1 to 4 and Comparative Example 1
(Water-solubilization of 1'-Acetoxychavicol acetate by high-speed vibration grinding method)
1'-Acetoxychavicol acetate (ACA) is a hydrophobic compound having anti-ulcer and insecticidal activity derived from ginger. As β-1,3-1,6-D-glucan, Daiso Corporation (Aqua β) or Mitsui Sugar Co., Ltd.'s Schizophylan (SPG) is used as “H. Azuma, K. Miyasaka, T. Yokotani, T. Tachibana, ACA (racemate) synthesized by the method described in “A. Kojima-Yuasa, I. Matsui-Yuasa, K. Ogino, Bioorg. Med. Chem., 14, 1811 (2006)”, and shown in Table 2 below. The mixture was mixed in a predetermined amount, enclosed in an agate pulverization jar together with agate pulverized balls, and pulverized at 25 Hz for 60 minutes with a mixer mill (MM200) manufactured by Lecce.

Next, 10,000 parts by weight of water was added to 1 part by weight of the pulverized product, and the mixture was stirred at room temperature for 2 days using a magnetic stirrer. Further, the suspension was stirred with a vortex mixer for 1 hour, and then centrifuged (25 ° C., 10,000 g, 10 min), and the supernatant was filtered through a 0.22 μm membrane filter.
A portion of the filtrate was lyophilized, and the residue was measured for ¹ H NMR in D ₂ O, and the glucose proton (CH X 4, CH ₂ X 1 total 6H) and the acetyl group (3H X 2) of ACA or By calculating the integral value of protons on the benzene ring (2H X 2), the composition ratio of β-1,3-1,6-D-glucan / ACA (glucose / ACA ratio) in the complex was calculated. The composition ratio is shown in Table 3 below.

The ratio of glucose / ACA in the complex to the glucose / ACA ratio supplied is shown in FIG. As the amount of ACA used per unit glucose of aqua β used for high-speed vibration crushing is increased, the glucose / ACA molar ratio calculated from ¹ H NMR decreases, and the internal hydrophobicity of aqua β forming a triple helix structure It became clear that ACA was taken in densely by the holes.
In addition, even when the amount of ACA used is 0.33 equivalent or more per glucose unit, the composition ratio of the complex is almost constant, and it is estimated that the calculated glucose / ACA = 6 is the maximum filling state when aqua β is used. Is done. Note that 6 glucoses correspond to 1 unit of triple helical repeating unit.
On the other hand, when the preparation ratio glucose / ACA = 5 was prepared, the complex composition ratio (glucose / ACA) was 10 for schizophyllan and 12 for aqua β. In other words, the triple helical repeating unit occupied by one molecule of ACA is 2.5 for schizophyllan and 2 for aqua β, and it was found that aqua β is more efficiently complexed than schizophyllan under the same conditions.

Example 5 (Evaluation of cytotoxicity of aqua β / ACA complex)
A 96-well plate is seeded with a cell suspension of RAW264.7 cells (mouse macrophage cells) at 5 × 10 ³ cells, and 37 ° C., 5 v using RPMI medium (containing 10 w / v% fetal calf serum). The cells were cultured under / v% CO ₂ for 24 hours. Remove the medium with an aspirator, add 100 μL of the medium with the ACA concentration of aqua β / ACA complex with glucose / ACA = 12, adjusted to a predetermined concentration, and incubate for 22 hours. WST reagent (Dojindo Laboratories) Cell-counting Kit-8) (see M. Ishiyama, Y. Miyazono, K. Sasamoto, Y. Ohkura, K. Ueno, Talanta, 44, 1299 (1997)) was added, and the cells were further cultured for 2 hours. A color reaction was performed.
Using a plate reader (ThermoLab Systems, MultiSkan Acent BIF), the absorbance at 450 nm (reference 650 nm) was measured, and the cell viability was calculated (M. Ishiyama, Y. Miyazono, K. Sasamoto, Y. Ohkura, K Ueno, Talanta, 44, 1299 (1997)).
The results are shown in FIG. The vertical axis in FIG. 4 indicates the cell viability, and each concentration in the figure indicates the concentration of ACA.
ACA is already known to have apoptotic activity (K. Ito, T. Nakazato, A. Murakami, H. Ohigashi, Y. Ikeda, M. Kizaki, Biochem. Biophys. Res. Commun., 338, 1702 (2005)). RAW264.7 cells also showed significant cytotoxicity at 10 μM with a cell viability of 20%. On the other hand, the complex with aqua β or schizophyllan did not show significant cytotoxicity even at 20 μM, and it was revealed that the cytotoxicity of ACA can be suppressed by complexing with β-1,3-glucan.

