JP6045784B2 - Preparation containing hard-to-color pyrroloquinoline quinones - Google Patents

Description

  The present invention relates to a pyrroloquinoline quinone preparation that does not color cells.

  Pyrroloquinoline quinone (hereinafter sometimes referred to as PQQ) is a substance present in molds, yeasts, and eukaryotes such as mammals, and is known to function as a redox coenzyme. Yes. PQQ can usually be isolated and identified from a metabolite of a methanol-utilizing bacterium. At present, PQQ is not approved for use as a pharmaceutical or food, but it promotes cell proliferation (Patent Document 1), prevents liver disease (Patent Document 2), and suppresses melanin production (Patent Document 3). It has been revealed that it has various actions such as nerve growth factor production promoting action (Patent Document 4). Therefore, PQQ is promising as an active ingredient of pharmaceuticals and functional foods.

JP 61-058584 A JP-A-63-192717 Japanese Patent Laid-Open No. 08-020512 Japanese Patent Laid-Open No. 06-21660

  However, when the present inventors have been studying a pyrroloquinoline quinone preparation, when it is contained in the mouth, there arises a problem that the oral cavity turns dark, and further, the oral discoloration continues for several weeks. It became clear that there was a problem that. Moreover, although a metal component is often added as nutrition enhancement, there also exists a problem which discoloration arises also in that case.

  Therefore, as a result of intensive research to solve the above problems, the present inventors have found that the discoloration in the oral cavity due to the ingestion of the PQQ preparation is such that PQQ taken into the oral mucosal cells and the epidermal cells of the tongue is other than the intracellular alkali metal. It has been found that the color changes in various colors such as black-green, brown, dark brown, black, and the like, and as a result, the appearance color of the cells changes. Furthermore, the present inventors have found that if a chelating agent is contained in the PQQ preparation, the cells can be prevented from being colored and the problem can be solved.

That is, the present invention provides the following general formula (I):

(Wherein R ₁ , R ₂ and R ₃ are the same or different and each represents a hydrogen atom, a phenyl group, or an alkyl group, aralkyl group, alkylaryl group, alkenyl group or alkynyl group having 1 to 6 carbon atoms). The above-described problems are solved by providing a hardly-colorable pyrroloquinoline quinone-containing preparation characterized by containing a pyrroloquinoline quinone or its alkali metal salt and a chelating agent.

  According to the present invention, it is possible to provide a pyrroloquinoline quinone-containing preparation that does not discolor in cells or color cells even when ingested or administered. The pyrroloquinoline quinone-containing preparation of the present invention does not color cells even when taken orally, for example, when a tablet is taken in the mouth or taken as a beverage, or when administered dermally, and thus the oral cavity is not colored. And no discoloration of the skin, so it can be taken or administered with confidence.

Effect of EDTA on coloring of model intracellular solution by PQQ. A: Fetal bovine serum, B: α-MEM + 10% FBS.

  The pyrroloquinoline quinone-containing preparation of the present invention (hereinafter referred to as the present preparation) is a metal cation coexisting with pyrroloquinoline quinone (PQQ) in the preparation when ingested or administered, for example, in the preparation or the PQQ. Since the binding to the metal cation present in the cell into which the species is taken up is dissociated or suppressed by the action of the chelating agent in the preparation, discoloration of the PQQs due to the binding with the metal cation occurs. As a result, coloring of the cells into which the PQQs have been taken up is prevented. That is, the preparation of the present invention is a preparation that is difficult to color without coloring cells, more specifically, a preparation that is difficult to color cells.

