JP5672751B2 - Liposomes containing pyrroloquinoline quinone - Google Patents

Description

The pyrroloquinoline quinone (hereinafter referred to as PQQ) handled in the present invention is a substance having a structure represented by the formula 1, and relates to the technology of liposomes containing this free form or salt.

PQQ has been proposed as a new vitamin (see, for example, Non-Patent Document 1), and has attracted attention as a useful substance for health supplements, cosmetics, and the like. Furthermore, it exists not only in bacteria but also in eukaryotic molds and yeasts and plays an important role as a coenzyme. In addition, PQQ has many cell growth promoting effects, anti-cataract effects, liver disease preventive and therapeutic effects, wound healing effects, anti-allergic effects, reverse transcriptase inhibitory effects and glyoxalase I inhibitory effects-anticancer effects, etc. Physiological activity has been elucidated.

This PQQ is obtained by subjecting PQQ obtained by a method such as an organic chemical synthesis method (Non-patent Document 2) or a fermentation method (Patent Document 1) to chromatography, concentrating the PQQ section in the effluent, and by crystallization. It can be obtained by crystallization (Patent Document 2) and drying.

Although this PQQ is expected to be used as a food with enhanced nutritional function, it is chemically stable under acidic conditions, but easily decomposes under alkaline conditions. It also has reactivity with carbonyl compounds. Furthermore, it is easy to deposit in strong acidity. Therefore, there is a demand for a method that can cope with changes in pH and the like. There is also a need for a method of increasing absorbability and improving the function of pyrroloquinoline quinone.

Liposomes usually have a capsule structure made of a lipid membrane composed of phospholipids, in which an aqueous phase is confined. The phospholipid molecule is shaped like a pine needle, and the head part is hydrophilic, and the part that looks like a leaf is hydrophobic, so it is hydrophilic when released into water. The part attracts water and forms liposomes. Liposomes can contain water-soluble components in their hydrophilic portions and oil-soluble components in their hydrophobic portions. Liposomes are attracting attention mainly in the field of medicine as a method of administering drugs, and the advantages of improved absorption, dispersibility, and stability are generally known. Widely used in. Therefore, there is a demand for liposomes containing PQQ.

Furthermore, although PQQ is expected to improve brain function, it is expected to be taken together with phosphatidylcholine and phosphatidylserine (non-patent document 4) having similar functions. Since it is contained in the liposome raw material, provision of a liposome containing a free form or salt of pyrroloquinoline quinone, a composition with improved functionality thereby, and a method for producing the same are desired.

So far, a fuel cell using PQQ as an electron mediator and immobilizing an enzyme as a liposome has been developed (Patent Document 3). Moreover, although it has been proposed as a composition for administering an S-nitrosylated compound to a mammal, there is no specific example, and there is no description of a production method (Patent Document 4). Phospholipids have a high viscosity as they are, and in order to make liposomes, it is common to dissolve in a solvent such as chloroform, form a liquid film inside the flask, and disperse with ultrasonic waves. Low and there is a risk of residual solvent. As described above, there is no known method for providing a specific solution or a manufacturing method therefor.

JP-A-1-218597 Japanese Patent No. 2072284 JP 2006-508519 A JP-T-2001-518096

nature, vol422, 24April, 2003, p832 JACS, 103, 5599-5600 (1981) JACS, Vol.111, pp.6822-6828 (1989) Fine Chemicals, Volume 34, No. 11, pp. 30-41 (2005)

The subject of this invention is providing the liposome containing the free body and / or salt of pyrroloquinoline quinone, and providing an efficient manufacturing method.

As a result of earnest research to solve the above-mentioned problems, the present inventor has found that the properties of pyrroloquinoline quinone and the conditions for liposome formation can be solved by the following method.
(1) The following formula (1);
A liposome composition comprising a free form and / or a salt of pyrroloquinoline quinone represented by the formula:
(2) A method for producing a solution containing a liposome composition, comprising a step of heating a free form of pyrroloquinoline quinone and / or a salt and a liposome at a pH of 1 to 8 at 60 ° C. to 190 ° C. .

