KR840000377B1 - Dermatic medicament - Google Patents

Abstract

A dermatic medicament is produced by fermn. with Trichosporon kashiwayama FERM-p4821. Thus, T. kawashiyama FERM-p4821 was inoculated into 800 L medium contg. glucose 0.3%, skim milk 0.5%, and yeast ext. 0.5% and incubated at 28≰C for 70 hr with stirring and aeration. The culture broth was filtered and concd. The resulting conct. lg was mixed with stearic acid lg, wax 7g, glycerol monostearate 4g, paraffin 30g, ranoline 0.1g and purified water 40g to give an ointment, which showed bactericidal effect and prevent acne.

Description

Skin disease pharmaceutical

Figure 1: Attachment of Segmented Spores and Military Cells Under Aerobic Conditions

Figure 2: Enlarged Photograph of Segmented Spores

Figure 3: Mycelial cells under anaerobic conditions and their germination

The present invention is a new monarch identified as a genus belonging to the genus Geotricum or endomicrosis, Trichosporon Kashiwaya monarch (deposited under the deposit number 4821 in the FRI) (FERM-P4821) according to a conventional procedure Skin medicinal products consisting solely of sterile supernatants produced by filtration and sterilization, or concentrates or dried products produced by conventional methods from sterile supernatants, or sterile supernatants or concentrates or dry products as active ingredients, It relates to a skin disease agent mixed with the same carrier.

It belongs to the microorganism used in the present invention, tricosphorone Kashiwayama strain, silver yeast, but sometimes classified as incomplete fungi. Therefore, it can be classified as a strain belonging to the genus Geotricum or endomicrosis is based on the professional concept of experts in this field.

As shown in the figure (photo), the features of the strains used in the present invention are globular spores and pseudomycelia. Another feature is that the fermentation product of the strain, ie the supernatant of the strain culture itself, exhibits excellent medical effects on human skin.

Active substances that have a medical effect have not yet been identified. However, when the sterile supernatant or concentrate or dry product of the strain culture solution is mixed with itself or with a suitable carrier, and formulated into, for example, an ointment suitable for human skin, the following effects can be expected.

(1) The fermentation broth is weakly acidic (pH 4.5 to 5.0) and is non-toxic to the skin and enhances the skin protective action of the fat membrane, in particular, the bacterial inhibition of the fat membrane.

(2) Preventing severe peeling of keratin from the stratum corneum and at the same time maintaining the moisture content of the stratum corneum appropriately, it is possible to maintain the skin properties such as moisture and elasticity in a satisfactory state.

(3) It regulates fat metabolism of surface cells to promote keratin production and smooth proliferation and differentiation of surface cells.

(4) In particular, in fatty skin, excessive sebum secretion is suppressed and fat film thickness is prevented from increasing. Thus, sebum diffusion from the hair follicles to the skin surface is promoted to prevent fatty dermatitis such as acne.

(5) Strengthen or swell collagen upset of the reticular body to properly maintain the amount of water in the upper layer, thereby maintaining the hardness and elasticity of the skin normally.

(6) Since the blood circulation is improved, the metabolism of the skin is promoted, the transport of skin waste to the veins and the excretion thereof are promoted. At the same time, the excretion of melanin is smoothed in vitro.

(7) For skin diseases caused by oxidation under actinicray, the fermentation broth or its sterile supernatant acts as an antioxidant to the fat film and promotes the discoloration of abnormally deposited melanin pigments in the skin spots. When fermentation broth or its sterile supernatant is administered in combination with infrared irradiation treatment or vitamin E oral administration, a synergistic effect of prevention or treatment of abnormal pigmentation due to dermatitis, skin spots and heliosensitivity can be obtained.

The fungal characteristics of the tricosphorone Kashiwayama strain used in the present invention are as follows.

(1) morphological characteristics

1) Form and size (malt extract-MY liquid medium)

After incubation at 25 ° C. for 3 days, cells of rectangular or ovoid size (3 to 4 μ) × (3 to 20 μ) are observed, and if culture is continued for 5 days or more, the cell morphology becomes irregular and final. To become myceloid cells.

2) Creation of gastric mycelia (Potato extract agar culture slide slide)

The cells are gastromycelial or gastromycelial, with characteristic zigzag segmented spores but no conidia.

