JP6429372B2 - Dispersant for poorly water-soluble substances containing hydrophobic peptides - Google Patents

Description

  The present invention relates to a dispersant for a poorly water-soluble substance containing a hydrophobic peptide.

  When a food and / or medicine is taken orally, the nutrients, functional ingredients contained in the food or medicine or the active ingredient of the medicine are absorbed from the digestive tract (for example, the intestinal tract). Needs to be dissolved in the solution in the digestive tract. However, when a nutrient, a functional ingredient or a drug contained in a food or drug is poorly water-soluble, the solubility and dissolution rate in the digestive juice is very small. In this case, dissolution in the digestive juice is the rate-limiting step of absorption of these components. Therefore, the absorption rate of orally ingested poorly water-soluble substances is very low, and the value may be in the order of several percent in some cases.

  As described above, when a poorly water-soluble physiologically active substance or drug is taken orally, the absorption rate and / or bioavailability is often lowered due to the low dissolution rate in the digestive fluid. . For this reason, a technique for combining a poorly water-soluble physiologically active substance or drug with various dispersants has been proposed and put into practical use. For example, clathrate formation with cyclodextrins, formation with water-soluble polymers or sugars, and microemulsification are well known.

  In addition to the above-described technique, a complex of a peptide (for example, a protein hydrolyzate) and a poorly water-soluble substance has been studied. For example, Patent Document 1 discloses a pharmaceutical preparation containing a poorly water-soluble active compound, wherein the poorly water-soluble active compound is dispersed and distributed in a non-crosslinked hydrophilic peptide having a molecular weight of more than 100 D. The formulation is characterized in that it releases the active compound in a dispersed form in aqueous solution with a particle size of less than 1 μm. According to the document, the peptide is selected from the group consisting of collagen, collagen inducer, collagen inducer mixture; gelatin, fractionated gelatin, collagen hydrolyzate, gelatin derivative; elastin hydrolyzate; and vegetable protein hydrolyzate Describe what can be done. This document does not describe the specific chemical structure and physical properties of the peptide.

  Non-Patent Documents 1 to 3 by the present inventors indicate that the hydrolyzate of casein and albumin, which are milk proteins, can improve dispersibility in water with respect to a specific poorly water-soluble physiologically active substance or drug. Is described. This document does not describe the specific chemical structure and physical properties of the milk protein hydrolyzate.

Japanese Patent No. 2975112

A. Inada, T. Oshima, H. Takahashi, Y. Baba, Enhancement of water solubility of indomethacin by complexation with protein hydrolysate, Int. J. Pharm., 453, p. 587-593 (2013) T. Oshima, A. Inada, Y. Baba, Evaluation of hydrophilic and hydrophobic balance for the complex between indomethacin and casein hydrolysate using aqueous two-phase system, Solvent Extr. Res. Dev. Jpn., Vol. 20, p. 71 -77, (2013) N. Matsushita, T. Oshima, H. Takahashi, Y. Baba, Enhanced water dispersibility of coenzyme Q10by complexation with albumin hydrolysate, J. Agric. Food Chem., 61, p. 5972-5978 (2013)

  As described above, it has been known that certain peptides can improve the dispersibility of a poorly water-soluble physiologically active substance or drug in water. However, the specific chemical structure and / or physical properties of peptides that can contribute to the improvement of dispersibility in water of poorly water-soluble substances have not been known. Moreover, the dispersibility improvement effect by the known peptide has not reached a satisfactory level.

  In view of the above problems, an object of the present invention is to provide means for stably dispersing a hardly water-soluble substance in water.

  As a result of various investigations on means for solving the above problems, the present inventors have found that a specific peptide having an amino acid sequence corresponding to a hydrolyzate of casein, which is a kind of milk protein, can hardly dissolve a water-insoluble substance in water. It was found that it can be stably dispersed. Based on the above findings, the present inventors have completed the present invention.

  That is, the gist of the present invention is as follows.

  (1) A dispersing agent for dispersing a poorly water-soluble substance containing a hydrophobic peptide.

(2) The hydrophobic peptide is:
(I) a peptide consisting of the amino acid sequence of SEQ ID NO: 1;
(Ii) a peptide consisting of the amino acid sequence of SEQ ID NO: 2;
(Iii) A peptide in which 1 to 5 amino acid residues are deleted, substituted or added in the peptide of (i) or (ii);
The dispersant according to (1) above, which is a peptide selected from the group consisting of:

  (3) A pharmaceutical composition comprising the dispersant according to (1) or (2) and at least one poorly water-soluble drug.

  (4) A food composition comprising the dispersant according to (1) or (2) and at least one poorly water-soluble substance.

  According to the present invention, it is possible to provide means for stably dispersing a hardly water-soluble substance in water.

  Problems, configurations, and effects other than those described above will be clarified by the following description of embodiments.