According to the present invention, it is possible to efficiently produce a highly stable liposome containing a free form and / or a salt of pyrroloquinoline quinone.

Liposome particle size Liposome particle size after dialysis Particle size of liposomes with added sugar Liposome particle size after freeze-thaw treatment Liposome particle size when using hydrogenated soybean phospholipids Absorption test results

The PQQ used in the present invention is not particularly limited as long as it is a free form or an alkali metal salt. It is particularly easy to use a free body, a dinatrim body and a dipotassium body, which are easily available.

Liposomes used in the present invention are prepared using phospholipids or glycolipids alone or in admixture. There is phosphatidylcholine as the main component of phospholipids contained in the living body, also called lecithin, egg yolk lecithin, soybean lecithin, purified soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, dicetyl phosphate, stearylamine, phosphatidylglycerol, Substances such as phosphatidic acid, phosphatidylinositol amine, cardiolipin, ceramide phosphoryl ethanolamine, ceramide phosphoryl glycerol, and mixtures thereof can be used. Commercially available phosphatidylcholine can be used in the present invention. For example, NOA COATSOME NC-21 (hydrogenated soybean phospholipid, PC content of 90% or more), Nikko Chemicals NIKKOL Resinol S-10EX (hydrogenated soybean phospholipid, PC content of 95% or more) can be used.

As the glycolipid, digalactosyl diglyceride, galactosyl diglyceride sulfate, galactosylceramide, galactosylceramide sulfate ertel, lactosylceramide, ganglioside G7, ganglioside G6, ganglioside G4, digalactosylceramide, and mixtures thereof can be used.

A sterol may be added to the base. The addition amount is 1/5 weight with respect to phospholipid or glycolipid, and the upper limit of the preferred amount is more preferable, and 1/10 weight is more preferable. As the sterol, cholesterol is most preferable, but other sterols may be used.

The liposome composition can be produced by a general method. For example, lecithin is dissolved in an organic solvent such as chloroform, and the solvent is distilled off with a rotary evaporator, and the lipid film adhered to the wall of the flask. Can be produced by adding a PQQ solution. However, this method is not optimal because the operation is complicated and there is a risk that a toxic organic solvent may remain, and the analysis is also required, which is expensive.

More preferably, a sterol, a polyhydric alcohol, and a pH adjuster are added to an aqueous solution in which PQQ is dissolved to a final concentration of 0.0001 to 2% by weight, if necessary, heated to 60 to 190 ° C., and then homogenized. It can be manufactured by dispersing. It can be made with high productivity by using a homogenizer. Here, the upper limit concentration of PQQ is the limit of solubility, and if it is higher than this, it tends to precipitate, and if it is lower than the lower limit concentration, the function of pyrroloquinoline quinone cannot be expected.

Since PQQ is alkaline and has low stability, the pH is preferably 8 or less, more preferably 2 to 6. When the pH is higher than 8, PQQ decomposes. Although there is no problem in terms of stability in making it acidic, it becomes difficult to make it high because solubility is lowered.

The size of the liposome of the present invention is 0.05 to 100 μm. The size of the liposome can be selected depending on its functionality. More preferably, lecithin is used, and a liposome solution having a PQQ weight concentration of 0.01 to 1% by weight, a pH of 2 to 6, and a size of 0.05 to 100 μm. When the weight concentration of PQQ is lower than this, an appropriate effect cannot be obtained, and when the concentration is high, the solution cannot be dissolved even when the temperature is raised, and therefore a uniform solution cannot be formed. In addition, when the pH becomes alkaline, decomposition of PQQ tends to occur. Further, in strong acidification, the solubility of PQQ is low, and the concentration that can be maintained in the aqueous layer in the liposome is low. The size of the liposome is in a range that can be easily formed. It is difficult to form liposomes smaller than 0.05 μm, and it is difficult to form liposomes larger than 100 μm. Large liposomes can be expected to be less effective in terms of absorbability and the like.