In this specification, the “colorability” of a substance refers to a property that changes the color of the appearance of an object having the substance. Further, in the present specification, “hard color” means that the above “color” is low.
For example, “colorability” relating to PQQs in the present specification means that PQQs combine with metal cations to produce colored PQQs metal salts, such as substances containing them, such as PQQs-containing liquids It refers to the properties of PQQs that bring about an appearance color change in PQQs-containing compositions or PQQs-containing cells.
In addition, “cell coloring” relating to PQQs in the present specification means that the PQQs attached to the cell surface or taken into the cells bind to metal cations to produce colored PQQ metal salts. It refers to the properties of PQQs that cause an appearance color change in cells.
Therefore, in the present specification, “hard color” and “hard cell color” relating to PQQs means that “color color” and “cell color” of PQQs described above are low.
For example, a hard-to-color pyrroloquinoline quinone-containing preparation containing pyrroloquinoline quinones or an alkali metal salt thereof according to the present invention and a chelating agent contains the same amount of pyrroloquinoline quinones or an alkali metal salt thereof but is chelated. Compared with a compound containing no agent, coloring of cells in the oral cavity is suppressed by 25% or more when administered orally. Preferably, the coloring is suppressed by 50% or more, more preferably 75% or more. It should be noted that the degree of cell coloring can be evaluated based on direct observation of a cell specimen, macroscopic observation with a microscope, etc., measurement with a chromaticity meter, and the like.

The pyrroloquinoline quinones (PQQ) contained in the preparation of the present invention are represented by the following general formula (I):

(Wherein R ₁ , R ₂ and R ₃ are the same or different and each represents a hydrogen atom, a phenyl group, or an alkyl group, aralkyl group, alkylaryl group, alkenyl group or alkynyl group having 1 to 6 carbon atoms). It is a compound represented. Preferably the carbon number of the said C1-C6 alkyl group is 1-4. 1-6 are preferable and, as for carbon number of the alkyl part of the said aralkyl group and an alkylaryl group, 1-4 are more preferable. 2-6 are preferable and, as for carbon number of the said alkenyl group and alkynyl group, 2-4 are more preferable.

Preferably, R ₁ , R ₂ and R ₃ are the same or different and are a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, more preferably the same or different, a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. And more preferably a hydrogen atom.

  The PQQs are more preferably alkali metal salts because of their high water solubility. Examples of the alkali metal include lithium, potassium, sodium and the like, and sodium is preferable.

The preparation of the present invention also contains a chelating agent. As the chelating agent, the following metal ions: magnesium ion, calcium ion, strontium ion, barium ion, cadmium ion, nickel (II) ion, zinc ion, copper (II) ion, mercury (II) ion, iron (II) Divalent metal ions such as ions, cobalt (II) ions, tin (II) ions, lead (II) ions, manganese (II) ions; trivalent ions such as aluminum ions, iron (III) ions, chromium (III) ions, etc. The compound is not particularly limited as long as it is a compound capable of forming a chelate complex with a metal ion; a tetravalent metal ion such as tin (IV) ion; or a monovalent metal ion such as silver ion, mercury ion, or gold ion. Preferably, it is a compound that is easily colored and can form a chelate complex with copper (II) ions, iron (II) ions, or iron (III) ions contained in many foods. Examples of preferred chelating agents include ethylenediaminetetraacetic acid (EDTA) and organic acids such as gluconic acid, heptogluconic acid, citric acid, phytic acid, phosphoric acid, malic acid, tartaric acid, phosphonic acid, and hydroxyacetic acid. Of these, EDTA, citric acid or malic acid is more preferred.
The preparation of the present invention may contain any one of the above chelating agents alone, or may contain any two or more in combination.

  The content ratio of the PQQs and the chelating agent in the preparation of the present invention varies depending on the desired anti-coloring effect and the type of the chelating agent, but the chelating agent may be 1 mg to 10 g with respect to 100 mg of the PQQs. . For example, when the chelating agent is citric acid, it is preferable that 1000 mg to 10 g of citric acid is contained per 100 mg of PQQs. When the chelating agent is malic acid, malic acid is 100 mg to 10 g per 100 mg of PQQs. Preferably, it is contained in 600 mg to 10 g.