FIG. 1 is a scheme showing a method for preparing a casein-derived peptide fraction. Figure 2 is a diagram showing the apparent solubility of coenzyme Q ₁₀ was a peptide complexed with contained in each fraction of casein-derived peptides. FIG. 3 is a diagram showing typical particle size distribution diagrams of peptide fraction A and coenzyme Q ₁₀ / peptide A complex. FIG. 4 is a diagram showing the apparent solubility of paclitaxel complexed with a peptide contained in each fraction of casein-derived peptides. FIG. 5 is a graph showing the apparent solubility of curcumin complexed with peptides contained in each fraction of casein-derived peptides. FIG. 6 shows the apparent solubility of retinoic acid complexed with peptides contained in each fraction of casein-derived peptides. FIG. 7 is a diagram showing MS spectra of a casein-derived peptide mixture and peptide fraction A. (A) MS spectrum of casein-derived peptide mixture; (b) MS spectrum of peptide corresponding to the peak of 1881 m / z contained in peptide fraction A; (c) 1718 m / z contained in peptide fraction A MS spectrum of peptide corresponding to peak; (d) MS spectrum of peptide corresponding to peak of 2460 m / z contained in peptide fraction A; (e) 1202 m / z peak contained in peptide fraction A MS spectrum of the corresponding peptide.

  Hereinafter, preferred embodiments of the present invention will be described in detail.

<1: Dispersant>
The present inventors have found that a peptide having a specific amino acid sequence and high hydrophobicity can stably disperse a poorly water-soluble substance in water. Therefore, the present invention relates to a dispersing agent for dispersing a poorly water-soluble substance containing a hydrophobic peptide.

  The dispersant of the present invention can be used for dispersing a hardly soluble substance in water. In the present invention, “dispersion” is a state in which a poorly water-soluble substance as a solute is not substantially dissolved in water as a solvent, and the hardly water-soluble substance is not substantially precipitated in water. The “dispersion liquid” means a liquid in which a poorly water-soluble substance as a solute is present in such a state. Here, water as a solvent may contain one or more other components such as a buffering agent, if desired. For example, a poorly water-soluble substance is usually present in a colloidal form in a dispersion obtained using the dispersant of the present invention, forming a complex with the dispersant of the present invention. In this case, the average particle size of the colloid is usually in the range of 50 to 1000 nm, typically in the range of 100 to 500 nm. By forming a colloidal form having an average particle diameter in the above range, the complex of the dispersant of the present invention and the hardly water-soluble substance can be stably present without substantially precipitating in water. In addition, the average particle diameter of the colloid of the complex of the dispersant of the present invention and the hardly water-soluble substance can be determined using, for example, a dispersion containing the colloid using a nanoparticle analyzer by dynamic light scattering (DLS) It can be determined by analyzing.

In the present invention, the “poorly water-soluble substance” means a normal condition, for example, a temperature in the range of usually 0 to 100 ° C., typically 0 to 60 ° C., particularly 0 to 30 ° C., usually about 1 atm. Means a substance that has substantially no solubility in water or aqueous buffer solutions, i.e. very low water solubility, at pressure and / or usually in the range of 5-8, typically in the range of 6-8 . For example, the solubility of a poorly water-soluble substance in water is usually less than 10 × 10 ⁻³ g / dm ³ and typically less than 5 × 10 ⁻³ g / dm ³ under the normal conditions. By applying the dispersant of the present invention to such a hardly water-soluble substance, the apparent solubility of the water-soluble substance can be greatly improved. In the present invention, the “apparent solubility” means the amount of a hardly soluble substance present in the dispersion state (usually in the colloidal form) in an aqueous dispersion. For example, in the dispersion obtained using the dispersant of the present invention, the apparent solubility of the poorly water-soluble substance in water is usually 5 × 10 ^{−3 to} 100 × 10 ⁻³ g / dm under the above normal conditions. The range is ³ , typically 10 × 10 ⁻³ to 50 × 10 ⁻³ g / dm ³ . Even if the dispersant of the present invention is a poorly water-soluble substance having the above-described characteristics, it can be stably dispersed in water with improved apparent solubility. In addition, the solubility and apparent solubility of a hardly water-soluble substance can be determined by the following method, for example. Under the normal conditions, only the poorly water-soluble substance, or the poorly water-soluble substance and the dispersant of the present invention are added and dispersed in water or an aqueous solution containing one or more other components such as a buffer as required. The obtained dispersion is treated by filtration or centrifugation to obtain a supernatant fraction. The poorly water-soluble substance contained in the supernatant fraction is quantified by analytical means such as high performance liquid chromatography (HPLC) analysis or spectrophotometric analysis to determine the solubility or apparent solubility.

Examples of poorly water-soluble substances to which the dispersant of the present invention can be applied include coenzyme Q ₁₀ , paclitaxel, curcumin, retinoic acid, β-carotene, α-tocopherol (vitamin E), folic acid, lecithin, aspirin, indomethacin, ibuprofen , Etazamide, tocopherol nicotinate, barbital, pentobarbital, diazepam, phenytoin, phenobarbital, prednisolone, tolbutamide, glibenclamide, doxorubicin, gentamicin, penicillin, streptomycin and erythromycin. All of these drugs or substances have high fat solubility and very low water solubility. Therefore, by applying the dispersant of the present invention to these hardly water-soluble substances, the apparent solubility of the hardly water-soluble substance can be improved and stably dispersed in water.

