JP3272743B2 - Placenta or liver extract having stable SOD activity and external preparation composition containing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an extract having stable SOD activity (hereinafter simply referred to as extract) obtained by enzymatically decomposing a placenta or liver extract with a protease to remove substances having a molecular weight of 10,000 or less. And a safe external preparation composition characterized by containing it.

[0002]

2. Description of the Related Art SOD which is a radical scavenger
(Superoxide dismutase) is a kind of biological defense enzyme that exists widely in animals and microorganisms, including humans, and is an active enzyme that is harmful and highly reactive in cells, such as superoxide radical (O ₂ ) and hydroxyl. Since it reduces the concentration of reactive oxygen species such as radicals (.OH), it has attracted attention as an important enzyme involved in maintaining homeostasis of life.

[0003] For example, it is known that placenta extract contains SOD (Japanese Patent Application Laid-Open No. 61-277626), and a production method for isolating SOD from human placenta is also known (Japanese Patent Application Laid-Open No. Sho 56). Japanese Patent Application Laid-open No.
No. 155991).

[0004] In addition, it has been confirmed that SOD has a strong anti-inflammatory effect and an inhibitory effect on erythema of the skin by rat carrageenin foot edema test and the like [Oyanagui.
Y. : Inhibition of superoxi
de anions atthe prostagla
ndin phase of carrageenan
foot-oedema. Biochem. Phar
macol. 25: 1465-1472, 1976: A
kira Y. , Yoshiki M .; , Miyach
i I. , J. et al. Dermtol. , 14, 569 (19
87)], and related techniques are also disclosed (for example, Japanese Patent Application Laid-Open No. 56-7720).

Further, cosmetics utilizing various characteristics of SOD are known (for example, Japanese Patent Publication No. 59-10324).
And Japanese Patent Application Laid-Open No. 55-87712).

[0006]

SUMMARY OF THE INVENTION However, SOD
Despite its excellent properties, it is inherently an unstable enzyme, and thus has problems such as loss of SOD activity and turbidity particularly when stored in an aqueous solution.

[0007] Therefore, even if the raw material obtained by the prior art is blended as an active ingredient of a cosmetic, for example,
Since there is a problem that the commercial value is impaired due to the effect of D itself not being exhibited or the color and odor changing over time, the development of raw materials and formulations having stable SOD activity has been desired.

[0008] The present invention has been made in view of such circumstances, and an object of the present invention is to provide a placenta or liver extract having stable SOD activity and an external preparation composition comprising the extract. It is assumed that.

[0009]

Means for Solving the Problems As a result of intensive studies, the present inventors enzymatically decompose a placenta or liver extract with a protease and remove substances having a molecular weight of 10,000 or less obtained by the enzymatic decomposition. It was found that an extract having stable SOD activity was obtained, and by blending this with an external preparation base, an external preparation composition in which the decrease in SOD activity was significantly improved was obtained, and the present invention was completed. .

The extract prepared in the external preparation composition of the present invention can be prepared by adding SOD activity to a liquid extracted from the placenta or liver by a general extraction method using water or a mixed solvent of water and an organic solvent such as alcohol. After a protease is allowed to act under a certain pH and temperature condition that does not cause inactivation,
It can be obtained by removing those having a molecular weight of 10,000 or less. The enzymatic degradation time of the protease is
As can be seen from the manufacturing examples described below, at least 5 hours
As a result, a product having stable SOD activity can be obtained.
Can be

The type of placenta or liver used in the present invention is not particularly limited. For placenta, human, cow, horse, pig and the like are preferable, and for liver, bovine, horse, and horse.
Pigs, birds and the like are preferably used.

When a protease is used, SOD
It is necessary to maintain the activity of
The reaction conditions are preferably 9, preferably 6.5 to 8.5, the temperature is 5 to 50 ° C, and preferably 20 to 40 ° C. As the enzyme used for decomposing the protein, neutral protease (eg, actinase E (manufactured by Kaken Pharmaceutical)), papain, and trypsin are preferably used.

