JPH0426630A - Stabilization of lysozyme salt in aqueous formulation - Google Patents

Abstract

PURPOSE:To stabilize lysozyme salt in an aqueous formulation by mixing a specific amount of phytic acid and polyoxyethylene fatty acid ether and adjusting pH in a specific range. CONSTITUTION:An aqueous agent containing 0.1-10.0wt.% lysozyme salt is mixed with 0.2-20.0wt.% phytic acid as a host and 0.5-5.0wt.% polyoxyethylene fatty acid ether (P.O. 20-50) and pH is adjusted to 4.0-7.0.

Description

[Detailed Description of the Invention] Lysozyme chloride has excellent anti-inflammatory properties for both internal and external use, so it is used as a single component in various preparations for the treatment of catastrophic diseases. It is an anti-inflammatory enzyme that is formulated as an anti-inflammatory enzyme.As with enzymes, it decomposes fairly quickly and loses its effectiveness in an aqueous medium, so it is generally prescribed as a dry preparation such as a powder or tablet. However, there are cases where it is desirable to use water-based preparations, such as dentifrices, gargles, inhalants, ointments, and poultices. Limited due to instability.

This invention relates to a method for stabilizing lysozyme salts in aqueous preparations, and the inventor has completed this after many years of research. This invention makes it possible to incorporate lysozyme salts into various aqueous preparations, which has been difficult until now, and opens the door to a wide range of treatment methods for Type 4, which have been desired but have not been achieved so far.

The stability of lysozyme in an aqueous medium can be improved to some extent by making the medium pH acidic, but this alone is insufficient to make it a commercially available product.Furthermore, lysozyme is a basic protein. Therefore, it is not uncommon for the compound to combine with acidic components, polymeric substances, or @J-soluble fine particle components that are often blended into aqueous media, to become poorly soluble, and to become ineffective.

Phytic acid is a meso-inositol hexaphosphate contained in rice bran in high concentration, and due to its structure, it has an extremely high chelating ability.In addition to its ability to chelate heavy metals, it also has the property of conjugating proteins, etc. It has been demonstrated that it binds to hemoglobin and has an allonuteric effect on it.

The inventor focused on this point and thought that phytic acid may stabilize lysozyme by conjugating it and blocking it from the surrounding environment, and as a result of intensive research, he completed this invention.

Lysozyme is extremely unstable in its free base form, but is stabilized in the form of a salt or an acidic substance, as typified by lysozyme chloride. That is, in aqueous preparations, it is generally stable in an acidic region, for example, each pH is 0.1M.
When lysozyme chloride dissolved in a 0.004% sodium sodium buffer solution was left at room temperature and the titer fluctuation was measured, the results shown in Table 1 and Figure 1 were obtained.

From this experiment, (1) the stability of lysozyme is determined by the TH of the medium.
It is relatively stable up to pH 7, but
p) (Exceeding 7 becomes extremely unstable, (2) I
) There are two points that can be observed: even if the product is below H7, it still lacks stability significantly as a marketed product.

However, when phytic acid 0.01% was added to the same yarn, the stability showed significant improvement as shown in Table 2 and Figure 2. In other words, in the case without phytic acid, decomposition was detected within minutes, but in the case with phytic acid, decomposition was barely detected when measured over days, indicating an order of magnitude better stability. Figure 2 shows that the conjugation effect of phytic acid is lost at pH below 4 and above 7.5; in the former case, phytic acid molecules disappear due to high hydrogen ion concentration, and in the latter case, phytic acid molecule decreases due to high hydroxyl ion concentration. The cause is thought to be acid decomposition.

Lysozyme forms poorly soluble complexes with certain acidic substances, which often poses a practical obstacle. That is, turbidity of the preparation, formation of a precipitate, and sometimes a decrease in the efficacy of lysozyme may occur. The combination of phytic acid and lysozyme is also a type of complex, but this complex is completely water-soluble at practical concentrations, and anti-emorgement tests have shown that it does not reduce the efficacy of lysozyme. Furthermore, it has been found that the conjugation effect of phytic acid is exerted not only on lysozyme alone, but also on the above-mentioned insoluble complex of lysozyme and an acidic substance.

For example, when dipotassium glycyrrhizinate solution is added to an aqueous solution of lysozyme chloride, it becomes cloudy, and when it is allowed to stand still, a resinous sedimentation occurs. IJe is formed, but when mW of phytic acid is further added to this mixture, this turbidity becomes clear and dissolved. A similar phenomenon is observed in the case of chlorhegicidine pruconate solution or sodium carboxymethyl cellulose solution. All of these phenomena are caused by the formation of a ternary complex between lysozyme, acidic substances, and phytic acid, in which the chelate-forming structure of phytic acid binds the two components, and the solubility of phytic acid residues increases. It is thought that solubilization occurs due to the

The possibility that the formation of such a binary or multicomponent combination with phytic acid as the main ingredient will negatively affect the effects of these medicinal ingredients is eliminated by adding polyoxyethylene fatty acid ether. For example, the results shown in Table 3 were obtained when the reproductive inhibitory concentration (MCC) for and was examined as an indicator.