3) Aspergillus spore 4) Injection spore

Do not create Do not create

(2) Characteristics of culture medium

1) Colonies on agar agar culture medium (malt extract-agar culture medium)

Dull, white, hairy, rough surface, very soft, non-flat, flat ciliary peripheral

2) Film on liquid culture medium (malt extract-liquid culture medium)

White, hairy, flocculent, tough film formed

(3) physiological characteristics

1) sugar fermentation

Glucose, galactose, sucrose, lactose, treharose, raffinose and inulin do not result from fermentation.

2) Use of sugar (fairy tale)

D-glucose +, D-galactose +, D-xylose +, erythritol-, D-mannitol +, L-sorbose +, inositol-, treharose-, D-arabinose-, L-arabinose- , Sodium succinate +, sodium citrate +, sodium acetate +, inulin-, soluble starch-, ethanol +, glycerol +, cellobiose-.

caution

+: Use 5) use of ethylamine hydrochloride

-: Not available.

3) Use of nitrates 6) Degradation of glucoside (arbutin)

Not available decomposition

4) Use of Nitrite 7) Production of Starch Analogs

Not available It is not generated.

8) Production of Pigments 15) Degradation of Urea

It is not generated. It does not decompose.

9) Formation of Ester 16) 50% (w / w) Glucose Agar or 60% (w / w) Phase

Is generated. Growth Yeast Extract of Agar:

10) Lit miss milk reaction does not grow.

Does not solidify 17) Growth at 37 ° C

11) Does not grow vitamin dependence.

I don't need it. 18) Acid Generation from Glucose

12) Salt resistance is not produced.

Critical concentrations 6 to 8 w / v% 19) Resistance to cyclohexamide

13) Optimum temperature tolerance

25 ℃ 20) Decomposition of oils and fats

14) Optimal Disassembly

5.0

(4) other fungal characteristics

1) Generation of aromatic substances

The characteristic of this strain is the production of aromatic components. By gas chromatography, it was confirmed that the aromatic component consisted of ethanol, ethyl acetate, isopropyl alcohol, isopropyl acetate, isobutyl alcohol, isobutyl acetate, isoalyl alcohol and isoamyl acetate. This strain is distinguished from known yeasts in that not only higher alcohols but also esters have higher aromatic-producing characteristics.

The optimum conditions for the generation of neutral aromatic components are as follows.

a) maintenance of initial pH 4.0 to 5.0 and low pH during fermentation

b) 400 to 500 mg / l total nitrogen culture medium

As the inorganic nitrogen source, ammonium sulfate, ammonium nitrate and ammonium chloride are preferred, and as the organic nitrogen source, yeast extract casamidoic acid and peptone are preferred. In addition, addition of amino acids such as leucine, isoleucine, valine and phenylalanine yields satisfactory results.

c) selection of carbon sources

Glucose, galactose, ethane chopped and glycerol are suitable. Preferred sugar concentration is 5%.

d) 1 to 2% inoculation ratio

e) incubation temperature 25-30 °

Fixed culture is preferred.

2) Generation of Organic Acids

Formic acid, propionic acid, n-butyric acid, succinic acid and citric acid are produced from glucose.

The tricosporone Kashiwayama strain according to the present invention can be used to produce a dermatological agent using a yeast culture medium. About 24 hours, 20 hours in a solid or book medium consisting of 0.3% (w / v) glucose, 0.5% (w / v) skim milk and 0.5% (w / v) yeast extract, pH 4.0-6.0 Shake or fix the culture at 30 to 30 ℃, inoculate the culture product in the same culture medium and shake or fix culture for 3 to 7 days at 20 to 30 ℃.

The culture is filtered and the cells are removed using a conventional method, such as a filtration membrane, and the supernatant is concentrated, for example, under reduced pressure, preferably at a concentration of 4 to 25. Concentrates are sterilized in conventional manner and stored at room temperature or in a freezer.

Cell-free supernatants prepared according to the invention have the characteristics of the table.

TABLE 1

Cell-free purified filtrate of the culture supernatant of the strain consists of small amounts of amino acids, sugars and fats.

In quantitative analysis of the filtrate, the total nitrogen is 0.005 to 0.015% and the total amount of phosphorus is 0.004 to 0.014%.

1. Appearance

The filtrate is a colorless to pale yellow transparent liquid with weak aromaticity

2. Confirmation test

(1) 20 ml of the filtrate is evaporated in a water bath until the volume is about 50 ml. 1 ml of ninhydrin solution is added to the concentrate. The mixture turns purple after 30 minutes of heating.