A high-pressure emulsifier can also be used. The high-pressure emulsifiers are Primix's thin-film swivel type high-speed homomixer (TK Filmix), Microfluidics ultra-high pressure homogenizer (Microfluidizer), MTechnic's internal shearing force mixer (Clairemix), Yoshida Machine Kogyo's wet medialess atomizer (nanomizer) and the like can be used.

After forming the liposome, purification of the liposome and control of the size can be performed by performing treatments such as dialysis, freeze-thawing, filter filtration, freeze-drying, and centrifugation.

Next, these operations will be described. Dialysis treatment is a method that utilizes the fact that liposomes are large molecular aggregates and cannot pass through an ultrafiltration membrane, and a solution that uses only the aqueous layer in liposomes can be produced. In the case of PQQ, since it is a low molecule, the ultrafiltration membrane to be used may be a membrane that passes a low molecular weight, and may be a general membrane. Although it is desirable to dialyze with a solution having the same osmotic pressure as much as possible, it is possible to actively change the size by using this effect by using solutions having different osmotic pressures.

Freezing and thawing is performed by cooling and freezing the liposome solution, and then dissolving it to take up substances outside the liposome and change the size according to this phase change. At this time, the operation can be performed without causing separation of the liposomal lipid by adding polyols such as glycerin, sorbitol, xylitol, sucrose and the like. Filter filtration is performed to sterilize and keep the size constant. Freeze-drying is an operation performed for pulverization and incorporation into liposomes, and the composition must withstand freezing. Centrifugation utilizes the difference in specific gravity and can be used for concentration and the like.

When preparing the liposome composition of the present invention, other sterols, polyoxyethylene sterol ethers, polyhydric alcohols, pH adjusters, nonionic surfactants, anionic interfaces are within the range not impairing the effects of the present invention. Contains active agents, cationic surfactants, amphoteric surfactants, oils, moisturizers, water-soluble polymers, antioxidants, UV absorbers, chelating agents, preservatives, antibacterial agents, colorants, flavors, etc. be able to. Moreover, it can also be set as the liposome which mix | blended plant extracts, such as coenzyme Q10, ascorbic acid derivative, tocopherol, arachidonic acid, DHA, retinol derivatives, etc. vitamins, a ginkgo extract, a kanka extract, etc.

The liposome composition of the present invention may be any of an aqueous solution in which liposomes are dispersed, an oil-in-water emulsion composition, a water-in-oil emulsion composition, a multiple emulsion composition, and a multilayer agent. To the solution thus prepared, other pharmaceutically acceptable formulation materials may be appropriately added and mixed by a conventional method. The preparation material that can be added is not particularly limited, and examples thereof include emulsifiers, tensioning agents, buffering agents, solubilizing agents, flavoring agents, preservatives, stabilizers, and antioxidants.

The method for storing the solution composition according to the present invention is not particularly limited, and low-temperature storage, anaerobic storage using a sealed container, light-shielding storage, and the like can be used. The solution of the present invention thus prepared can be stably stored without precipitation when stored at refrigeration or at room temperature.

Hereinafter, the present invention will be described in more detail with reference to examples and preparation examples. However, the present invention is not limited to these examples and preparation examples. Soy lecithin used JUNSEI, pyrroloquinoline quinone disodium (PQQ-Na2) used Mitsubishi Gas Chemical, and others used WAKO reagents.

Example 1
Preparation of Liposome Solution 3.0 g of soybean lecithin was added to 100 g of 3 g / L PQQ-Na2 aqueous solution. The pH at this time was 3.5. While warming to about 80 ° C., it was treated with a NISSI AM-3 homogenizer for 30 minutes at 7000 rpm, lowered to room temperature and treated for 10 minutes. After the treatment, water with reduced evaporation was added. The homogeneous solution was stable for more than 3 weeks.

Measurement of PQQ concentration An equal amount of dimethyl sulfoxide was added to the above-mentioned liposome solution and diluted with the following eluent to measure the PQQ concentration. Measurement is Shimadzu, high performance liquid chromatography, LC-20A, column is YMC-Pack ODS-TMS (5μm), 150x4.6mm ID at 40 ℃, eluent 100mM CH ₃ COOH / 100mM CH ₃ COONH ₄ (30 / 70, pH 5.1) was detected at 260 mL at 1.5 mL / min. As a result, no change in the concentration of PQQ was observed, and no decrease in the concentration of the PQQ-containing liposomes that could be produced under the conditions of the present invention occurred.