In carrying out the present invention, appropriate amounts of fragrances, sweeteners, urea, surfactants, abrasives, ethanol, gelling agents, etc. may be added depending on the intended use of the preparation.

In addition, the present invention is applied to toothpastes, gargles, mouth washes, oral liniments, poultices, etc. that have anti-inflammatory effects.
This invention, which includes liniments, lotions, creams, ointments, shampoos, packs, cleaning liquids, etc., will be further explained using Examples.

Example-1 Lysozyme chloride
. , 4-chlorhexidine curconate
0-05 dipotassium glycyl phosphate
0.2 phytic acid
1.0 POE Oreillete z+/)
20E10) 1.0 Sodium sarcosine laurate 0.5 Sodium carboxymethylcellulone 5.0 Propylene glycol 5.0 Cinnamon oil 0.021
-Mentor/I10 ・06 Chojimae 0・
01 Edible Kenshiki No. 4
0.001 potassium hydroxide
0.6 min water
86.159 total
100.0 The above ingredients are mixed according to a conventional method.1.6.00 Example-1 is a medicated toothpaste.

Example-2 Lysozyme chloride Gluconate Chlorhexidine Glycyrrhizinate Dipotassium Urea Phytate POE Cetyl ether Sodium sarcosine laurate Sodium carboxymethyl cellulose Propylene glycol Cinnamon oil L-Menthol T-shaped pre-edible blue No. 4 Potassium hydroxide Purified water meter The above ingredients are commonly used Mix according to law.

pH 5・05 Example-2 is a medicated toothpaste.

○Bow 0.05 2.0.2 5.0 1.O l.0 0.5 5.0 5.0 0.02 0.06 0@01 0.001 0.4 81.359 100.0 Implementation When the room temperature storage stability of lysozyme for Examples-1 and -2 was tracked using other companies' products as a control, the effects of the present invention were obvious as shown in Table 4.Example-3 Stratified lysozyme
2.0 Chlorhexidine gluconate
0025 Urea
10・Ophytin i2
5. OPOE Lau Ritv:
r-chill (2 0E, O, )
5.0 propylene glycol
5.04-Menthol
0.3 Potassium hydroxide 2.5
Purified water 69・
95 total
100.0 The above ingredients are mixed according to conventional methods.

pl(’5・25 Example-3 is a mouthwash. Dilute 5 times before use.

Example-4 Lysozyme chloride.

Chlorhexidine gluconate
. , 25 urea ,.

, O-phytic acid 15・OP
OE Rau Lil Ether (20F, O)
2.

Propylene glycol/L15. .

l-j 7 F-o, 3 cinnamon oil.・2 choji n out
. ,. Potassium pentahydroxide
. ,.

purified water ,
8.2 total
Yu. . ,.

Mix the above ingredients according to the negative method.

H 4.02 Example 4 is a mouthwash and is diluted 10 times before use.

[Brief explanation of drawings]

FIG. 1 shows the relationship between AFrr/day and H, and FIG. 2 shows the relationship between 4R days and H in the case of containing phytic acid. Example-5 Lysozyme chloride Gluconate Chlorhexidine Phytic acid POE Lauryl ether (50E10, ) Propylene glycol Sodium salicylate Sodium carboxymethyl cellulose Potassium hydroxide Purified water meter pH 6.05 Example-5 is for gums Example-6 Lysozyme chloride Phytic acid POE lauryl ether (20E, O,) propylene glycol sodium salicylate methyl paraoxybenzoate liniment. 0 ・ 4 0 ・ 01 2 ・ 0 1 ・ 0 5 Umbrella 0 0 ・ 5 8 ・ 0 1 ・ 2 81 ・ 89 100 ・ 0 ) 0 Mu 2 1 ・ 0 0 ・ 5 5 ・ 0 0 ・ 5 0 ・ 5 Carboxy methylcellulose sodium
5.○ Potassium hydroxide
0.5 purified water
86.8 total
100-OpH5・08 Example-6 is a senile applied adhesive Example-7 Lysozyme chloride phytate alpha-tocopherol acetate POE lauryl ether (50E, O,) Salicy/I/
Sodium varoxin benzoate Methylpropylene glycol Potassium hydroxide Purified water Meter pH 4.85 Example - 2 is lotion. ・ 0 ・ 2 1 ・ 0 0 ・ 5 0 ・ 5 0 ・ 5 5 ・ 0 0 ・5 90 ・ 8 0O-0 (1z) Table-1 Room temperature stability of lysozyme chloride at each pH Table-2 Anstability of lysozyme in 01 1M phosphate buffer containing 0201% phytic acid

Claims (1)

[Claims] In aqueous preparations containing 0.1% to 10.0% lysozyme salt, 0.2% to 20.00% phytic acid and polyoxyethylene fatty acid ether (P, O, 20-50)
Add 0.5 to 5.0% and further adjust the pH to 4.0 to 7.0.
A method for stabilizing lysozyme salt, characterized by: