KR102244368B1 - Ethosomal composition with tocopheryl acetate and dexpanthenol and method for preparing the same - Google Patents

Abstract

The present invention relates to an ethosome composition comprising ethosome in which vitamins and dexpanthenol are encapsulated, wherein the ethosome contains ethanol, peptides, phosphatidyl choline, and amino acid compounds, and to a method for preparing the same.

Description

Ethosome composition containing vitamin and dexpanthen and its manufacturing method {ETHOSOMAL COMPOSITION WITH TOCOPHERYL ACETATE AND DEXPANTHENOL AND METHOD FOR PREPARING THE SAME}

The present invention is an etosomal composition comprising a vitamin and dexpanthen encapsulated etosomal, by using an etosomal composition encapsulated with a poorly soluble substance to effectively permeate the poorly soluble substance to the skin, particularly the scalp, and to hair growth and wool. It has an effect, it relates to an etosomal composition capable of preventing hair loss and a method for preparing the same.

Human hair is about 100,000 to 150,000 per individual, and hair grows with its own cycle. This cycle is divided into anagen, catagen, and telogen, and according to this cycle, hair growth and dropout are repeated. In this process, it is a natural phenomenon that an average of about 100 hairs per day are dropped out.

Hair loss refers to a condition in which the hair has fallen out and is genital or lost. The mechanism of hair loss has not yet been accurately identified, but studies so far have shown that excessive sebum production caused by abnormalities in the circulatory system such as hormonal imbalance, autonomic nervous system and blood circulation disorders, nutrient deficiency of hair roots, allergies, bacterial infections, It is estimated that genetic factors and aging are the main causes.

In addition, in recent years, the cause of environmental factors such as mental stress, air pollution, food, etc. from complex living environments has emerged.

As the external hair loss factors increase, hair loss, which was mainly seen in middle-aged people in the past, can be easily found in young people in their 20s and 30s in recent years, and the number is also increasing.

Accordingly, many studies have been conducted to treat hair loss, and drug therapy and hair transplantation methods such as female hormones and blood flow promoters have been developed. Currently, the most commonly used drugs for the treatment of hair loss are FDA-approved minoxidil and finasteride.

Minoxidil is a drug that induces a vasodilating effect by inducing the opening of Ca-channels, and increases blood flow through this, thereby continuously supplying nutrients to the hair roots, thereby inducing hair growth. In particular, it is known to have a great effect on hair loss in the crown part. However, in the case of hair loss in areas other than the crown, it has a disadvantage that it does not have a good effect.

On the other hand, finasteride has an effect of inhibiting hair loss by inhibiting the function of type 2 5α-reductase, thereby preventing the conversion of testosterone to DHT (Dihydrotestosterone). However, finasteride has side effects of decreased libido and erectile dysfunction in some patients. In addition, in the case of such pharmacological treatment, it is possible to quickly treat a short period of time, but it has a fatal problem that hair loss proceeds immediately when the use of the therapeutic drug is stopped. However, in the case of male-type hair loss and late hair loss, finasteride alone does not have sufficient hair loss improvement effect, and it has a problem that it is difficult to show a continuous wool effect without the help of a DHT inhibitor.

Therefore, different woolen agents that can overcome female type alopecia, male pattern alopecia, and late alopecia are required, and there is a need to increase efficiency and reduce side effects by approaching differently depending on the type of hair loss.

Various formulation technologies have been developed for transdermal delivery of active ingredients, but there are still many limitations in efficiently delivering active ingredients.

After numerous experiments and repetitions, the inventors of the present invention have come to complete an etosomal composition capable of efficiently delivering an active ingredient having an effect on hair growth and wool, and a method for producing the same.

Korean Patent Registration No. 10-0352088 Republic of Korea Patent Publication No. 10-2015-0044029

An object of the present invention is to provide an etosomal composition capable of efficiently delivering poorly soluble active ingredients through the skin protective film and a method for preparing the same.

The present invention enables efficient delivery to the scalp by using an effective skin anti-aging active ingredient that has effects such as skin anti-aging, antioxidant, and collagen promotion, not a hair growth / wool functional active ingredient that is recognized as effective for hair growth / wool. It is an object of the present invention to provide a composition that promotes the wool effect and prevents hair loss.

The etosomal composition according to the present invention for solving the above problems is an etosomal composition comprising an etosomal encapsulated with vitamins and dexpanthen,

The etosomes include ethanol, peptides, phosphatidyl choline, and amino acid compounds.

