JPS6289623A - Dermal medical agent - Google Patents

Abstract

PURPOSE:To obtain a dermal medical agent effective against liver-spot skin roughening, dermatitis, etc., by cultivating novel Trichosporon kashiwayama strain (FERM-P No. 4821) under specific conditions and collecting the germ-free supernatant liquid of the above-mentioned culture fluid. CONSTITUTION:Trichosporon kashiwayama strain (hereinafter abbreviated to kashiwayama strain) is preferably cultivated in a yeast culture medium at 4.0-6.0pH consisting of 0.3 wt./vol.% glucose, 0.5 wt./vol.% skimmilk and 0.05 wt./vol.% yeast extract at 20-30 deg.C for 3-7 days. The resultant culture fluid is aseptically filtered through a membrane filter, etc., and the germ-free supernatant liquid is concentrated and dried at any time to give the aimed dermal medical agent. The fermentation product of the kashiwayama strain is found to have remarkable medical effect on the skin and the effective against liver-spot, skin roughening, fine wrinkles, contact-type dermatitis, burn, etc. The agent has no irritancy and contains no antibiotic substance.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel microorganism called Trichosporon.
A sterile supernatant obtained by filtering and closing a culture solution of the Kashiwayama strain (Sporon) Kashiwayama strain (Feikoken Bibori No. 4821: hereinafter referred to as the Kashiwayama strain) according to a conventional method, or the supernatant liquid further The present invention relates to a medical agent for the skin, which is made of a concentrated liquid or a dried product thereof, which is concentrated by a conventional method.

Incidentally, the term "dermatological medical agents" used in the present invention includes what is broadly called "cosmetics" from the viewpoint of the actions and effects they exhibit. That is,
Products containing a liquid or solid substance obtained by post-cultivation treatment as an active ingredient in a carrier such as a cream or ointment base material for dermatitis are only useful as medical agents for the skin. It is also useful as a cosmetic product.

The novel microorganism Trichosporon kashiwayama strain used in the present invention is considered to belong to yeast. However, when identifying bacteria, for example, the following documents, (1)
Key to the East (1974)
w key to TIIE Y[5TS to i
) (2) A Guide to Identifying and Classifying East (1979) (A
guide to identifying
and classifying yeasts), it may be classified as Deuteromycota, and it may also be identified as a bacterium belonging to the genus Geotricum or Endomycopsis.

As shown in the attached photograph, the novel microorganism used in the present invention is unique in that it has segmented spores and (M hyphae) arranged in a genus-like manner.

The fermentation products of the microorganisms, more specifically, the supernatant liquid of the culture fluid, have remarkable medical effects on the skin. Incidentally, the substance of the effective substance that exhibits the above-mentioned medical effects has not yet been completely elucidated. As mentioned above, the sterile supernatant of the microbial culture of the present invention, its concentrated or dried product, when used alone or when applied to the skin with an appropriate carrier, has the following usefulness (medical effects). ): (1) Although the intoxicating liquid exhibits weak acidity (pH 4.5-5.5), it is harmless to the skin and bacteria, and has a protective effect on the skin, including a bacterial control effect on the skin fat film. has.

(2) Prevent excessive exfoliation of horny pieces from stratum corneum cells and at the same time retain moisture in the stratum corneum. As a result, the properties of healthy skin itself ("moisture" and "elasticity") can be maintained.

(3) By controlling the lipid metabolism of epidermal cells, proper keratin production and smooth proliferation and differentiation of epidermal cells are promoted.

(4) Suppresses excessive sebum secretion, especially for oily skin,
It prevents the thickening of the fatty membrane and therefore promotes the diffusion of sebum from the hair 1 to the surface of the skin H, so it has a preventive effect on hyperlipidic dermatoses including acne.

(5) It strengthens or swells collagen fibers in the reticular layer and retains moisture in the upper layer, thereby promoting normal hardness and elasticity of the skin.

(6) Improve blood marriage. That is, it promotes skin metabolism and at the same time activates the circulation and excretion of skin wastes into the veins. Along with this, it also promotes the excretion of melanin pigment from the body.

(7) It acts as a fatty membrane antioxidant against skin diseases caused by oxidation under sunlight, and promotes the fading of melanin pigments that are abnormally deposited, such as age spots. In particular, when used in combination with infrared irradiation, oral vitamin E, etc., it works in a multi-layered manner, and has not only a preventive effect but also a therapeutic effect on contact dermatitis, age spots, and other pigment abnormalities caused by hypersensitivity to sunlight.

The Trichosporon strain Shiwayama used in the present invention is a novel strain obtained from a type of health drink conventionally used in folk medicine.

This drink is a type of lactic acid bacteria drink that has been handed down in local areas for more than 50 years, and is a fermented product of a mixed system consisting of lactic acid bacteria and several types of yeast. There was also some knowledge that this drink was effective when applied to the skin. Therefore, from the perspective of demonstrating its effectiveness on the skin, we conducted extensive research into the characteristics of the individual bacterial groups that make up this mixed fermentation product, and found that certain unknown ingredients produced by the Kanwayama strain were particularly effective on the skin. We have also discovered that this effect shows a significant effect.

The Kashiwayama strain of the present invention produces certain active ingredients only when it is stored for a long period of time in a highly concentrated lactic acid environment and has acid resistance and strong oxidative decomposition ability of lactic acid. That is, the Kashiwayama strain of the present invention was screened by repeating the purification of the strain from the lactic acid bacteria drink described above three or more times, and then purified with a whey medium containing a high concentration of lactic acid (lactic acid 2.5 to 2.5 times). 3. Culture with shaking at 25°C for 48 hours (Ow/V%, pH 3.0-3.3), and select strains again using lactate dehydrogenase activity or lactic acid oxidative decomposition ability as an indicator. It was created as a result.

The Kashiwayama strain thus obtained has significant clinical differences in its skin efficacy compared to coexisting or similar standard strains.

The mycological properties of the Kashiwayama strain used in the present invention are as follows: Morphological properties (1) Morphology and size Wort, MY liquid medium) 25°C
, 3 days later, a rectangle (re
The cells are ctangular or oval in shape, and after 5 days they take on an irregular shape and eventually become hyphal-like.

(2) Pseudohyphal formation (slide culture on potato extract agar medium)
domycereal). It has characteristic zigzag-shaped arthrospores, but no conidiophores.

(3) Ascospores not present (4) Exjected spores not present IBI culture Cultural characteristics) Colonies on agar slant medium (wort and MY agar medium) Dull, white, Pinnate (
It has a rough surface and a very soft surface. It is flat with no protuberances, and the periphery is ciliate.

(2) Film in liquid medium (wort and MY liquid medium) Forms a white, hairy, wrinkled, hard film (3) Forms aroma from nutrient sources in the medium (4) ) Suitable growth pH: 4.0 to 6.0, suitable growth temperature: 20 to 30°C tel Physiological characteristics) Non-fermentable to all sugar fermentable glucose, galactose, nuclose, lactose, trehalose, raffinose and inulin.

(2) Class a assimilation (assimilation) D-glucose (D-G Iucose) +
D-Galactose
+ Maltose
-Raffinose
-Sucr
ose) - Breasts
Sugar (Lactose)
-Sodium Lactate
+D-Xylose (D-Xylose) −
“Elite (total ythrit)
ol) -D-7 unit (D-Ma
nnitol) +L -7/L/Bose (L
-5orbose) + innoshift (I
nositol) -trehalose (Tr
eharose) -D-7 Rabbit/-su(D
-Arabinose) -L-arabinose (
L-Arabinose) - Sodium succinate + Sodium citrate (Na C1trate) - 1- Sodium acetate (Na Acetate) + I
lnulin
-Soluble Denzo 7 (So
lible 5tarch) -Ethanol (
Bthanol) + glycerin (G
lycerol) + Ce
llobiose) - (Note) 10 Assimilation - Not assimilation (3) Nitrate assimilation Not assimilation (4
) Assimilation of nitrite Not assimilated (5) Assimilation of ethylamine hydrochloride Not assimilated (7) Formation of starch-like substances Not formed (8) Pigment Not formed (9) Ester Formation Formation of αO lidmus milk reaction Non-coagulating Ill vitamin requirement
Not required (12) Salt tolerance limit concentration 6~
8 (w/v)% (13) Optimal temperature
25℃ (14) Optimum pH 5.0 +151 Urea degradability No decomposition+
Growth at +7137℃ ′ No growth (
18) Cycloheximide resistance Resistant [I
EI fat degradability Decomposes (D+
Other mycological characteristics (1) The aroma-producing Kashiwayama strain has remarkable characteristics in producing neutral aroma components. According to gas chromatographic analysis of its neutral aroma components, it is a so-called apple drink whose main components are ethanol, ethyl acetate, isopropyl alcohol, propyl isoacetate, isobutyl alcohol, butyl isoacetate, isoamyl alcohol, and amyl isoacetate.