In the dispersant of the present invention, the hydrophobic peptide is:
(I) a peptide consisting of the amino acid sequence of SEQ ID NO: 1;
(Ii) a peptide consisting of the amino acid sequence of SEQ ID NO: 2;
(Iii) A peptide in which 1 to 5 amino acid residues are deleted, substituted or added in the peptide of (i) or (ii);
A peptide selected from the group consisting of The peptide of (i) has an amino acid sequence (amino acid sequence: QEPVLGPVRGPFPIIV; SEQ ID NO: 1) corresponding to a peptide fragment located 16 residues from the C-terminal side of β-casein, which is a milk protein. The peptide (ii) has an amino acid sequence (amino acid sequence: YQEPVLGPVRGPFPIIV; SEQ ID NO: 2) corresponding to a peptide fragment at a position of 17 residues from the C-terminal side of β-casein, which is a milk protein. The peptides (i) and (ii) have high hydrophobicity. Also, these peptides can be present in a colloidal form in water, complexed with a poorly water-soluble substance. Therefore, the peptide having the amino acid sequence can stably disperse the poorly water-soluble substance in water.

  The hydrophobic peptide that can be used as the dispersant of the present invention has a higher hydrophobicity than other peptides. For example, the hydrophobic peptide typically has a grand average of hydropathicity (GRAVY) score of 0 to 2.5, and typically has a GRAVY score in the range of 0.4 to 1.5. Hydrophobic peptides having a GRAVY score in the above range can be present in colloidal form in water, complexed with poorly water-soluble substances. Therefore, a hydrophobic peptide having a GRAVY score in the above range can stably disperse a poorly water-soluble substance in water. The GRAVY score is a known index that quantitatively expresses the hydrophobicity / hydrophilicity of a peptide. For the GRAVY score of hydrophobic peptides, refer to known literature (J. Kyte and RF Doolittle, “A simple method for displaying the hydropathic character of a protein,” Journal of Molecular Biology, 157, 105-132 (1982)). Can be calculated.

  In the peptide of (iii), the deleted, substituted or added amino acid residue is usually in the range of 1 to 5, preferably in the range of 1 to 3, preferably 1 to 2. More preferably, it is in the range of 1 and more preferably 1. The peptide (iii) preferably has the above deletion, substitution or addition and has high hydrophobicity. The preferred peptide (iii) is the peptide of (i) or (ii), wherein the amino acid residues at positions 1-5, 1-3, 1-2, or 1 from the N-terminal side are deleted or substituted It is a peptide having high hydrophobicity. The peptide of (iii) having high hydrophobicity has a GRAVY score in the range described above. By using the peptide (iii), high dispersibility substantially equivalent to the peptides (i) and (ii) can be expressed.

  The dispersant of the present invention is a peptide having the specific amino acid sequence described above, which corresponds to a hydrolyzate of milk protein. Therefore, the dispersant of the present invention can be produced by using a peptide forming means usually used in the art based on the specific amino acid sequence described above. As a means for peptide formation, for example, a solid phase or liquid phase peptide synthesis method can be used. Alternatively, in a mammal (for example, bovine) capable of producing a peptide having a predetermined amino acid sequence or a full-length protein thereof, a DNA encoding the peptide or the full-length protein is used to transform in a transformation system such as E. coli or budding yeast. A method of expressing a large amount of a modified peptide or protein may be used. Alternatively, from a mammal (eg, bovine) product (eg, milk protein such as casein or raw milk containing milk protein), tissue or cells capable of producing a peptide having a predetermined amino acid sequence or a full-length protein thereof, A method for purifying a peptide or its full-length protein may be used. Alternatively, a peptide having a predetermined amino acid sequence produced in advance or a full-length protein thereof may be purchased and used as it is or after purification or the like. Either case is included in the embodiment of the method for producing a dispersant of the present invention.

  For example, when the dispersant of the present invention is produced from a milk protein using the method for purifying a peptide having the specific amino acid sequence described above, it can be carried out by a method including the following steps. Milk protein is hydrolyzed by enzymatic digestion with protein digestive enzymes or hydrolysis with acid or alkali. The obtained mixture of hydrolyzed peptides is separated by means commonly used in the art such as ammonium sulfate fractionation, ultrafiltration and chromatography (eg gel filtration chromatography), and the desired peptide is separated from the mixture. Purify and isolate. For example, when the peptides (i) and (ii) are produced from casein, it can be carried out by a method including the following steps. From the casein hydrolyzed peptide mixture, the fraction precipitated with ammonium sulfate (ammonium sulfate) in the range of 10.2 to 19.5% by mass is separated by ammonium sulfate fractionation, and then the fraction having a molecular weight of 5,000 or more is separated by ultrafiltration. . Thereby, the peptides (i) and (ii) can be obtained.

<2: Use of dispersant>
The dispersant of the present invention can be used to disperse various poorly water-soluble substances in water. Therefore, the present invention relates to a food composition containing the dispersant of the present invention and at least one poorly water-soluble substance.

In the food composition of the present invention, the at least one poorly water-soluble substance is a substance included in the poorly water-soluble substance described above, and is a substance usually used as a food ingredient. Examples of the at least one poorly water-soluble substance include coenzyme Q ₁₀ , curcumin, retinoic acid, β-carotene, α-tocopherol (vitamin E), folic acid and lecithin. All of these poorly water-soluble substances have high fat solubility and very low water solubility. Therefore, by applying the dispersant of the present invention to these hardly water-soluble substances, the apparent solubility of the hardly water-soluble substances can be improved and stably dispersed in water. Thereby, the absorption rate of a poorly water-soluble substance and / or bioavailability (bioavailability) can be improved.