(2) To the concentrate produced by evaporation as described in (1) above, 5 ml of nitric acid is added and the mixture is boiled for 20 minutes. After the mixture has cooled down, it is neutralized with 10% sodium hydroxide solution. 2 ml ammonium molybdate test solution is added to the mixture. Heating the mixture turns yellow. A 10% solution of sodium hydroxide is added to the liquid to make it colorless.

(3) 10 ml of indole and 2 ml of hydrochloric acid are added to 5 ml of the filtrate and the mixture is shaken sufficiently. The mixture is heated to red for 10 minutes.

3. Physical features

: 1.000 내지 1.015 (3) pH(25℃) : 4.5 내지 5.5(1) specific gravity

: 1.000 to 1.015 (3) pH (25 ° C): 4.5 to 5.5

: 1.330 내지 1.340(2) refractive index

: 1.330 to 1.340

4. Evaporation residue: 0.5 to 2.0%

Table 2 shows the analysis values of the concentrate (concentration ratio 2) of the cell-free supernatant prepared according to the present invention.

TABLE 2

Water content (according to atmospheric pressure drying method): 98.7% Sugar content: 1.0%

Protein (factor 6.25): Trace gross solids ^* : 1.3%

Fat content (according to Soxhlet extraction method): 0.2% Amino nitrogen content: 10%

Fiber content: 0% Proper acid value: 0.6 ^**

Ash content: 0.1% Nitrogen in volatile basic form: 1%

Arsenic amount (as AS ₂ O ₃ ): Not detected (detection limit: 0.1ppm)

Heavy metal amount (as pb): Not detected (detection limit: 1ppm)

Mercury total amount: Not detected (detection limit: 0.01ppm)

Calcium content: 14.7mg% Citric acid content: 0.02%

Phosphorus content: 9.8mg% Number of living cells: Less than 30 per ml

Formic acid content: Not detected (detection limit: 0.1%) Coli: (-)

Acetic acid content: Undetected (detection limit: 0.01%) Staphylococcus Oreus: (-)

Butyric acid content: Undetected (detection limit: 0.01%) Fungi: (-) / ml

Lactic acid content: 0.06% Yeast water: (-) / ml

Propionic acid content: Not detected (detection limit: 0.02g / kg)

Sorbolic acid content: Undetected (detection limit: 0.005g / kg)

Benzoic acid: 0.42 g / kg

P-hydroxybenzoic acid ester: Undetected (detection limit: 0.005 g / kg)

Vitamin B ₁ content: Undetected (detection limit: 0.01mg%)

Vitamin B ₂ Content: 0.02mg%

Total amount of vitamin C: Not detected (detection limit: 2mg%)

Non-mineral B ₆ content: Undetected (detection limit: 5㎍%)

Vitamin B ₁₂ content: Undetected (detection limit: 0.05㎍%)

Pantothenic Acid Content: 0.09mg%

Choline content: Not detected (detection limit: 0.03%)

Hydrochloric acid content: Undetected (detection limit: 1㎍%)

Niacin content: Not detected (detection limit: 0.03mg%)

Total amount of carotene: Undetected (detection limit: 0.02mg%)

PH: 4.9 Valine: 0.01 or less

Amino acid composition ^*** : Alanine: 0.01 or less

Arginine: 0.01 or less Glycine: 0.01 or less

Lysine: 0.01 or less Proline: 0.01 or less

Histidine: 0.01 or less Glutamic acid: 0.01 or less

Phenylalanine: 0.01 or less Serine: 0.01 or less

Tyrosine: 0.01 or less Threonine: 0.01 or less

Leucine: 0.01 or less Aspartic acid 0.01 or less

Isoleucine: 0.01 or less Tryptophan 0.01 or less

Methionine: 0.01 or less Cystine: 0.01 or less

caution

*:% Based on water

**: ml number of 1N alkali consumed to neutralize 100g sample

***: number of amino acids g in 100 g sample

(Cystine: performic acid oxidation, tryptophan: microbial analysis) Of course, the cell-free sterile supernatant can be used as a dermatological agent without concentration. However, from the viewpoint of storage, it is preferable to concentrate the cell-free sterile supernatant. It is also possible to use concentrates or dry products of 25 or more concentrations. Such concentrates, high concentrates or dry products can themselves be used as dermatological agents. The dermatological agents of the present invention may also be formulated by mixing such concentrates, high concentrates or dry products with creams, ointments or other carriers.