Further, in order to remove the factors that destabilize SOD from the proteolytic enzyme solution, a molecular weight of 10,000 is obtained by a usual operation such as ultrafiltration or filtration using a dialysis membrane.
Remove the following materials:

The extract of the present invention thus obtained has a stable SOD activity and hardly causes irritation to the skin. Therefore, when the extract is added to an external preparation base, the odor and the color tone change over time. It is possible to provide a safe external preparation composition having a low SOD activity and a stable SOD activity.

[0015] The dosage form of the preparation containing the extract of the present invention is
There is no particular limitation as long as it is an external use form, and various bases such as a lotion, a cream, an emulsion, a pack, an essence, an ointment, and an emulsion which are usually used for pharmaceuticals and cosmetics can be used. . However, during the production process of cream or the like, long-time high-temperature heating during emulsification is not preferable because the activity of SOD is impaired, and it is desirable to always add and stir the extract under conditions of 45 ° C. or lower.

When the extract of the present invention is mixed with the above-mentioned base, it is used in an amount of 0.01 to 50.0% (weight), preferably 0.1 to 30% (weight), based on the total amount of the external preparation. used.

Hereinafter, the present invention will be described in detail with reference to examples.

[0018]

【Example】

Production Example 1 1 kg of bovine placenta is washed with cold physiological saline, cut into small pieces, 2 kg of purified water is added, and the mixture is extracted with stirring at 30 ° C. for 3 hours. Next, this was centrifuged (8,000 rpm, 20 minutes, 5 ° C.), and the supernatant was adjusted to weak basicity (pH 7.3), actinase E (manufactured by Kaken Pharmaceutical Co., Ltd.) was added, and 30 ° C.
And stir for 5 hours. Further, the solution was acidified (pH 4.
5) and centrifugation (8,000 rpm, 10 minutes, 5 minutes)
℃) and then neutralized (pH 7.0), and treated with a dialysis membrane to have a molecular weight of 1
SOD activity of 10,5 or less
A bovine placenta extract having 00 units / ml was obtained.

Production Example 2 1 kg of horse liver was washed with cold physiological saline, cut into small pieces, 3 kg of purified water was added, and the mixture was extracted with stirring at 40 ° C. for 3 hours. Next, this was centrifuged (8,000 rpm, 20 minutes, 5 ° C.), and the supernatant was taken to adjust to weak basicity (pH 8.0), and actinase E (manufactured by Kaken Pharmaceutical) was added.
And stir for 6 hours. Further, the solution was acidified (pH 4.
5) and centrifugation (8,000 rpm, 10 minutes, 5 minutes)
℃) and then neutralized (pH 7.0), ultrafiltration with molecular weight 1
Remove SOD activity of 25.0 or less
An equine liver extract with 00 units / ml was obtained.

Production Example 3 1 kg of pig placenta is washed with cold physiological saline, cut into small pieces, 2 kg of purified water / ethanol mixture (1: 1) is added, and the mixture is extracted at 30 ° C. for 3 hours with stirring. Next, this is centrifuged (8,000 rpm, 20 minutes, 5 ° C.), the supernatant is taken, papain is added, and the mixture is stirred at 25 ° C. for 5 hours. Further, the solution was acidified (pH 4.5) and centrifuged (8,000).
The mixture was neutralized (pH 7.0) after 0 rpm for 10 minutes at 5 ° C., and substances having a molecular weight of 10,000 or less were removed by ultrafiltration to obtain a swine placenta extract having SOD activity of 3,000 units / ml.

Production Example 4 1 kg of bovine liver was washed with cold saline, cut into small pieces, 3 kg of purified water was added, and the mixture was extracted with stirring at 35 ° C. for 3 hours. Next, this was centrifuged (8,000 rpm, 20 minutes, 5 ° C.), the supernatant was taken out, and trypsin was added thereto.
Stir at 8 ° C. for 8 hours. Further, the solution was acidified (pH 5.
0), and after centrifugation (8,000 rpm, 10 minutes,
5 ° C.) Neutral (pH 7.0), remove substances having a molecular weight of 10,000 or less using a dialysis membrane, and
A bovine liver extract having 000 units / ml was obtained.