  In the food composition of the present invention, the dispersant of the present invention is usually in a mass ratio of more than 1, for example, a mass ratio of 2 or more, typically a mass of 5 or more, with respect to the substance to be dispersed. In particular, it is contained in a mass ratio of 10 or more. In the food composition of the present invention, the content of the dispersant of the present invention is preferably in the range of 2: 1 to 500: 1 in terms of mass ratio to the substance to be dispersed, and is 5: 1 to 200: 1. More preferably, it is in the range of 10: 1 to 150: 1, more preferably in the range of 50: 1 to 150: 1. When content of the dispersing agent of this invention is below the said lower limit, it may become difficult to make the dispersing agent of this invention and a hardly water-soluble substance complex. Therefore, by containing the dispersant of the present invention in the above content, the food composition of the present invention combines the dispersant of the present invention with a poorly water-soluble substance to form a poorly water-soluble substance. It can be contained in a stably dispersed form.

  The food composition of the present invention is processed into various food forms usually used in the art, for example, solid (eg, powder, tablet or granule), paste, or liquid food. Can do. In addition to the above ingredients, the food composition of the present invention comprises one or more food ingredients, and one or more food-acceptable preservatives, stabilizers, leavening agents, surfactants, oily liquids, and buffering agents. , Antioxidants, sweeteners, flavoring agents, dyes and pigments may be included.

  The food composition of the present invention may be used as a food as it is, or may be used as a food raw material by mixing with other foods or food ingredients. The form of the food composition of the present invention may be, for example, a form of a health supplement such as a supplement in addition to a normal food or beverage. Examples of the form of the health supplement include tablets, capsules, elixirs, microcapsules, tablets, syrups, suspensions, and the like with sugar coating or a soluble coating as necessary. Additives that can be incorporated into tablets or capsules are not limited, but include, for example, binders such as gelatin, corn starch, gum tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch Swelling agents such as gelatin and alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry. When the preparation is a capsule, it may further contain a liquid carrier such as fats and oils.

  The dispersant of the present invention contained in the food composition of the present invention is a peptide having an amino acid sequence corresponding to a milk protein hydrolyzate. For this reason, the food composition containing the dispersant of the present invention is safe and has low toxicity. Therefore, the food composition of the present invention can be used without substantially affecting the health of the intaker.

  The present invention also relates to a pharmaceutical composition comprising the dispersant of the present invention and at least one poorly water-soluble drug.

In the pharmaceutical composition of the present invention, at least one poorly water-soluble drug is a substance included in the poorly water-soluble substance described above and having a specific pharmacological activity. Examples of at least one poorly water-soluble drug include coenzyme Q ₁₀ , paclitaxel, aspirin, indomethacin, ibuprofen, etenzaamide, tocopherol nicotinate, barbital, pentobarbital, diazepam, phenytoin, phenobarbital, prednisolone, tolbutamide, glibenclamide, Mention may be made of doxorubicin, gentamicin, penicillin, streptomycin and erythromycin. All of these poorly water-soluble drugs have high fat solubility and very low water solubility. Therefore, by applying the dispersant of the present invention to these poorly water-soluble drugs, the apparent solubility of the poorly water-soluble drug can be improved and stably dispersed in water. Thereby, the absorption rate and / or bioavailability (bioavailability) of a poorly water-soluble drug can be improved.

  In the pharmaceutical composition of the present invention, the dispersant of the present invention is usually in a mass ratio of more than 1, for example, a mass ratio of 2 or more, typically a mass of 5 or more, with respect to the drug to be dispersed. In particular, it is contained in a mass ratio of 10 or more. In the pharmaceutical composition of the present invention, the content of the dispersant of the present invention is preferably in the range of 2: 1 to 500: 1 by mass ratio to the drug to be dispersed, and is 5: 1 to 200: 1. More preferably, it is in the range of 10: 1 to 150: 1, more preferably in the range of 50: 1 to 150: 1. When the content of the dispersant of the present invention is not more than the lower limit value, it may be difficult to complex the dispersant of the present invention with a poorly water-soluble drug. Therefore, by containing the dispersant of the present invention in the above content, the pharmaceutical composition of the present invention combines the dispersant of the present invention with a poorly water-soluble drug to form a poorly water-soluble drug. It can be contained in a stably dispersed form.

  The pharmaceutical composition of the present invention can be formulated into various dosage forms commonly used in the art depending on the desired administration method. In addition to the above components, the pharmaceutical composition of the present invention comprises one or more pharmaceutically acceptable carriers, excipients, binders, vehicles, solubilizers, preservatives, stabilizers, swelling agents, lubricants. , Surfactants, oily liquids, buffers, soothing agents, antioxidants, sweetening agents, flavoring agents, and the like.

  The pharmaceutical composition of the present invention is usually a preparation for use in oral administration. Examples of the preparation for use in oral administration include tablets, capsules, elixirs, microcapsules, tablets, syrups, suspensions, and the like with sugar coating and a soluble coating as necessary. . Additives that can be incorporated into tablets or capsules are not limited, but include, for example, binders such as gelatin, corn starch, gum tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch Swelling agents such as gelatin and alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry. When the preparation is a capsule, it may further contain a liquid carrier such as fats and oils.

  The pharmaceutical composition of the present invention can contain a poorly water-soluble drug in a stably dispersed form. Therefore, the pharmaceutical composition of the present invention can also be formulated as a depot preparation for use in parenteral administration. In this case, the pharmaceutical composition of the present invention in the form of a depot preparation can be administered, for example, by subcutaneous or intramuscular implantation or by intramuscular injection. By applying the pharmaceutical composition of the present invention to a depot preparation, a slightly water-soluble drug can be continuously released over a long period of time.