Example 6 (Confirmation of NF-κB inhibitory effect of aqua β / ACA complex)
Using the aqua β / ACA complex sample prepared in Example 1 (glucose / ACA = 1), the effect of inhibiting NF-κB, which is the activity of ACA, was evaluated by Western blotting.
1 ml of RAW264.7 cell suspension is spread to 5 × 10 ⁵ cells in a 35 mm dish, cultured for 24 hours, each sample is added so that the final ACA concentration is 0, 1, 5 μM, and incubated for 30 minutes. . Furthermore, 10 μL of LPS (10 mg / ml) was added and cultured for 15 minutes, and then the cells were collected. LPS was sonicated for 5 minutes before use.
Add 5% 2-mercaptoethanol solution, heat treatment at 95 ° C for 5 minutes, and perform SDS-PAGE (20 mA), transcription (70 mA, 90 minutes), blocking, antibody (IκBα antibody) reaction with 12% gel. went.
The results are shown in FIG. NF-κB is a transcription factor for various inflammatory cytokines. When various inflammatory signals are transmitted to cells, NF-κB, which is an inhibitor of NF-κB in the cytoplasm, is phosphorylated and decomposed to activate NF-κB and translocate into the nucleus to function as a transcription factor. That is, when macrophage cells are stimulated with LPS, which is an inflammatory substance, IκBα is degraded and NF-κB is activated (FIG. 5, second lane from the left). In the absence of stimulation, macrophage cells have IκBα present to detect NF-κB (FIG. 5, control c lane). Alkaline is already known to suppress IκBα activation (H. Matsuda, T. Morikawa, H. Managi, M. Yoshikawa, Bioorg. Med. Chem. Lett., 13, 3197 (2003)). 5 μM ACA was confirmed to suppress the degradation of IκBα (FIG. 5, third lane from the left).
However, when ACA is used as an immunosuppressant, cytotoxicity becomes a problem as described above. On the other hand, it was found that aqua β / ACA also inhibited IκBα degradation, that is, activation of NF-κB in a concentration-dependent manner (FIG. 5, 4th and 5th lanes from the left). Aqua β / ACA is expected to function as a useful immunosuppressive agent since cytotoxicity is not a problem.