Analysis of the concentrate produced by concentrating the cell-free sterile supernatant at concentration ratio 4 shows that several amino acids and vitamins are contained. However, since the generic term is analyzed, even if each component is mixed according to the analysis value, the dermatological agent of the present invention cannot be prepared. In other words, the activity of the dermatological agent of the present invention is due to an unknown component that is not identified by analysis.

Toxicity tests using chicken embryos The above-mentioned sterile supernatant (concentration ratio 4) is nontoxic. In addition, human patch tests and subsequent body skin irritation tests have demonstrated that the concentrate is completely nontoxic to human skin. In the rabbit eye irritation test, the concentrate was completely non-irritating. Antibiotic testing also revealed that the concentrate did not contain any antibiotics.

The dermatological agents of the present invention have been demonstrated to have therapeutic effects on skin spots, skin cracks, contact dermatitis and burns.

The therapeutic effect is explained by the following experiment.

Human Stomach Test

Fifty women aged 23 to 44 years old (who have a skin disease and are seeking dermatology) are selected for testing.

A bandage measuring 4 × 4 cm and containing a concentrate of about 0.2 (concentration ratio 2) is applied to the medial and paravertebral areas of the left upper limb, and after 48 hours the bandage is removed and tested. In each patient, there are no side effects inside the left upper arm or the spinal chamber.

Continuous human skin irritation test

A 10 cm ² sized sterile gauze was soaked in 5 ml of the concentrate (concentration ratio 2) of the present invention and placed on the right cheek area of 45 women aged 23 to 45 years (35 patients had a skin disease and received medical treatment from a dermatologist. 10 patients are at this hospital). This treatment is continued for 40 days.

None of these reactions occur and no abnormalities such as erythema occur at all.

Rabbit Eye Irritating Test

Three white rabbits are used as test animals. Before doing this, make sure there is nothing wrong with their eyes. Per rabbit, 0.1 ml of the concentrate of the present invention (concentration ratio 2) is added to the right eye and 0.1 ml of blank liquid is added to the left eye. Immediately and after 3, 6, 24, 48 and 72 hours after dropping, the eyes are irradiated using a Slitlamp.

Abnormalities such as hyperemia of the corona, iris or conjunctiva are absent.

Skin spot treatment effect

Except for liver or heterosexual patients and pregnant women, 621 women with skin spots on the face are selected to seek dermatology to treat skin spots.

After washing, the skin pores are opened with ozone and steam, and the gauze soaked in the cell-free supernatant concentrate (concentration ratio 2) is attached to the spot-generating site. The applied gauze is attached with a saran-wrap tape and irradiated with infrared light for 15 to 20 minutes to allow the concentrate to penetrate into the skin. This treatment is continued for three months, once a week. The results improved dramatically in 363 (58.4%) and improved in 122 (19.8%), no improvement in 133 (21.4%), and contact dermatitis in the remaining 2 (0.03%). (The incidence of contact dermatitis is believed to be unrelated to this treatment).

The proportion of patients with very or relatively improved conditions reaches 80%. In addition, skin spots disappear completely in 60% of 365 people, who are feeling better.

Treatment test for skin cracks and fine lines

Usually 25 to 30 years old women care about skin cracks and fine lines, especially in women aged 35 to 40 years, this tendency is obvious, and women over 40 are also sensitive to skin cracks and fine lines. 962 women are selected to seek dermatology in this trial (150 for 25 to 30 years, 218 for 30 to 35 years, 248 for 35 to 40 years, 172 for 40 to 45 years and 174 for 45 years) That's it.

After washing, the skin pores are opened with ozone and steam, the gauze soaked in the cell-free supernatant concentrate (concentration ratio 2) of the present invention is brought into close contact with the face, and the gauze is fixed with Saran-Wrap tape. Infrared radiation is irradiated for 15-20 minutes to allow the concentrate to penetrate into the skin. After one treatment, the cracked skin improved significantly in 698 (73%), relatively improved in 156 (16%), unchanged in 72 (7%), and none in the remaining 36 (4%). It doesn't get worse. Considering the fact that the skin cracks turn into fine lines, the concentrate of the present invention, which is effective for treating cracked skin, has the effect of preventing the formation of fine lines.

Contact dermatitis treatment effect test

The 21-year-old woman, who had been diagnosed with contact dermatitis, selects a rash and itching on both eyelids from the day after switching to A's eye shadow while using A's eye shadow. The gauze soaked in the concentrate (concentration ratio 2) of the subcellular supernatant of the present invention is applied to the infected site. Steroid contracts, anti-inflammatory and antihistamines were not used at all. Approximately 20 ml of concentrate is applied to the patient and then taped or applied twice a day in the morning and evening for about 30 minutes. After 48 hours of treatment, the itch disappears and after 96 hours, the rash almost disappears. One week later, no rash is observed.