In addition, the Kashiwayama strain is characterized not only by its production of higher alcohols but also by its high esterification rate, compared to yeasts that have been known to have aromatic productivity.

The optimal conditions for the production of these neutral aroma components are: a) initial 1114.0-5.0 and low p++ during fermentation;
b) Total nitrogen content in the medium; 400-500 mg/β
; As nitrogen sources, ammonium sulfate, ammonium nitrate, and ammonium chloride; as organic nitrogen RiFR, yeast extract, casamino acid, and peptone are suitable. Furthermore, the effect of adding amino acids such as leucine, isoleucine, valine, and phenylalanine is remarkable.

C) Selection of carbon source: glucose, galactose, ethanol, glycerol, etc. are suitable. P! Concentration is 5%
d) Bacterial inoculation rate 1-2% e) Culture at 25-30°C, static culture 2) Organic acid production Formic acid, propionic acid, n-acetic acid, succinic acid, and citric acid are produced from glucose.

Next, the differences between the Trichosporon strain Shiwayama of the present invention and known species are listed below.

Morphologically, it closely resembles Trichosporon penicillatum (currently Geotrichum penicillium) in terms of colony characteristics, hyphae, and segmental spore morphology; Has no fermentability; ■Assimilates D-mannitol and citric acid; ■Higher alcohols and their esters, such as ethyl acetate, isopropyl alcohol, propyl isoacetate, isobutyl alcohol, butyl isoacetate, isoamyl alcohol, and amyl isoacetate. They are different in that they produce neutral aroma components, with the main component being

(Old) Similarly, it is morphologically similar to Geotrichum candidum, but ■It has no glucose fermentability, ■Arbutin (
Arbutin, 1lydroquinone-β-
It differs from known species in that it has the ability to decompose 〇-Glucopyranoside), and ③ it has a strong ability to assimilate DL-lactic acid and succinic acid.

[C] Furthermore, Endomycopsis opetensis (currently Tsendera opetensis): ■ Has arbutin decomposition ability; ■ Has no vitamin requirement; ■ Assimilates D-1-sylose, D-mannitol, and citric acid. ■They differ in that their budding morphology is different, they do not form ascospores, and their pond morphology is different.

Conventional yeast culture media can be used to produce novel dermatological medicinal agents using the Kashiwayama strain.

A suitable example is a medium with the following composition: glucose 0.
pH 4 containing 3% (W/V), skim milk 0.5% (W/V), yeast extract 0.05% (W/V),
Using a solid or liquid medium of 0 to 6.0, it is
The seed culture is cultured with shaking or standing at ~30°C for about 24 hours, and cultured with standing or shaking in the same medium at 20-30°C for 3-7 days.

The culture fluid is then sterilized by filtration using a membrane filter in a conventional manner, and the sterile supernatant is concentrated (for example, under reduced pressure) to obtain a 2- to 25-fold concentrated solution, preferably.

The concentrate is further sterilized by a conventional method and then stored at room temperature or refrigerated.

An example of a sterile supernatant produced according to the present invention had the properties shown in Table 1.