Specifically, the peptide is pal-GHK, Pal-GQPR, Pal-KTTK, Pal-KTTKS, Biotin-GHK, PAL-KVK (Palmiotyl Tripeptide-5), Palmitoyl tripeptide-38, Palmitoyl-Tripeptide (Pal-RFK) , Human Oligopeptide-20(Val-Gly-Val-Ala-Pro-Gly), GHK-Cu, (AHK)2Cu, AHK-Cu, Myristic acid-KLAKK-CONH2, Hexapeptide-11, Octapeptide-2, Palmitoyl-hexapeptide , Acetyl tetrapeptide-15, Acetyl Hexapeptide 3, Acetyl Tetrapeptide-2, Acetyl Tetrapeptide-5, Acetyl-Decapeptide-3, Ac-KGHK, rh-Oligopeptide-1(humanEGF) (NSDSECPLSWEHDGYCLHDGVCRDMYIEALDKYACNCVVVG YIGERCakeYIEALDKYACNCVVVG YIGERCakeY Pro-Dab-NHBzl), peptide CK+(Ac-Arg-Lys-Arg-NH2), peptide CK(Arg-Lys-Arg), tripeptide-3(Ala-His-Lys.HCl), Ac-KGHK-NH2, It may be one or more selected from the group consisting of Ac-Tyr-Arg-hexadecylester and Argireline.

In the present invention, the vitamin may be one or more selected from the group consisting of vitamin E, vitamin C, vitamin B, salts thereof, and analogs thereof.

In the present invention, the amino acid compound may be one or more selected from the group consisting of amino acids and amino acid analogs, and the amino acid and amino acid analogs are NAC (N-Acetyl Cysteine), arginine, lolysine, lysine ethyl ester, It may be one or more selected from the group consisting of histidine and histidine ethyl ester.

In the present invention, the ethosome composition is vitamin 20 to 70 mg/100ml, dexpanthenol 150 to 250 mg/100ml, ethanol 5 to 15 ml/100ml, peptide 0.1 to 10 mg/100ml, phosphatitylcholine 10 to 1000 It may be one containing mg/100ml, 10 to 1000 mg/100ml of an amino acid compound.

In the present invention, the etosomal may have an average particle diameter of 90 to 200 nm.

In the present invention, the ethosome may have an absolute value of a surface charge of 10 to 100 mV.

The present invention also provides a method for preparing the above-described ethosome composition,

(a) solubilizing an amino acid compound in purified water;

(b) solubilizing dexpanthenol in purified water;

(c) solubilizing ethanol, vitamins, phosphatidylcholine, and phenoxyethanol;

(d) adding the solution of step (b) to the solution of step (a) and then mixing the solution;

(e) adding the solution of step (c) to the solution of step (d) and then mixing the solution;

(f) adding a pH adjuster to the solution of step (e) and then mixing; It provides a method for preparing an etosomal composition comprising a.

The step (c) may further include at least one selected from the group consisting of surfactants and other wool active ingredients.

The pH adjusting agent may include one or more selected from the group consisting of citric acid and salts thereof.

The step (a) may be adding 80 to 120 g of an amino acid compound to 50 to 70 L of purified water.

The step (b) may be adding 100 to 300 g of dexpanthenol to 25 to 35L of purified water.

The present invention also provides a composition for promoting hair growth and/or wool comprising an etosomal composition according to the present invention.

The present invention also provides a composition for preventing telogen hair loss comprising an etosomal composition according to the present invention.

More specifically, the composition for preventing telogen hair loss may be to prevent telogen hair loss through scalp anti-aging and/or promoting collagen production.

The present invention also provides a composition for promoting hair growth and / or hair growth, characterized in that the etosome composition is a male composition further comprising oleanolic acid (Oleanolic acid) and / or apigenin (Apigenin).

In addition, the etosomal composition provides a composition for preventing telogen hair loss, characterized in that the composition for men further comprises oleanolic acid and/or apigenin.

According to the present invention, it is possible to efficiently deliver poorly soluble active ingredients through the skin protective film.

According to the present invention, effective skin anti-aging ingredients, which have effects such as skin anti-aging, antioxidant, and collagen promotion, are efficiently delivered to the scalp, thereby promoting hair growth, hair growth, and preventing hair loss.

According to the present invention, by using the etosomal morphology, it is possible to efficiently deliver various compositions in addition to the active ingredient for hair growth, thereby increasing the hair growth effect.

1 is a schematic diagram showing a hair cycle;
2 to 5 are results of measuring the size and surface charge values of etosomes of Examples 1, 5, 10 and 14;
6 is a graph showing the test results confirming the skin penetration rate of Dexpantenol;
Figure 7 is a graph showing the test results confirming the skin penetration rate of Vitamin E acetae;
8 is a graph showing the result of measuring skin tissue thickness;
9 is a photograph of the skin tissue of the experimental group in which the hair was H&E dyed after application of the test substance of Experimental Example 4-1;
10 is a photograph of a regrowth state of hair after application of a test substance to the hair during the experiment period of Experimental Example 4-1;
11 is a photograph of a skin tissue after H&E staining of Experimental Example 4-2;
12 is a photograph of the regrowth state of hair during the experiment period of Experimental Example 4-2;
Figure 13 is a graph showing the thickness of the skin tissue in the growth phase sustained / resting phase inhibition effect test;
14 is a graph showing the growth phase hair follicle ratio in the growth phase sustained / resting phase inhibition effect test;
Fig. 15 is a photograph showing the difference between each group on the 19th day after the start of the test in the growth phase sustained / resting phase inhibition effect test;
FIG. 16 is a graph of scoring the growth phase maintenance state of the growth phase sustaining/resting phase inhibition effect experiment.