The therapeutic effect of recall

Dermatologists visited a dermatologist about 1 hour after a burn of a 47-year-old woman who had a dermatitis (2 degree burn) rash, flam swelling, and herpes outbreak in hot water. The burn site is immediately cooled with running water, and a gauze soaked in the cell-free sterile supernatant (concentrate concentration ratio 2) of the present invention is applied to the burn site and an oil paper is applied thereon. Bandage the burn. After 24 hours, the herpes is completely gone, but the rash and swelling still remain, and the pain disappears.

The 30-year-old man, who suffered a dermal rash (2 degree burn) rash and egg size swelling in hot water, was treated in the same manner as the cell-free sterile supernatant concentrate (concentration ratio 2) of the present invention. After 24 hours, the rash remains but the bloat disappears and does not complain of pain.

Therefore, it can be seen that the concentrate of the present invention has a great effect on the burn.

The skin name Ide and cosmetics of this invention are demonstrated to the following example.

Example 1

800 liter of culture medium having the composition as described above was filled into a 1 kL aeration high-fermentation tank, heat sterilized and naturally cooled, and then the culture medium was subjected to about 30 in 10 liters of the same culture medium in a small aeration stirred fermentation tank at 28 ° C. Inoculate the inoculum obtained by preculture for a period of time. Subsequently, the cells are ventilated, plated and incubated at 28 ° C. for about 70 hours. After the fermentation was completed, the culture was passed through a Naigai Shokuhin Kogyo Kabushiki Kaisha strainer model MP293-16 (Toyo Roshi Sine GL-90: 0.5μ) and a light filter model MP293-4 (Toyo Roshi apple membrane: 0.45μ). Remove solid particles and cells. The obtained solution was concentrated by a reversible osmosis membrane method using Bioengineering Co.'s reversible osmosis system model RO-T10 to reduce the volume to 200 l. The resulting concentrate is sterilized in a plate heat tester (Iwai Kikai Kogyo Kabushiki Kaisha), sterilized and filled in a glass container and stored in a freezer (5 ° C).

Example 2

The sterile concentrate obtained in Example 1 was lyophilized and the powder obtained was mixed with sterile meat bodies to prepare a skin ointment.

Example 3

1 g of the sterile concentrate obtained in Example 1 is mixed with a cosmetic base consisting of 1 g of 7 g of stearic acid and 4 g of self-emulsifying glycerol monostearate, 30 g of liquid paraffin of 0.1 g of hydrous lanolin and 40 g of purified water, Prepared by mixing the mixture in a conventional manner. The composition is filled in ampoules and the packaged ampoules are swashed.

Example 4

An appropriate amount of pyridoxine hydrochloride, γ-orizanol, allantoin, DL-α-tocopherol (vitamin E) and mink oil are mixed with the composition prepared in Example 3 to prepare a medicament for treating skin diseases.

The figure (photo) illustrates the growth of the tricosphorone Kashiwayama monarch used in the present invention when slide culture was carried out at 25 ° C. for 3 days using a potato extract agar culture medium.

FIG. 1 is a micrograph (70 times magnification) showing the state of adhesion of segmental spores with military cells under aerobic conditions. In FIG. 1, it can be seen that the segmental spores do not adhere to the conidia and extend laterally in a chain form from the septal portion of the mycelia.

Figure 2 is an enlarged (300x) micrograph of the segmental spores.

FIG. 3 is a micrograph (300x magnification) showing cells and their budding state under anaerobic conditions.

Claims (1)

Tricosporon Kashiwayama strain (deposited in Deposit No. 48213-FRI), a new strain identified as a genus belonging to the genus Geotricum or endomicrosis, was deposited on or in a culture medium having a pH of 4.0 to 6.0 or 20 to 30 ° C. Culture to obtain a culture or culture medium, optionally removing cells from the culture to obtain a cell-free sterile solution, a sterile filtrate or a sterile supernatant and optionally to concentrate the cell-free sterile solution, a sterile filtrate or a sterile supernatant Characterized in that the drying, the culture or culture of the new strain, cell-elimination sterilization solution, sterile filtrate or sterile supernatant, a method for producing a concentrate or dry product thereof.

Images