The preparation of the present invention may further contain a metal cation. Examples of the metal cation include metal cations having a valence selected from monovalent, divalent, trivalent and tetravalent.
Examples of monovalent metal cations include lithium ions, sodium ions, potassium ions, silver (I) ions, mercury (I) ions, and gold ions.
Examples of divalent metal cations include magnesium ion, calcium ion, strontium ion, barium ion, cadmium ion, nickel (II) ion, zinc ion, copper (II) ion, mercury (II) ion, iron (II) Ions, cobalt (II) ions, tin (II) ions, lead (II) ions, manganese (II) ions, and the like.
Examples of trivalent metal cations include aluminum ions, iron (III) ions, chromium (III) ions, and the like.
Examples of tetravalent metal cations include tin (IV) ions.
The preparation of the present invention may contain any one of the above metal cations alone, or may contain any two or more in combination.

  The present invention also provides a medicine or quasi-drug containing the preparation of the present invention. The medicament or quasi-drug is an medicament or medicament for obtaining effects based on the physiological action of any PQQ, for example, effects such as cell proliferation promotion, liver disease prevention treatment, melanin production suppression, nerve growth factor production promotion, etc. It can be a quasi-product.

The drug or quasi-drug may further contain a pharmaceutically acceptable carrier in addition to the above-mentioned components of the preparation of the present invention. Examples of the carrier include excipients, coating agents, binders, extenders, disintegrating agents, lubricants, diluents, surfactants, osmotic pressure adjusting agents, pH adjusting agents, dispersing agents, emulsifiers, preservatives. , Stabilizers, antioxidants, wetting agents, thickeners, alcohol, water, water-soluble polymers, or colorants, flavors, flavoring agents, flavoring agents, etc. It is done.
These carriers can be appropriately used alone or in any combination depending on the pharmaceutical or quasi-drug formulation.

The pharmaceutical or quasi-drug includes the pyrroloquinoline quinones, a chelating agent, and if necessary, the metal cation or other active ingredient or medicinal ingredient, and if necessary, the pharmaceutically acceptable carrier. It can mix | blend and can be manufactured in accordance with a conventional method.
As the dosage form of the preparation of the present invention, a dosage form for oral or transdermal administration is preferable. Examples of dosage forms for oral preparations include tablets, capsules, granules, powders, syrups, dry syrups, solutions, suspensions, etc. Examples of dosage forms for transdermal preparations include lotions and gels. , Cream, spray, ointment, patch, patch and the like.

  Since the pyrroloquinoline quinone-containing preparation according to the present invention has an effect of preventing coloring of the skin and oral mucosa during ingestion and administration, the medicament or quasi-drug of the present invention is the Alternatively, it is useful not only as a preparation for transdermal administration but also as a preparation for administration forms in which pyrroloquinoline quinones can come into contact with a site where coloring is not preferred, such as skin and oral mucosa, at the time of administration. Examples of such administration forms include nasal drugs that pass through the nose and nasal mucosa, inhalants or enteral drugs that pass through the oral cavity, eye drops, suppositories, injections, drops, and the like.

  The content of the preparation of the present invention in the pharmaceutical or quasi drug varies depending on the dosage form, but may be 1 to 100% by mass, preferably 5 to 50% by mass with respect to the drug or quasi drug. It is.

  The present invention also provides a food or drink or feed containing the preparation of the present invention. The food or drink or feed of the present invention is intended to have effects based on the physiological action of any PQQ, such as cell proliferation promotion, liver disease prevention treatment, melanin production suppression, nerve growth factor production promotion, etc. Health foods, functional foods and drinks, foods and drinks for specific health use, foods and drinks for sick people, livestock, racehorses, feed for appreciation animals, pet foods, and the like.

  The above-mentioned food or drink or feed is the above-mentioned preparation of the present invention, other food and drink materials used for the production of food and drink or feed, various nutrients, various vitamins, minerals, amino acids, various fats and oils, various additives (for example, taste ingredients) , Sweeteners, acidulants such as organic acids, surfactants, pH adjusters, stabilizers, antioxidants, dyes, flavors) and the like, and the like, and can be produced according to conventional methods. Or the food / beverage products or feed of this invention can be manufactured by mix | blending the said this invention formulation with the food / beverage products or feed currently eaten normally.