  The dispersant of the present invention contained in the pharmaceutical composition of the present invention is a peptide having an amino acid sequence corresponding to a milk protein hydrolyzate. For this reason, the pharmaceutical composition containing the dispersant of the present invention is safe and has low toxicity. Therefore, the pharmaceutical composition of the present invention can be applied to various subjects having various symptoms, diseases and / or disorders that are prevented or treated by at least one poorly water-soluble drug. Examples of the subject include a human or non-human mammal (for example, a warm-blooded animal such as a pig, dog, cow, rat, mouse, guinea pig, rabbit, chicken, sheep, cat, monkey, baboon or chimpanzee). Or a patient can be mentioned. By administering the pharmaceutical composition of the present invention to the subject, various symptoms, diseases and / or disorders that are prevented or treated by at least one poorly water-soluble drug can be prevented or treated.

  As used herein, “prevention” means substantially preventing the occurrence (onset or onset) of symptoms, diseases and / or disorders. In the present specification, “treatment” means to suppress (e.g., suppress progression), relieve, repair, and / or cure a symptom, disease, and / or disorder that has occurred (onset or onset).

  The dosage form of the pharmaceutical composition of the present invention may be a unit dosage form or a multiple dosage form. Moreover, the administration route and frequency | count of administration of the pharmaceutical composition of this invention are not specifically limited, Orally, it may administer once or multiple times.

  When the pharmaceutical composition of the present invention is administered to a subject, particularly a human patient, the exact dosage and number of doses depends on the subject's age, sex, the exact state of the condition, disease and / or disorder to be prevented or treated. In view of many factors such as (eg severity) and route of administration, the attending physician should ultimately determine a therapeutically effective dose and number of doses. Therefore, in the pharmaceutical composition of the present invention, at least one poorly water-soluble drug as an active ingredient is usually contained in a therapeutically effective amount.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

<Experiment I: Preparation of casein-derived peptide fraction>
Distilled water (500 cm ³⁾ was adjusted to 40 ° C., and 25 g of casein was added to the distilled water little by little while stirring (50 g / dm ³ ). To the mixture, 5 mol / dm ³ sodium hydroxide was added dropwise little by little, and the mixture was stirred for 2 hours while maintaining the pH at around 7.8. As a result, casein was dissolved to obtain a uniform suspension.

The temperature of the resulting suspension was set to 45 ± 1 ° C. While stirring the suspension, 125 mg of α-chymotrypsin (enzyme) and 1.5 g of calcium chloride dihydrate were added. Using a pH controller, 1 mol / dm ³ sodium hydroxide was added dropwise to the enzyme reaction solution to maintain the pH at around 7.8. Thereby, casein was digested with enzyme. After 6 hours from the addition of the enzyme, the enzyme reaction solution was maintained at 80 ° C. for 5 minutes to inactivate the enzyme. Thereafter, the enzyme reaction solution was freeze-dried to obtain 24.5 g of a white powder. The above series of operations was performed again to obtain 24.6 g of white powder. From the two operations, 49.1 g of the peptide mixed sample was obtained.

15.0 g of the obtained peptide mixed sample was added to 300 cm ³ of distilled water and stirred. The mixture was stirred for 30 minutes under ice cooling. Subsequently, the mixture was centrifuged at 10,000 × g for 10 minutes at 4 ° C. to obtain a supernatant. The supernatant was fractionated as follows in accordance with the fractionation procedure shown in FIG.

Ammonium sulfate was added to the supernatant to make a 10.2% by mass ammonium sulfate solution. The resulting ammonium sulfate solution was stirred for 30 minutes. Thereafter, the ammonium sulfate solution was centrifuged at 4 ° C. and 20,000 × g for 10 minutes, and the supernatant was recovered (supernatant a). The precipitate (precipitate a) obtained by the 10.2 mass% ammonium sulfate fraction was dissolved in water to obtain a 250 cm ³ aqueous solution. The obtained aqueous solution was subjected to ultrafiltration with an ultrafiltration membrane having a molecular weight cut off of 1,000 (Ultracel 1 KDa manufactured by Millipore). The obtained filtration residue was collected and lyophilized to obtain a small amount of white powder.

  Next, ammonium sulfate was further added to the supernatant a to obtain a 19.5 mass% ammonium sulfate solution. The obtained ammonium sulfate solution was centrifuged to separate into supernatant b and precipitate b. The precipitate b was dissolved in water to obtain an aqueous solution. The obtained aqueous solution was ultrafiltered through an ultrafiltration membrane (Ultracel 5 KDa manufactured by Millipore) having a molecular weight cut-off of 5,000. The obtained filtration residue was collected and lyophilized to obtain a white powder (peptide fraction A). Subsequently, the ultrafiltration permeate was further ultrafiltered through an ultrafiltration membrane (Ultracel 3 KDa manufactured by Millipore) having a fractional molecular weight of 3,000. The obtained filtration residue was recovered and lyophilized to obtain a white powder (peptide fraction B). Similarly, the ultrafiltration permeate was further ultrafiltered with an ultrafiltration membrane having a molecular weight cut off of 1,000 (Ultracel 1 KDa manufactured by Millipore). The obtained filtration residue was collected and lyophilized to obtain a white powder (peptide fraction C).