Production Example 5 1 kg of chicken liver was washed with cold physiological saline, cut into small pieces, and 2.2 kg of purified water and 0.8 kg of ethanol were added.
Extract with stirring at 3 ° C. for 3 hours. Next, this is centrifuged (8,000 rpm, 20 minutes, 5 ° C.), and the supernatant is collected, actinase E is added, and the mixture is stirred at 30 ° C. for 5 hours.
Further, the solution was acidified (pH 4.5), centrifuged (8,000 rpm, 10 minutes, 5 ° C.), and neutralized (pH
7.0), and a substance having a molecular weight of 10,000 or less was removed with a dialysis membrane to obtain an avian liver extract having an SOD activity of 17,000 units / ml.

Next, test results (effect data) of the enzyme-decomposed solution of the present invention and a formulation containing the same are shown.

Test Examples (1) SOD Activity of Placenta or Liver Extract The following experiment showed that the SOD activity of each organ extract of the present invention was very stable.

A) Extraction method of organ extract 1 kg of an organ (placenta or liver) is minced and purified water 2
Then, the mixture is stirred and extracted at 40 ° C. for about 3 hours. This extract is centrifuged (8,000 rpm, 20 minutes, 5 ° C.)
The supernatant was adjusted to a slightly basic pH (7.5) with a 10% aqueous sodium hydroxide solution, and actinase E was added thereto.
The enzyme is decomposed while stirring at 30 ° C. for 5 hours. This is made weakly acidic (about pH 5.0), left overnight, and centrifuged (8,000 rpm, 10 minutes, 5 ° C.) to collect the supernatant. Further, the pH of the solution is adjusted to neutral (7.0), and substances having a molecular weight of 10,000 or less are removed to prepare a test solution.

B) SOD activity measurement method In 2.4 ml of 0.05 M sodium carbonate buffer (pH 10.2), 3 mM xanthine, 3 mM disodium edetate, 0.15% bovine serum albumin, 0.75 mM
Add 0.1 ml of each test solution of nitro blue tetrazolium. To this, an organ extract extract sample appropriately diluted with purified water.
After adding 1 ml and leaving at 25 ° C. for 10 minutes, 0.1 ml of xanthine oxidase (about 1 unit / ml) is added, and the mixture is incubated at 25 ° C.

After 20 minutes, 0.1 ml of a 6 mM copper chloride solution is added to stop the reaction, and the absorbance at 560 nm is measured.

The concentration that inhibits the absorbance by 50% is defined as one unit.

C) Stability over time of SOD activity of organ extract The above-mentioned test solution (extract of the present invention) and a comparative solution (the above-mentioned test example, which was not subjected to enzymatic decomposition and molecular weight fractionation, was referred to as Comparative Example 1; The compound which decomposed but did not undergo molecular weight fractionation was designated as Comparative Example 2) was stored at 5, 20, 45 ° C for 1 month, and changed its appearance (precipitation, turbidity, off-odor) on days 2, 10, 16, and 30. And the change in SOD activity were measured.

The results are shown in Tables 1 to 4.

[0031]

[Table 1]

[0032]

[Table 2]

[0033]

[Table 3]

[0034]

[Table 4]

As described above, each organ extract of the present invention contains
Under all temperature conditions, a decrease in SOD activity was hardly observed, and it was confirmed that the SOD activity was stable.

No change in appearance was observed.

Next, specific formulation examples of the external preparation composition will be shown, but the present invention is not limited to only these examples.