The form of the food or drink or feed is not particularly limited, and may be, for example, solid, semi-solid or liquid, or for tablets, chewable tablets, powders, capsules, granules, drinks, gels, syrups, enteral nutrition Various forms, such as a liquid food, are mentioned.
Specific examples of the form of food and drink include tea drinks such as green tea, oolong tea and tea, coffee drinks, soft drinks, jelly drinks, sports drinks, milk drinks, carbonated drinks, fruit juice drinks, lactic acid bacteria drinks, fermented milk drinks, Powdered beverages, cocoa beverages, alcoholic beverages, beverages such as purified water, butter, jam, sprinkles, margarine spreads, mayonnaise, shortening, custard cream, dressings, breads, cooked rice, noodles, pasta, miso soup, tofu , Milk, yogurt, soup or sauce, confectionery (for example, biscuits and cookies, chocolate, candy, cake, ice cream, chewing gum, tablet) and the like.
Since the feed of the present invention can be used in almost the same composition and form as foods and drinks, the description relating to the foods and drinks in this specification can be applied to feeds as well.

  Although content of this invention formulation in the said food / beverage products and feed changes with forms of foodstuff, it should just be 1-100 mass% with respect to the said food / beverage products and feed, Preferably it is 5-50 mass%.

The above-mentioned medicine, quasi-drug, food and drink, and feed are administered or ingested in the range of 5 to 100 mg per day in terms of the amount of PQQs contained in the preparation of the present invention. In the case of oral administration or ingestion, the general daily dose is preferably 10 to 20 mg. The daily administration or intake may be administered or ingested once, but may be administered or ingested in several times.
The dosage form or dosage regimen of the medicament or quasi-drug of the present invention, or the form of food, drink or feed can be managed in the dosage or intake per day so that the daily dose can be appropriately administered or consumed. It is desirable to make it.

  Further, according to the present invention, the preparation of the present invention contemplates effects based on the physiological action of any of the above PQQs, for example, effects such as cell proliferation promotion, liver disease prevention treatment, melanin production suppression, nerve growth factor production promotion, etc. It can be used for the manufacture of manufactured medicines, quasi drugs, foods and drinks, feeds and the like.

Furthermore, according to the present invention, even when PQQs are ingested or administered by the action of a coexisting chelating agent and adhere to the cell surface or are taken into the cell, the metal cation present on the cell surface or in the cell Since the production of colored PQQ-type metal salts due to binding with PQQs is hindered, coloring due to the PQQ-type metal salts of the cells can be prevented.
That is, according to the present invention, cell coloring by pyrroloquinoline quinones characterized in that pyrroloquinoline quinones represented by the above general formula (I) or alkali metal salts thereof coexist with a chelating agent in cells. A prevention method is provided.

  In the above method, the structure of PQQs, the type of chelating agent, and the abundance ratio of PQQs and chelating agent are as described above. The cell type is not particularly limited as long as it is a cell that can contact PQQs, but a mammalian cell is preferable, and a human cell is more preferable. Effective cells are cells that are present in the lips, oral cavity, esophagus, intestine, and skin that come into contact during oral administration. Moreover, it is effective not only in living bodies but also in cultured cells.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited only to these Examples.

Example 1 Verification of coloring properties of PQQs 2.5 mg of PQQ (4,5-dioxo-1H-pyrrolo [2,3-f] quinoline-2,7,9-tricarboxylate: manufactured by Mitsubishi Gas Chemical) / ML aqueous solution was prepared, and this was mixed with an equal volume of various metal cation aqueous solutions shown in Table 1 and allowed to stand for 10 minutes, and then the color of the solution, the presence or absence of precipitation, and the color of the precipitation were visually observed. Table 1 shows the results.