Table 1 The sterile filtration purified product of the culture supernatant obtained by the Kashiwayama strain contains small amounts of amino acids, carbohydrates, and sushi substances. Furthermore, total nitrogen is 0.0 O5 to 0.015%, and total phosphorus is 0.05%. OO
Contains 4-0.014%.

■Properties: Colorless to slightly yellow, transparent liquid with a slightly unique aroma.

2. Confirmation test (2) Evaporate 20 ml of crystal on a water bath until it becomes about 5-, add 1 rri of ninhydrin test solution to this liquid, and heat for 30 minutes, the liquid will take on a purple color.

Add nitric acid to the evaporated liquid in the same way as ■■. 51 Thi! Add and boil for 20 minutes.

After neutralizing with cold 10% sodium hydroxide solution, ammonium molybdate test solution 21Tll! When heated, the liquid turns yellow. When cold, when adding 10% sodium hydroxide solution to this liquid, the liquid becomes colorless.

■5ml! Take 10 mg of indole and 21 mg of hydrochloric acid.
T11! After adding and shaking well, heat for 10 minutes, the liquid will turn red.

3. Physical properties ■Specific gravity (d): L, 000~
1, O15 ■Refractive index (n) ・1.330-1,340 ■pH (25°): 4.5-5.5 ・1 Evaporation residual 05-20% Sterile supernatant liquid produced by the present invention Concentrate (2x concentrate)
- Examples had analytical values as shown in Table 2.

The resulting sterile supernatant can be used as a dermatological medical agent without being concentrated. However, concentrating as described above has storage and other advantages. Further, it is also possible to use a thick material having a degree of shrinkage of 25 times or more, or a dry material. Furthermore, they can also be used in combination with creams, ointment bases and other carriers.

An example of the results of an analysis of the above-mentioned sterile 4-fold concentrate revealed that it contained various amino acids and vitamins. However, since these analyzes only target general items, it is not possible to produce the medical agent for skin of the present invention even if the components are blended according to the analytical values. That is, it is presumed that the effective substance of the medical drug for skin is due to unknown components outside the analysis items.

The above-mentioned sterile concentrate (4x concentrate) was proven to be harmless as a result of a chicken embryo toxicity test, and was found to be harmless to the skin in a human patch test and a continuous human skin irritation test using a 2x concentrate. It was proven to be harmless and non-irritating in rabbit eye irritation tests. Furthermore, an antibacterial test revealed that it did not contain any antibiotics.

Furthermore, it has been found that it has a therapeutic effect on skin spots, rough skin, small wrinkles, contact skin irritation, and burns.

The dermatological medical agents and cosmetics of the present invention will be explained in more detail below with reference to Examples.

Reference Example 1 A medium (pH 5,0) containing 2.4 J of glucose, 4.0 kg of skim milk, and 0.4 kg of yeast extract in 8001 pure water was sterilized in an ill-volume aerated fermentation tank by a conventional method, and then added to the medium. Same composition medium 101
The seed culture was pre-cultured in a small aerated and agitated fermentation tank at 28°C for about 30 hours and then inoculated as a seed culture, followed by aerated agitation culture at 28°C for about 70 hours.

After fermentation, the culture solution was transferred to MP2 manufactured by Naigai Shokuhin Koki Co., Ltd.
93-16 type filter (filter: Toyo Roshi GL-90
, 0,5μ) and MP293-4 type filtration machine manufactured by the same company (
Menpuran filter: Toyo Roshi TM-2,0,45μ
), the solid particles and further the bacterial cells were removed. The obtained filtrate was subjected to reverse osmosis membrane method (manufactured by Bio Engineering Co., Ltd.,
Concentrate to 400A using an R○-TIO type reverse osmosis membrane device and obtain a 2-fold concentrate.

Repeat the above method, concentrate to Tazo 2001 and make a 4x concentrate.

The obtained concentrates were sterilized using a plate heat exchanger (manufactured by Iwai Kikai Kogyo Co., Ltd.), filled aseptically into glass containers, and stored under refrigeration (5° C.).