Example 7 (Evaluation of collagen production promoting action)
Using the aqua β / ACA complex (glucose / ACA = 6) prepared in Example 1, the ability to enhance collagen production in human fibroblasts was evaluated by the method disclosed in JP-A-2009-079004.
Specifically, human normal skin-derived fibroblasts (CCD-1059SK, Dainippon Pharmaceutical Co., Ltd.) were subcultured 3 to 6 times in EMEM medium containing 10% FBS (fetal calf serum). Next, it is prepared on a culture slide (manufactured by Falcon) so that the number of cells becomes 1 × 10 ⁶ cells, cultured for 24 hours in EMEM medium containing 10% FBS, and the cells are fixed to the slide, and further the cell cycle In order to synchronize, only EMEM medium was cultured for 4 hours. Subsequently, the medium was replaced with an EMEM medium containing 10% FBS, and each sample was added so that the final ACA concentrations were 0, 0.1, and 1.0 μM, and cultured for 24 hours. As a control experiment, ACA (racemic) was used, and the activity was compared with the aqua β / ACA complex.
The culture slide was washed 3 times with PBS solution for a total of 3 times, 4% formalin solution was added, and the plate was allowed to stand overnight at 4 degrees to be fixed. After washing with PBS solution containing 0.1% Triton-X for 5 minutes for a total of 3 times, endogenous peroxidase was blocked with 3% H ₂ O ₂ solution for 5 minutes. The non-specific reaction was then blocked using 10% standard goat serum for 5 minutes. And the reaction of the primary antibody was performed for 60 minutes using the anti-rat type I collagen antibody (Anti-rat type I collagen antibody (made by LSL) 200 times dilution). After washing with PBS solution for 5 minutes for a total of 3 times, a secondary antibody reaction was performed for 30 minutes using a biotin-labeled goat anti-rabbit immunoglobulin antibody (Biotinylated Goat anti-rabbit immunogloblins antibody (DAKO) diluted 400 times). After washing with PBS solution for 5 minutes 3 times in total, a reaction with an enzyme solution (400-fold diluted solution of Horseradish peroxidase-labelled streptavidin-biotin complex (manufactured by DAKO)) was performed for 30 minutes. After washing with PBS solution for 5 minutes 3 times in total, DAB (3,3-diaminobenzidine tetrahydrochloride) solution was reacted for 5 minutes to perform peroxidase coloring reaction. After washing with PBS solution for 5 minutes for a total of 3 times, it was sealed with a water-soluble mounting medium to prepare a specimen.
The micrograph and dyeing | staining figure of each obtained sample are shown in FIG. Aqua β / ACA complex (glucose / ACA = 6) has higher ability to promote collagen production at both low concentration (0.1 μM) and high concentration (1.0 μM) than ACA alone. As a result, it is expected as cosmetics and pharmaceuticals that increase the amount of collagen in the skin.

Example 8 (Solubilization of curcumin by high-speed vibration grinding method)
Curcumin is a hydrophobic compound that has antioxidant, anti-inflammatory and anti-tumor effects. As a β-1,3-1,6-D-glucan, 10 mg of Daiso (Aqua β) and 0.75 mg of Curcumin manufactured by Wako Pure Chemical Industries, Ltd. are mixed together and enclosed in an agate grinding jar together with agate grinding balls. The mixture was pulverized at 25 Hz for 60 minutes with a Lecce mixer mill (MM200). Next, 10,000 parts by weight of water was added to 1 part by weight of the pulverized product, and the mixture was stirred at room temperature for 2 days using a magnetic stirrer. The suspension was further stirred for 1 hour with a vortex mixer and then centrifuged (25 ° C., 10,000 g, 10 min) to obtain a supernatant.
The absorption spectrum of the supernatant was measured (FIG. 7B). In addition, the absorption spectrum of a 21.7 μM ethanol solution of curcumin was measured (FIG. 7A), and the molar extinction coefficient ε = 5.56 × 10 ⁴ at a maximum absorption wavelength of 420 nm was used, and an aqueous curcumin / aqua β complex solution was used. The curcumin concentration in the medium was calculated. As a result, an aqueous solution with [curcumin] = 20 μM (water solubility 9.8%) could be prepared. This water-solubilization rate is a value obtained from the molar extinction coefficient.

Example 9 (Solubilization of Coenzyme Q10 by High-Speed Vibration Crushing Method)
Coenzyme Q10 is a poorly water-soluble compound having an antioxidant action. 14.1 mg of Daiso Co. (Aqua β) is used as β-1,3-1,6-D-glucan, and 3.67 mg of Coenzyme Q10 manufactured by Wako Pure Chemical Industries, Ltd. is mixed, and agate grinding balls are added to an agate grinding jar. Then, the mixture was subjected to high-speed vibration pulverization at 25 Hz for 20 minutes using a Lecce mixer mill (MM200). Thereafter, 10,000 parts by weight of ultrapure water was added to 1 part by weight of the pulverized product. Further, the suspension was stirred with a vortex mixer for 1 hour, and then centrifuged (25 ° C., 10,000 g, 10 min) to obtain a supernatant.
The absorption spectrum of the supernatant and the absorption spectrum of an aqueous solution subjected to the same treatment in the absence of aqua β were measured (FIG. 8). Using the molar extinction coefficient ε = 1.33 × 10 ⁴ at the maximum absorption wavelength of 275 nm, the coenzyme Q10 concentration in the coenzyme Q10 / aqua β complex aqueous solution was calculated. As a result, an aqueous solution having [Coenzyme Q10] = 119 μM (water solubility 28%) was successfully prepared. This water-solubilization rate is a value obtained from the molar extinction coefficient.