Example 2 Effect of malic acid addition 3.75 mg / mL of PQQ (4,5-dioxo-1H-pyrrolo [2,3-f] quinoline-2,7,9-tricarboxylate disodium: manufactured by Mitsubishi Gas Chemical) An aqueous solution and a 1.5 mg / mL aqueous solution of iron sulfate were prepared. Separately, 5, 25, 50, 100, 500 mg / mL malic acid aqueous solution was prepared. An equal volume of each aqueous solution of PQQ, iron sulfate and malic acid was mixed and allowed to stand for 10 minutes, and then the color of the solution was visually observed, and coloring was scored according to the criteria in Table 2. As a control, the same observation was made on a sample without malic acid added. Table 3 shows the results.
Malic acid suppressed coloring of the PQQ-iron sulfate solution, and the effect was more remarkable as the concentration of malic acid was higher.

Example 3 Effect of adding various chelating agents 3.75 mg / day of PQQ (4,5-dioxo-1H-pyrrolo [2,3-f] quinoline-2,7,9-tricarboxylate disodium: manufactured by Mitsubishi Gas Chemical) An aqueous solution of mL and a 1.5 mg / mL aqueous solution of iron sulfate were prepared. Separately, malic acid, citric acid and ascorbic acid were each prepared in 45 mg / mL aqueous solution and EDTA in 22.5 mg / mL aqueous solution. An equal volume of each aqueous solution of PQQ, iron sulfate and each chelating agent was mixed and allowed to stand for 10 minutes, and then the color of the solution was visually observed, and coloring was scored according to the criteria in Table 2. As a control, the same observation was made on a sample without addition of a chelating agent. Table 4 shows the results.

Example 4 Reduction of coloring property of model intracellular solution by EDTA Coloring by PQQ using fetal bovine serum (FBS) or α-MEM + 10% FBS which is a medium for animal cells as a model intracellular solution close to the intracellular composition. The effect of the chelating agent on was measured.
In the experiment, EDTA trisodium manufactured by Dojindo Chemical was used as a chelating agent. 100 μL of the above model solution was added to a 96-well plate, and 100 μL of PQQ: EDTA3 sodium = 1: 6 (wt) PQQ 2.5 mg / mL mixture was added thereto. Next, the operation of taking 100 μL of this and adding it to the hole containing the next 100 μL of the model solution was repeated, and the PQQ concentration in the model solution was diluted stepwise by half. As a control, a model solution to which only the same concentration of PQQ was added was prepared in the same procedure. This was treated at 37 ° C. for 1.5 hours in a mixed state of 5% carbon dioxide and air under saturated water vapor pressure. Thereafter, the absorbance at 600 nm was measured with a plate reader to examine the coloring of the model solution.

  The results are shown in FIGS. 1A and B. In both fetal bovine serum (A) and α-MEM + 10% FBS (B), which are intracellular model solutions, the absorbance increased in a PQQ concentration-dependent manner, indicating that coloration by PQQ occurred. In the fetal calf serum, saturation was finally observed. On the other hand, the absorbance of each solution was greatly reduced by adding EDTA, indicating that coloring was suppressed.

Claims (4)

The following general formula (I):
(Wherein R ₁ , R ₂ and R ₃ are the same or different and each represents a hydrogen atom, a phenyl group or an alkyl group having 1 to 6 carbon atoms, an aralkyl group, an alkylaryl group, an alkenyl group or an alkynyl group)
Pyrroloquinoline quinones or alkali metal salts thereof represented by:
Chelating agents,
1 or 2 types or more of monovalent, divalent, trivalent and tetravalent metal cations selected from valences, containing a difficult-to-oral cell coloring pyrroloquinoline quinones Oral formulation (unless the oral formulation is a cider) .   The oral preparation according to claim 1, wherein the alkali metal salt is a sodium salt.   The oral preparation according to claim 1 or 2, wherein the chelating agent is an organic acid and / or EDTA. The following general formula (I):
(Wherein R ₁ , R ₂ and R ₃ are the same or different and each represents a hydrogen atom, a phenyl group or an alkyl group having 1 to 6 carbon atoms, an aralkyl group, an alkylaryl group, an alkenyl group or an alkynyl group)
Oral cell coloring of pyrroloquinoline quinone-containing oral preparations (except when the oral preparation is a cider) , characterized in that a chelating agent is added to the pyrroloquinoline quinones or alkali metal salts thereof represented by Sexual decline method.