A powder product obtained by freeze-drying the sterilized concentrate was mixed with a sterile ointment base material to produce a medical material for the skin.

A creamy cosmetic was also produced from the concentrate.

These have shown excellent effects as dermatological medical agents and cosmetics. This point will be explained in more detail below with reference to Examples.

Example 1 Human Patch Test Approximately 0.2 g of 2x concentrate was applied to a 4 cm square patch, and this was applied to 50 women aged 23 to 44 (who had some skin symptoms and were referred to a dermatologist). It was applied to the vertebrae on the inside of the left upper arm and near the back of a patient attending a specialized hospital. The adhesive bandage was removed after 48 hours. As a result of the evaluation performed 30 minutes after removal, there were no cases in which the inside of the left upper arm and near the back of the vertebrae showed positive results.

Human body continuous skin II! rIrritation test Approximately 5 ml of the 2x concentrate was placed in 1 Q cut of sterile gauze and administered to 45 women aged 23 to 45 (35 of whom were outpatients at a dermatology hospital with some kind of skin condition).
It was applied to the right facial cheek area of 0 female employees of the hospital for 15 minutes. This coating treatment was continued for 40 days. As a result, all the subjects showed no reaction, and none of them exhibited abnormal symptoms such as erythema.

Rabbit eye ill intense test: 0.1 ntf of 2x concentrated solution for the right eye and a blank test solution for the left eye of 3 white domestic rabbits that have been confirmed to have no abnormalities in their eyes! Immediately after instillation, 3
．． 6.24. After 48 and 72 hours both eyes were examined using a slit lamp. As a result, no hyperemia or other abnormalities were observed in the cornea, iris, or conjunctiva of both eyes during any observation.

Treatment trial for age spots Excluding patients with liver disease, gynecological patients, and pregnant women. Sixty-two women with facial skin spots attending a dermatology hospital.
After washing one person's face, the pores were opened using ozone and steam, and the product of the present invention (a 2-fold concentrate of the sterile supernatant liquid) was made into gauze and applied closely to the spot where the stain was generated. I covered it with saran wrap. Then infrared irradiation for 15 minutes
This was carried out for ~20 minutes to allow the nuclear crystals to penetrate into the skin. This treatment was continued once every day for three months.

As a result, 363 people (58
,4%), and 122 people (19.8%) who improved slightly.
), 133 people (21.4%) with no improvement recognized,
1 patient (0.01%) had worsening symptoms, and 2 other patients (0.03%) (recognized to have contact I3 inflammation due to another cause)
Met. The total of those who showed great improvement and those who showed slight improvement reached about 80%, and of the 363 patients who showed great improvement, about 60% had no dark spots at all.

Treatment test for rough skin and small wrinkles The product of the present invention (double concentrate) was used on a trial basis for the treatment of rough skin and small wrinkles in a female patient over 25 years of age attending a dermatology hospital.

Normally, women over the age of 30 are concerned about rough skin and fine wrinkles. In particular, people between the ages of 35 and 40 are most concerned about rough skin and fine wrinkles, followed by those between the ages of 30 and 35, and those over 40 tend to be less concerned about it as there is no significant difference.

Therefore, 150 women were examined, 25 to 30 years old, and 2 were 30 to 35 years old.
After washing the facial skin of a total of 962 people (18 people, 248 people aged 35-40 years, 172 people aged 40-45 years, and 174 people aged 45 years or older), the pores were opened with ozone and steam, and then the product of the present invention ( Place gauze soaked with sterile supernatant (2x concentrate) and cover the face with Saran wrap, and irradiate with infrared rays for 15 to 20 minutes to peel off the nuclear crystals. I tried a method of letting it seep through.

- 698 people whose rough skin has been greatly improved after several treatments
(73%), 156 (16%) who showed slight improvement after one treatment, 72 (7%) who showed no change, and 36 (4%) who showed no worsening. The result was obtained.

As skin becomes rougher, it is more likely to develop small wrinkles.
It has been found that the product of the present invention, which is effective in treating rough skin, is useful as a cosmetic product for preventing fine wrinkles.