Liposome particle size measurement The liposome particle size is an important factor in the absorption and stability of cells and living bodies. Therefore, the particle diameter prepared was measured.
Using SEISHIN LMS-350, the particle size distribution was obtained by dispersing in water. In this apparatus, 0.1 μm is the lower limit of detection. The results are shown in FIG. A vertical axis | shaft shows the ratio in volume conversion. The x90, which is 90% of the particle diameter as a percentage of the graph in which the particle diameter is accumulated with a normal distribution of 50% 0.6 μm in terms of volume, was 2.0 μm. It was possible to produce stable liposomes maintaining the PQQ concentration.

Example 2
Dialysis of liposome solution In the liposome solution of Example 1, PQQ exists in the aqueous solution inside the liposome and the aqueous solution outside. Therefore, dialysis was performed to make it exist only inside.
About 3 ml of dialysis kit Slide-A-Lyzer Dialysis Cassetes (Thermo) was added and dialyzed with water. After 3 days at room temperature, the solution was recovered and increased to 6 ml. The solution changed from red to yellow turbid liquid. As a result of breaking liposomes with DMSO and analyzing the PQQ concentration in the same manner as in Example 1, it was 0.23 g / L PQQ. 85% of the original solution was dialyzed. The rest is considered internal. Furthermore, the particle diameter of the liposome was measured in the same manner as in Example 1. The results are shown in FIG. The particle size was large due to the difference in osmotic pressure.

Example 3
5 g of sorbitol and 3.4 g of soybean lecithin were added to 100 g of 3 g / L PQQ-Na2 aqueous solution. The pH at this time was 3.5. While warming to about 80 ° C., it was treated with a NISSI AM-3 homogenizer for 30 minutes at 7000 rpm, lowered to room temperature and treated for 10 minutes. After the treatment, water with reduced evaporation was added. The homogeneous solution was stable for more than 3 weeks. The particle diameter of the liposome was measured in the same manner as in Example 1. The results are shown in FIG. The normal distribution was 50%, 0.6 μm, and x90 was 2.0 μm.

Example 4
Freeze-thaw treatment The liposome solution obtained in Example 3 was cooled to -20 ° C and frozen, and it was thawed at room temperature. This operation was repeated three times. The particle diameter of the liposome was measured in the same manner as in Example 1. The results are shown in FIG. The normal distribution was 50%, 2.8 μm, and x90 was 4.5 μm. The particle size of the liposome was increased by freezing and thawing.

Example 5
To 100 g of 3 g / L PQQ-Na2 aqueous solution, 3.0 g of COATSOME NC-21 (hydrogenated soybean phospholipid) was added. The pH at this time was 3.5. While warming to about 80 ° C., it was treated with a NISSI AM-3 homogenizer for 30 minutes at 7000 rpm, lowered to room temperature and treated for 10 minutes. After the treatment, water with reduced evaporation was added. The particle diameter of the liposome was measured in the same manner as in Example 1. The results are shown in FIG. The normal distribution was 50%, 3.8 μm, and x90 was 6.8 μm. The size could be changed by changing the type of lecithin.

Example 6
To 100 g of 3 g / L PQQ-Na2 aqueous solution, 3.0 g of egg yolk lecithin was added. The pH at this time was 3.5. While heating to about 80 ° C., it was treated with a NISSEEI AM-3 homogenizer at 7000 rpm for 30 minutes, lowered to room temperature and treated for 10 minutes. After the treatment, water with reduced evaporation was added. A liposome solution could be made.

Example 7
To 100 g of 3 g / L PQQ-Na2 aqueous solution, 3.0 g of soybean lecithin was added, and 0.06 g of coenzyme Q10 (Mitsubishi Gas Chemical) was added. The pH at this time was 3.5. While warming to about 80 ° C., it was treated with a NISSI AM-3 homogenizer for 30 minutes at 7000 rpm, lowered to room temperature and treated for 10 minutes. After the treatment, water with reduced evaporation was added. A liposome solution could be made.