Formulation Example Formulation Example 1 (Cream) (% by weight) Polyoxyethylene oleyl ether (POEO) 1.60 Glycerin monostearate 3.00 Salami beeswax 2.80 Cetanol 3.00 Stearic acid 1.75 Liquid paraffin 7.10 Parsol 1789 0.50 Squalane 4.50 Glycerin 2.50 Carbopol 940 0.08 Crewat N 0.01 Pig placenta extract prepared in Production Example 3 0.10 Purified liquid 73 0.06 In addition, small amounts of perfume and preservatives were added.

Formulation Example 2 (Emulsion) (% by weight) Squalane 5.00 Vaseline 2.00 Beeswax 0.50 Sorbitan sesquioleate 1.20 Propylene glycol 5.00 Ethanol 5.00 Carboxyvinyl polymer (1% aqueous solution) 20.00 Potassium hydroxide 0.10 Bovine placenta extract prepared in Production Example 1 10.00 Purified liquid 51.60 In addition, small amounts of fragrances, preservatives and antioxidants were added.

Formulation Example 3 (Emulsion) In Formulation Example 2, a formulation using “Horse liver extract manufactured in Preparation Example 2” instead of “Bovine placenta extract manufactured in Preparation Example 1” was prepared. And

Formulation Example 4 (Emulsion) In Formulation Example 2, instead of "Bovine placenta extract manufactured in Preparation Example 1", a formulation using "Avian liver extract manufactured in Preparation Example 5" was prepared. And

Formulation Example 5 (Lotion) (% by weight) Citric acid 0.40 Calcium carbonate 0.20 Ethanol 5.00 Propylene glycol 9.00 Bovine liver extract manufactured in Production Example 4 30.00 Purified solution 55. 40 In addition, small amounts of fragrances and preservatives were added.

Formulation Example 6 (pack) (% by weight) Vegum 5.00 Squalane 2.00 Propylene Glycol 5.00 Zinc Oxide 10.00 Ethanol 5.00 Horse Liver Extract Produced in Production Example 2 2.00 Purified Water 61.00 A small amount of perfume and preservative were added.

Formulation Example 7 (Essence) (% by weight) Carboxyvinyl polymer (1% aqueous solution) 10.00 Glycerin 20.00 Ethanol 1.00 Sodium hyaluronate (1% aqueous solution) 10.00 Horse produced in Production Example 2 Liver extract 10.00 Purified water 49.00 In addition, a small amount of flavor and preservative were added.

Formulation Example 8 (hair tonic) (% by weight) Ethanol (95%) 75.00 Glycerin 5.00 Bovine liver extract prepared in Production Example 4 5.00 Purified water 15.00 In addition to a small amount of fragrance And preservatives were added.

[0046]

According to the present invention, various placenta or liver extracts having stable SOD activity can be obtained, and by blending them with a trivial external preparation base, the original action of SOD can be improved. The present invention provides an external preparation composition that is safe, does not lose its commercial value, and is exhibited without any problems.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12N 9/50 C12N 9/76 9/76 C12P 21/06 C12P 21/06 A61K 37/50 (56) References JP-A-64 JP-A-2-300137 (JP, A) JP-A-63-284133 (JP, A) JP-A-62-135434 (JP, A) JP-A-64-20084 (JP, A) JP-A-62-130684 (JP, A) JP-A-56-32422 (JP, A) JP-A-56-7720 (JP, A) JP-A-55-87712 (JP, A) 155991 (JP, A) JP-A-56-102787 (JP, A) JP-A-64-63383 (JP, A) JP-A-61-277626 (JP, A) JP-B-59-10324 (JP, B2) Tokuyo Sho 62-501472 (JP, A) Tokuyo Sho 63-501473 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 35/50 A61K 7/00 A61K 7/06 A61K 35/407

Claims (2)

(57) [Claims] 1. Placenta or liver extract at least 5 hours
An extract having stable SOD activity, obtained by enzymatically decomposing with an intermediate protease to remove substances having a molecular weight of 10,000 or less. 2. Placenta or liver extract at least 5 hours
And enzymatic degradation between protease, external composition characterized by containing an extract having a stable SOD activity obtained by removing a molecular weight of 10,000 or less of material.