Example 10 (Solubilization of vitamin D3 by high-speed vibration grinding method)
Vitamin D3 (VD3) is a poorly water-soluble compound having various physiological activities. 13.9 mg of Daiso Corporation (Aqua β) is used as β-1,3-1,6-D-glucan, 3.62 mg of vitamin D3 made by Wako Pure Chemical Industries, Ltd. is mixed, and agate grinding balls are added to an agate grinding jar. Then, the mixture was subjected to high-speed vibration pulverization at 25 Hz for 20 minutes using a Lecce mixer mill (MM200). Thereafter, 10,000 parts by weight of ultrapure water was added to 1 part by weight of the pulverized product. Further, the suspension was stirred with a vortex mixer for 1 hour, and then centrifuged (25 ° C., 10,000 g, 10 min) to obtain a supernatant.
The absorption spectrum of the supernatant and the absorption spectrum of an aqueous solution subjected to the same treatment in the absence of aqua β were measured (FIG. 9). Using the molar extinction coefficient ε = 1.50 × 10 ³ at the maximum absorption wavelength of 271 nm, the concentration of vitamin D3 in the aqueous solution of vitamin D3 / aqua β complex was calculated. As a result, an aqueous solution having [vitamin D3] = 149 μM (water solubility: 16%) was successfully prepared. This water-solubilization rate is a value obtained from the molar extinction coefficient.

Example 11 Water-solubilized paclitaxel (taxol) by high-speed vibration pulverization of paclitaxel (taxol) is a poorly water-soluble compound having antitumor activity. 15.0 mg of Daiso (Aqua β) was used as β-1,3-1,6-D-glucan, and 3.84 mg of paclitaxel (Taxol) manufactured by Wako Pure Chemical Industries, Ltd. was mixed. The mixture was encapsulated with pulverized balls and subjected to high-speed vibration pulverization at 25 Hz for 20 minutes using a Lecce mixer mill (MM200). 10,000 parts by weight of ultrapure water was added to 1 part by weight of the pulverized product. Further, the suspension was stirred with a vortex mixer for 1 hour, and then centrifuged (25 ° C., 10,000 g, 10 min) to obtain a supernatant.
The absorption spectrum of the supernatant and the absorption spectrum of an aqueous solution subjected to the same treatment in the absence of aqua β were measured (FIG. 10). Using the molar absorption coefficient ε = 2.96 × 10 ⁴ at the maximum absorption wavelength of 230 nm, the concentration of paclitaxel (taxol) in the aqueous solution of paclitaxel (taxol) / aqua β complex was calculated. As a result, an aqueous solution of paclitaxel (taxol) = 186 μM (water solubility 41%) was successfully prepared. This water-solubilization rate is a value obtained from the molar extinction coefficient.

  Since the complex of the present invention has water solubility while maintaining the pharmacological activity of the poorly soluble pharmacologically active substance, it can be practically used as health food, functional food, medicine, cosmetics and the like.