Example 8
10 ml of the solution prepared in Example 3 was added to a stainless steel vat and frozen at −80 ° C. This was dried overnight in a freeze dryer to obtain a powder.

Example 9 Mixing Test NOF PS50-phosphatidylserine 50 wt% containing powder (starch containing) 1.2 g and the solution of Example 1 2 g were mixed. The powder was uniformly dispersed.

Comparative Example 1
3.0 g of soybean lecithin was added to 100 g of 3 g / L PQQ-Na2 aqueous solution. The pH at this time was 3.5. This was treated with 7000 rpm for 30 minutes with a NISSI AM-3 homogenizer at room temperature. After the treatment, water with reduced evaporation was added. There was a precipitate that was not uniform throughout.

Comparative Example 2
A potassium hydroxide solution was added to 100 g of 3 g / L PQQ-Na2 aqueous solution to adjust the pH to 10 or more. After standing overnight at 30 ° C., the PQQ was 60% and was degraded before liposome formation.

Comparative Example 3
While adding 3.0 g of soybean lecithin to 100 g of water and heating to about 80 ° C., the mixture was treated with a NISSEI AM-3 homogenizer at 7000 rpm for 30 minutes and lowered to room temperature for 10 minutes. After the treatment, water with reduced evaporation was added. Liposomes without PQQ were made.

Comparative Example 4
Concentrated hydrochloric acid was added to 100 g of 3 g / L PQQ-Na2 aqueous solution to adjust the pH to 0.8. PQQ precipitated and the solution became clear. Since PQQ is not dissolved, it cannot be taken into the liposome even when the liposome is formed.

Absorbency test PQQ suppresses cell growth at very high concentrations relative to cultured animal cells. Using this, it was examined how cell absorption changes using animal cells by liposome formation.
Example 10
Chinese hamster ovary cells (CHO-DHFR) were cultured in an α-MEM + 10% fetal bovine serum medium at 5% CO 2 and 37 ° C. Using a 96-well plate made of Iwaki, it was added together with 100 μl of the medium so that 6500 cells would be in one hole, and cultured overnight. The culture solution was removed and a medium having a predetermined test concentration was added. After culturing for one day, the medium was changed and reacted for 1 hour using the Dojindo WST assay kit, and the absorbance at 450 nm was measured. The absorbance at this time is proportional to the number of cells.

As test samples, PQQ disodium, soybean lecithin-PQQ liposome (Example 1) and soybean lecithin liposome (Comparative Example 3) were used. These samples were diluted with media and tested. Each sample was run twice and averaged.
Test concentrations were 500, 250, 125, 62, 31, 16, 8, 4, 2, 1, 0.5 0 μM.
The addition amount at which the total number of cells was reduced by 10% as compared with no addition was 500 μM for PQQ disodium and 31 μM for the liposome solution of Example 1. In the case of the liposome of Comparative Example 3 alone, there was no rapid decrease in cell concentration. It is considered that the absorbability is increased by 16 times due to liposome formation. FIG. 6 is a graph showing the concentration under each condition where the average cell concentration when PQQ is not added is 100.

The solution of the present invention can be used in a wide range such as medical use, cosmetic use, food use, horticulture use, and dairy use. More specific forms include injections, infusions, solutions, eye drops, liquids for internal use, lotions, hair tonics, cosmetic emulsions, sprays, aerosols, drinks, liquid fertilizers, preservatives, etc. .

Claims (3)

Following formula (1);
Heating the pyrroloquinoline quinone disodium salt represented by phospholipid and / or glycolipid at a pH of 1 to 8 at 60 to 190 ° C;
A step of dispersing using a homogenizer,
A method for producing pyrroloquinoline quinone-containing liposomes. The production method according to claim 1, wherein the phospholipid is lecithin. The production method according to claim 2, wherein the lecithin is soybean lecithin or egg yolk lecithin.