  Ammonium sulfate was further added to the supernatant b to obtain a 28.1% by mass ammonium sulfate solution. The obtained ammonium sulfate solution was centrifuged to separate into supernatant c and precipitate c. The precipitate c was subjected to sequential ultrafiltration using ultrafiltration membranes with a molecular weight cut off of 5,000, 3,000, and 1,000 in the same procedure as the precipitate b to obtain peptide fractions D, E, and F.

  Ammonium sulfate was further added to the supernatant c to obtain a 35.9% by mass ammonium sulfate solution. The obtained ammonium sulfate solution was centrifuged to separate into supernatant d and precipitate d. The precipitate d was sequentially ultrafiltered in the same procedure as the precipitate b to obtain peptide fractions G, H and I.

  Ammonium sulfate was further added to the supernatant d to obtain a 43.4% by mass ammonium sulfate solution. The obtained ammonium sulfate solution was centrifuged to separate into supernatant e and precipitate e. Precipitate e was sequentially ultrafiltered in the same procedure as precipitate b to obtain peptide fractions J, K and L. The supernatant e was not used.

In all the fractionation operations described above, the pH of the solution was adjusted while dropping 6 mol / dm ³ hydrochloric acid or 5 mol / dm ³ sodium hydroxide so that the pH was around 7.0.

  The yield of each peptide fraction obtained by the fractionation operation is shown in Table 1. In 10.2 mass% ammonium sulfate solution, the peptide hardly precipitated. In contrast, increasing the ammonium sulfate concentration to 19.5 mass%, 28.1 mass%, 35.9 mass% or 43.4 mass% gradually precipitated the peptide. Among all fractions, the peptide fraction (peptide fraction G) that precipitated as a 35.9 mass% ammonium sulfate fraction and was obtained as a filtration residue by separation with an ultrafiltration membrane with a molecular weight cut off of 5,000 was the highest yield. It was big.

<Experiment II: Coenzyme Q ₁₀ · peptide preparation of conjugates, and dispersible Evaluation>
Peptide fractions A to L obtained in Experiment I were weighed 20 mg each, and 10 cm ³ of distilled water was added thereto to obtain an aqueous solution (2.0 g / dm ³ ). To the resulting aqueous peptide solution, 1.0 cm ^{3 of} an acetone solution of 1.0 g / dm ³ of coenzyme Q ₁₀ was added and mixed. By the operation, each peptide aqueous solution, a peptide and coenzyme Q _10, 20: including at a mass ratio. The mixed aqueous peptide solution was shaken at 30 ° C. for 2.0 hours. Acetone contained in the obtained liquid mixture was distilled off under reduced pressure. The resulting mixture was lyophilized to give coenzyme Q ₁₀ · peptide complex as a powder of yellowish color.

The resulting coenzyme Q ₁₀ · peptide complex was 17 mg weighed. To the weighed complex, 10 cm ^{3 of} 0.010 mol / dm ³ phosphate buffer adjusted to pH 7 was added. The resulting mixture was stirred for 15 seconds using a vortex mixer. Thereafter, the mixed solution was filtered using a Φ0.8 μm membrane filter (DISMIC 25CS080AN manufactured by Advantech). To the obtained filtrate 0.3 cm ³ , methanol / ethanol mixed solution (volume ratio 13: 7) 0.7 cm ³ was added for dilution. The diluent, high performance liquid chromatography (HPLC, column: Waters Ltd. XBridge BEH130 C ₁₈ 3.5 μm; flow rate: 0.5 mL / min; measurement wavelength: 275 nm) by analyzing at, the coenzyme Q ₁₀ that has passed through the filter Apparent solubility was quantified.

Figure 2 shows the apparent solubility of coenzyme Q ₁₀ complexed with peptide contained in each peptide fraction. In the figure, the apparent solubility represents the concentration of coenzyme Q _10, which has passed through the membrane filter Φ0.8 μm. As shown in FIG. 2, coenzyme Q ₁₀ precipitated with 19.5 mass% ammonium sulfate fraction and complexed with peptide fraction A obtained as a filtration residue by separation with an ultrafiltration membrane with a molecular weight cut off of 5,000, Compared with the complex with other peptide fractions, the apparent solubility was more than 10 times higher. The results of this experiment, relatively transparent hard peptide fraction ultrafiltration membrane with a hydrophobic have been shown to be an excellent dispersing agent for coenzyme Q _10.

<Experiment III: the particle size of the peptides and coenzyme Q ₁₀ · peptide complex>
Weigh 5.0 mg of peptide fraction A obtained in Experiment I, add 5.0 cm ^{3 of} 0.010 mol / dm ³ phosphate buffer (pH 7.0) to prepare a 1.0 g / dm ³ peptide dispersion. did. Using a nanoparticle analyzer (Horiba Nano Partica SZ-100) by the dynamic light scattering method (DLS), the particle size of the peptide dispersion filtered using a Φ0.8 μm membrane filter (DISMIC 25CS080AN manufactured by Advantech) Measured.