Treatment test for contact dermatitis I: °1 The day after switching from eyeshadow made by company A to eyeshadow made by company B, redness and itching occurred on the eyelids on both sides of the face, and the patient was diagnosed with contact dermatitis. A 21-year-old woman who had been diagnosed with this disease was treated with gauze soaked with the product of the present invention (double concentrate) and applied to the affected area by a dermatologist.

At that time, only the product of the present invention was used without using steroid preparations, anti-inflammatory agents, or antihistamines.

That is, about 20 ml of the product of the present invention was given to the patient, and the patient was allowed to apply or paste the product at home for about 30 minutes twice in the morning. 48
The itching disappeared after an hour, the redness almost disappeared after 96 hours, and no redness was observed after 1 week.

Burn Treatment Trial Prisoner: A woman with degree 47 burns caused by boiling water who had palm-sized redness and swelling on the outside of her left forearm (TT!) and the formation of small chicken pox on the outside of her left forearm (tt!) was referred to a dermatologist about 1 hour after the injury from boiling water. After immediately cooling the affected area with tap water (running water), gauze soaked with the product of the present invention (double concentrate) was placed on the affected area, and the top was covered with oiled paper and a bandage was applied. After 24 hours, residual redness and swelling were observed in the affected area, but the small varicella disappeared and the pain disappeared.

tB) The same treatment was given to a 30-year-old man who suffered from grade 1 burns, which caused redness and swelling on the dorsum of his right leg to the size of a hen's egg due to boiling water. As a result, 24 hours later, although some redness remained, the swelling was gone and the pain was gone.

Example 2 Preparation of creamy cosmetics 1 g of stearic acid 1 7 g of beeswax 4 g of self-emulsifying monostearic acid-glycerin, 30 g of liquid paraffin 1 0.1 g of lanolin and knead by a conventional method,
A creamy cosmetic was obtained. Dispense this into regular small bottles,
It was filled and sold.

Example 3 Preparation of a medical agent for skin A 4-fold concentrated solution was prepared for the following base materials: 1 g of stearic acid, 7 g of beeswax, 4 g of self-emulsifying monostearic acid-glycerin, 30 g of cast-iron paraffin, 0.1 g of phosphorus, and 40 g of purified water. A medical agent for skin was obtained by adding appropriate amounts of pyridoxine hydrochloride, gamma-oryzanol, allantoin, DL-alpha tocopherol (vitamin E), and mink oil.

[Brief explanation of drawings]

The attached photograph shows the morphology of the Trichosporon kashiwayama strain of the present invention when it was extracted from halibut and cultured on a slide agar medium (25°C, 3 days later). FIG. 1 is a micrograph (×75) showing the state of adhesion of segmented spores from hyphal cells under aerobic conditions. The segmental spores do not settle on the conidiophores, but are derived from the septum of the hyphal cells and linked to laterals. Figure 2 is an enlarged micrograph of segmented spores (X30
0). FIG. 3 is a micrograph showing cells and their budding morphology under aerobic conditions (X300).・, 1F1 [S: Happy, °Fig.

Claims (5)

[Claims] (1) Trichosporon Kashiwayama strain (Feikoken Bacillus No. 4
No. 821) at a pH of 4.0 to 6.0 and a temperature of 20 to 30°C.
1. A method for producing a medical agent for skin, which comprises culturing on or in a medium, and obtaining a substance having a medical effect on the skin from the obtained culture solution. (2) The method for producing a medical agent for skin according to claim (1), wherein the medium comprises glucose, skim milk, and yeast extract. (3) The method for producing a medical agent for skin according to claim (2), wherein the culturing is carried out under aerobic conditions. (4) A means of obtaining a substance that has a medical effect on the skin,
A method for producing a medical agent for skin according to claim (1), which comprises filtering and sterilizing the culture solution and collecting the resulting sterile supernatant. (5) A method for producing a medical agent for skin according to claim (4), which comprises evaporating water from a sterile supernatant liquid to obtain a concentrated liquid or a dried product thereof.