Claims (7)

Β-1,3-1, wherein the poorly water-soluble pharmacologically active substance of any one of the following (a) to (e) and the degree of branching of β-1,6 bond to β-1,3 bond is 85-100% , A complex with 6-D-glucan.
(A) The following general formula (1)
(In the formula, the 2′-3 ′ bond is a single bond or a double bond. R ¹ , R ² , R ³ and R ⁴ may be the same or different and each represents a hydrogen atom, a methoxy group, or an RCOO— group (R is An alkyl group having 1 to 4 carbon atoms).
(B) Curcumin (c) The following general formula (2)
[Wherein n represents an integer of 6 to 10. ]
Ubiquinone (d) Vitamin D
(E) Pakuritakise Lumpur   The complex according to claim 1, wherein β-1,3-1,6-D-glucan is obtained by neutralization after alkali treatment at pH 12 or higher.   The ratio of poorly water-soluble pharmacologically active substance to β-1,3-1,6-D-glucan (poorly water-soluble pharmacologically active substance: β-1,3-1,6-D-glucan) was 1: 0.1. The complex according to claim 1 or 2, which is -20. Β-1,3-1, wherein the poorly water-soluble pharmacologically active substance of any one of the following (a) to (e) and the degree of branching of β-1,6 bond to β-1,3 bond is 85-100% , 6-D-glucan in a solid state and a step of adding water to the mixture and stirring the mixture, thereby imparting water solubility while maintaining the pharmacological activity of the poorly water-soluble pharmacologically active substance Method.
(A) The following general formula (1)
(In the formula, the 2′-3 ′ bond is a single bond or a double bond. R ¹ , R ² , R ³ and R ⁴ may be the same or different and each represents a hydrogen atom, a methoxy group, or an RCOO— group (R is An alkyl group having 1 to 4 carbon atoms).
(B) Curcumin (c) The following general formula (2)
[Wherein n represents an integer of 6 to 10. ]
Ubiquinone (d) Vitamin D
(E) Pakuritakise Lumpur Β-1,3-1, wherein the poorly water-soluble pharmacologically active substance of any one of the following (a) to (e) and the degree of branching of β-1,6 bond to β-1,3 bond is 85-100% , 6-D-glucan in a polar solvent, and a method of adding water to the resulting mixture and ripening the mixture to impart water solubility while maintaining the pharmacological activity of the poorly water-soluble pharmacologically active substance .
(A) The following general formula (1)
(In the formula, the 2′-3 ′ bond is a single bond or a double bond. R ¹ , R ² , R ³ and R ⁴ may be the same or different and each represents a hydrogen atom, a methoxy group, or an RCOO— group (R is An alkyl group having 1 to 4 carbon atoms).
(B) Curcumin (c) The following general formula (2)
[Wherein n represents an integer of 6 to 10. ]
Ubiquinone (d) Vitamin D
(E) Pakuritakise Lumpur Β-1,3-1, wherein the poorly water-soluble pharmacologically active substance of any one of the following (a) to (e) and the degree of branching of β-1,6 bond to β-1,3 bond is 85-100% , 6-D-glucan in a solid state and a step of adding water to the mixture and stirring the mixture, a poorly water-soluble pharmacologically active substance and β-1,3-1,6-D -A method for producing a complex with glucan.
(A) The following general formula (1)
(In the formula, the 2′-3 ′ bond is a single bond or a double bond. R ¹ , R ² , R ³ and R ⁴ may be the same or different and each represents a hydrogen atom, a methoxy group, or an RCOO— group (R is An alkyl group having 1 to 4 carbon atoms).
(B) Curcumin (c) The following general formula (2)
[Wherein n represents an integer of 6 to 10. ]
Ubiquinone (d) Vitamin D
(E) Pakuritakise Lumpur Β-1,3-1, wherein the poorly water-soluble pharmacologically active substance of any one of the following (a) to (e) and the degree of branching of β-1,6 bond to β-1,3 bond is 85-100% , 6-D-glucan in a polar solvent, and aging by adding water to the resulting mixture and β-1,3-1,6-D- A method for producing a complex with glucan.
(A) The following general formula (1)
(In the formula, the 2′-3 ′ bond is a single bond or a double bond. R ¹ , R ² , R ³ and R ⁴ may be the same or different and each represents a hydrogen atom, a methoxy group, or an RCOO— group (R is An alkyl group having 1 to 4 carbon atoms).
(B) Curcumin (c) The following general formula (2)
[Wherein n represents an integer of 6 to 10. ]
Ubiquinone (d) Vitamin D
(E) Pakuritakise Lumpur