In the same procedure as in Experiment II, an aqueous solution of peptide fraction A (2.0 g / dm ³ ) and 1.0 g / dm ³ of coenzyme Q ₁₀ in acetone (1.0 g / dm ³ ) were mixed at a volume ratio of 10: 1. Te, peptides and coenzyme Q ₁₀ 20: including at a mass ratio to prepare a coenzyme Q ₁₀ · peptide complex. 10 mg of this complex was mixed with 5.0 cm ^{3 of} 0.010 mol / dm ³ phosphate buffer (pH 7.0) to prepare a 1.0 g / dm ³ complex dispersion. The obtained complex dispersion was centrifuged at 3,310 × g for 5 minutes, and further filtered using a membrane filter of Φ0.8 μm. The particle diameter of the obtained composite dispersion was measured using a nanoparticle analyzer (nano Partica SZ-100) by a dynamic light scattering method as described above.

Figure 3 shows a typical particle size distribution diagram of Fai0.8 [mu] m membrane filter the transmitted peptide fraction A, and coenzyme Q ₁₀ · Peptide A complex. The dispersion of peptide fraction A was a slightly collided stable colloidal solution, and the cumulant average particle size was about 180 nm. In contrast, dispersions of coenzyme Q ₁₀ · Peptide A complex is a colloidal solution tinged slightly yellowish white, cumulant average particle diameter was about 250 nm. The results of this experiment, coenzyme Q ₁₀ · Peptide A complex be stably dispersed in an aqueous solution as colloidal particles was suggested. Moreover, it was suggested that the particle diameter becomes larger than the peptide of a raw material.

<Experiment IV: Preparation of paclitaxel-peptide complex and evaluation of dispersibility>
Peptide fractions A to L obtained in Experiment I were dissolved in distilled water to obtain an aqueous solution (50 g / dm ³ ). The resulting peptide solution 0.50 cm ^3, was added and mixed ethanol solution of paclitaxel by 0.50 cm ^3. Through the above operation, each aqueous peptide solution contains the peptide and paclitaxel in a mass ratio of 50: 1. The mixed aqueous peptide solution was shaken at 30 ° C. for 1.0 hour. Ethanol contained in the obtained mixture was distilled off under reduced pressure. The obtained mixed solution was freeze-dried to obtain a paclitaxel / peptide complex as a white powder.

To the total amount of the obtained paclitaxel peptide complex, 0.500 cm ^{3 of} 0.100 mol / dm ³ phosphate buffer adjusted to pH 7.2 was added. The resulting mixture was shaken at 30 ° C. for 1.0 hour. Thereafter, the mixed solution was filtered using a centrifugal filter unit (Ultrafree manufactured by Millipore, filtration membrane: Durapore PVDF membrane 0.45 μm) having a diameter of 0.45 μm. The pH of the obtained filtrate was measured. The filtrate 0.040 cm ³ was mixed with 0.060 cm ^{3 of} acetonitrile. The resulting mixture was centrifuged at 3,000 rpm for 3 minutes. The obtained supernatant was subjected to high performance liquid chromatography (HPLC, column: Waters XBridge BEH130 C ₁₈ 3.5 μm; mobile phase: phosphate buffer (pH 7.2, 10 mM): acetonitrile = 50: 50; flow rate: 1.0 mL / min; measurement wavelength: 227 nm), the apparent solubility of paclitaxel contained in the supernatant was quantified.

  FIG. 4 shows the apparent solubility of paclitaxel complexed with peptides contained in each peptide fraction. In the figure, the apparent solubility represents the concentration of paclitaxel that has passed through a centrifugal filter unit of Φ0.45 μm. As shown in FIG. 4, paclitaxel precipitated with a 19.5 mass% ammonium sulfate fraction and complexed with peptide fraction A obtained as a filtration residue by separation with an ultrafiltration membrane having a molecular weight cut off of 5,000, The apparent solubility was more than 3.9 times higher than the complex with the peptide fraction. From the results of this experiment, it was shown that the peptide fraction A is an excellent dispersant for paclitaxel, similar to the results of Experiment II.

<Experiment V: Preparation of Curcumin / Peptide Complex and Evaluation of Dispersibility>
Peptide fractions A to L obtained in Experiment I were dissolved in distilled water to obtain an aqueous solution (10 g / dm ³ ). 0.20 cm ³ of 0.1 g / dm ³ of a solution of curcumin in acetone was added to 0.20 cm ^{3 of the} obtained peptide aqueous solution and mixed. By the above operation, each peptide aqueous solution contains the peptide and curcumin in a mass ratio of 100: 1. The mixed aqueous peptide solution was shaken at 30 ° C. for 1.0 hour. Acetone contained in the obtained liquid mixture was distilled off under reduced pressure. The obtained mixed solution was freeze-dried to obtain a curcumin / peptide complex as a pale yellow powder.

To the total amount of the obtained curcumin peptide complex, 0.010 mol / dm ³ phosphate buffer adjusted to pH 6.8 was added in an amount of 1.2 cm ³ . The resulting mixture was shaken at 30 ° C. for 1.0 hour. Thereafter, the mixed solution was filtered using a Φ0.8 μm membrane filter (DISMIC 25CS080AN manufactured by Advantech). The apparent solubility of curcumin contained in the filtrate was quantified by measuring the absorbance of the obtained filtrate using an ultraviolet-visible absorptiometer.

  FIG. 5 shows the apparent solubility of curcumin complexed with peptides contained in each peptide fraction. In the figure, the apparent solubility represents the concentration of curcumin that has passed through a membrane filter of Φ0.8 μm. As shown in FIG. 5, curcumin precipitated with a 19.5% by mass ammonium sulfate fraction and complexed with peptide fraction A obtained as a filtration residue by separation with an ultrafiltration membrane with a molecular weight cut off of 5,000, Compared with the complex with the peptide fraction, the apparent solubility was 3.8 times higher. From the results of this experiment, it was shown that the peptide fraction A is an excellent dispersant for curcumin, similar to the results of Experiments II and IV.

<Experiment VI: Preparation of Retinoic Acid / Peptide Complex and Evaluation of Dispersibility>
Peptide fractions A to K (25 mg each) obtained in Experiment I were dissolved in distilled water to obtain an aqueous solution (10 g / dm ³ ). To the obtained aqueous peptide solution (2.5 cm ³⁾ , 0.10 g / dm ³ of an acetone solution of retinoic acid was added 2.5 cm ^{3 at a} time and mixed. By the above operation, each peptide aqueous solution contains the peptide and retinoic acid in a mass ratio of 100: 1. The mixed aqueous peptide solution was shaken at 30 ° C. for 1.0 hour. Acetone contained in the obtained liquid mixture was distilled off under reduced pressure. The obtained mixed solution was freeze-dried to obtain a retinoic acid / peptide complex as a yellowish white powder.

To the total amount of the obtained retinoic acid / peptide complex, 5.0 cm ^{3 of} 0.100 mol / dm ³ phosphate buffer adjusted to pH 7 was added. The resulting mixture was shaken at 30 ° C. for 1.0 hour. Thereafter, the mixed solution was filtered using a Φ0.8 μm membrane filter (DISMIC 25CS080AN manufactured by Advantech). The apparent absorbance of the retinoic acid contained in the filtrate was quantified by measuring the absorbance of the obtained filtrate using an ultraviolet-visible absorptiometer.

  FIG. 6 shows the apparent solubility of retinoic acid complexed with peptides contained in each peptide fraction. In the figure, the apparent solubility represents the concentration of retinoic acid that has passed through a membrane filter of Φ0.8 μm. As shown in FIG. 6, retinoic acid precipitated with a 19.5% by mass ammonium sulfate fraction and complexed with peptide fraction A obtained as a filtration residue by separation with an ultrafiltration membrane with a molecular weight cut off of 5,000, Compared with the complex with the peptide fraction, the apparent solubility was 10.3 times higher. From the results of this experiment, it was shown that the peptide fraction A is an excellent dispersant for retinoic acid, similar to the results of Experiments II, IV and V.

<Experiment VII: Analysis of molecular weight and amino acid sequence of peptide contained in peptide fraction A>
Peptide fraction A obtained in Experiment I was dissolved in distilled water to obtain an aqueous solution (0.5 g / dm ³ ). This aqueous solution was centrifuged at 3,000 × g for 3 minutes. The obtained supernatant was used as a peptide solution. This was mixed with the peptide solution using α-cyano-4-hydroxycinnamic acid (HCCA) as the matrix. The mixture was dried and the resulting samples were analyzed using a MALDI-TOF MS mass spectrometer (Bruker autoflex III TOF / TOF). Peptide Calibration Standard II (Bruker) was used as a calibration standard sample. The peptide contained in the sample was identified using a database search engine of Mascot (Matrix Science) based on the fragment pattern of the MS / MS spectrum obtained by MALDI-TOF MS mass spectrometry. Swiss-Prot was used for the amino acid sequence database.

FIG. 7 shows the MS spectra of casein-derived peptide mixture and peptide fraction A. Peaks at 1202, 1632, 1718, 1881, and 2460 m / z were confirmed as main peaks of the casein-derived peptide mixture (FIG. 7 (a)). In addition, 1718 and 1881 m / z peaks were confirmed as the main peaks of peptide fraction A. As a result of analyzing the amino acid sequence of the peptide contained in the peptide fraction A by MALDI LIFT-TOF / TOF MS measurement, the peaks at 1202, 1632, 1718 and 1881 m / z are respectively in the β-casein fragment, The 2460 m / z peak was shown to correspond to the α _S1 -casein fragment, respectively.

  Peptides corresponding to the 1718 and 1881 m / z peaks detected as the main peak of peptide fraction A are each 16 residues (amino acid sequence: QEPVLGPVRGPFPIIV; SEQ ID NO: 1) from the C-terminal side of β-casein. The peptide and 17 residues (amino acid sequence: YQEPVLGPVRGPFPIIV; SEQ ID NO: 2) were identified. As an index of hydrophobicity / hydrophilicity of identified peptides, each peptide's grand average of hydropathicity (GRAVY) (J. Kyte and RF Doolittle, “A simple method for displaying the hydropathic character of a protein,” Journal of Molecular Biology , 157, 105-132 (1982)). The GRAVY scores for peptides corresponding to 1718 and 1881 m / z were 0.59 and 0.48, respectively, suggesting that they are more hydrophobic than the other peptides. As shown in Experiments II, IV, V and VI, it is presumed that such a hydrophobic peptide contained in peptide fraction A contributes to the improvement of dispersibility of the poorly water-soluble substance.

Claims (3)

following:
(I) a peptide consisting of the amino acid sequence of SEQ ID NO: 1;
(Ii) a peptide consisting of the amino acid sequence of SEQ ID NO: 2;
A dispersing agent for dispersing a poorly water-soluble substance, comprising a hydrophobic peptide selected from the group consisting of : A pharmaceutical composition comprising the dispersant according to claim 1 and at least one poorly water-soluble drug. A food composition containing the dispersant according to claim 1 and at least one poorly water-soluble substance.

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