Hereinafter, each configuration of the present invention will be described in more detail so that those of ordinary skill in the art can easily implement the present invention, but this is only an example, and the scope of the present invention is based on the following contents. Is not limited.

The etosome composition according to the present invention has an effect of efficiently transmitting a poorly soluble substance such as vitamins and dexpanthenol through the skin.

Hydrophilic substances are well soluble in solvents such as water, but the skin is composed of a hydrophobic protective film, so hydrophilic active ingredients are difficult to pass into the skin, and in the case of hydrophobic substances, aggregates are formed in hydrophilic solvents such as water. Therefore, there is a problem that the transfer efficiency using a solvent is inferior because it is not dissolved.

Ethosome is a phospholipid nano vesicle used for the skin and transdermal delivery of molecules.The invention uses this to form a vesicle like an ethosome using a phospholipid called phosphatidylcholine, and the active ingredient is encapsulated in the vesicle. It is configured to move to the protective film, and to transmit the active ingredient efficiently by transmitting the active ingredient into the skin protective film.

In particular, the present invention uses an active ingredient for improving skin aging, which has an effect on skin anti-aging, antioxidant, and collagen promotion, not an ingredient known to be effective for hair growth and wool, and effectively delivers it to the scalp, thereby preventing the scalp. By inducing aging and antioxidant effects, it promotes hair growth and wool, and has the effect of preventing hair loss.

The vitamin may be one or more selected from the group consisting of vitamin E, vitamin C, vitamin B, salts thereof, and analogs thereof. Anything that functions as a vitamin can be used irrespective of its form, and various forms such as vitamin E acetate, fat-soluble vitamin C, and the like can be used.

In the present invention, the ethosome includes ethanol, peptide, phosphatidylcholine, and amino acid compounds, thereby improving the flexibility of the etosomal shell, thereby increasing the skin penetration rate, thereby enhancing the effect of efficient delivery of the active ingredient. Have.

Specifically, the peptides are pal-GHK, Pal-GQPR, Pal-KTTK, Pal-KTTKS, Biotin-GHK, PAL-KVK (Palmiotyl Tripeptide-5), Palmitoyl tripeptide-38, Palmitoyl-Tripeptide (Pal-RFK), Human Oligopeptide-20(Val-Gly-Val-Ala-Pro-Gly), GHK-Cu, (AHK)2Cu, AHK-Cu, Myristic acid-KLAKK-CONH2, Hexapeptide-11, Octapeptide-2, Palmitoyl-hexapeptide, Acetyl tetrapeptide-15, Acetyl Hexapeptide 3, Acetyl Tetrapeptide-2, Acetyl Tetrapeptide-5, Acetyl-Decapeptide-3, Ac-KGHK, rh-Oligopeptide-1(humanEGF) (NSDSECPLSWEHDGYCLHDGVCMYIEALDKYACNCVVVG YIGERCLake, Snake-LakePro -Dab-NHBzl), peptide CK+(Ac-Arg-Lys-Arg-NH2), peptide CK(Arg-Lys-Arg), tripeptide-3(Ala-His-Lys.HCl), Ac-KGHK-NH2, Ac -Tyr-Arg-hexadecylester, one or more selected from the group consisting of Argireline may be used. Although the peptide is known to have an effect on skin antioxidants, etc., it is not a component known to be effective in hair growth, hair growth, etc., but in the present invention, by sufficiently efficiently delivering the component into the scalp, the scalp antioxidant / By maximizing the anti-aging effect, it regulates the growth cycle of the hair to shorten the resting period, and by sustaining the growth period, it induces hair growth and wool effects. Preferably, the peptide may be pal-GHK.

The phosphatidyl choline is one of lecithin, and lecithin refers to a group of fatty substances occurring in animal and plant tissues consisting of phosphoric acid, choline, fatty acids, glycerol, glycolipids, triglycerides, and phospholipids.

Among them, phosphatidyl choline is a class of phospholipids containing choline as a headline, and as it is made of phospholipids such as a skin protective film, it has the advantage of being easy to penetrate the skin membrane when the active ingredient is delivered.

The amino acid compound may be one or more selected from the group consisting of amino acids and amino acid analogs, and the amino acid and amino acid analogs are specifically, NAC (N-Acetyl Cysteine), arginine and lysine, lysine ethyl ester, histidine, histidine. It may include one or more selected from the group consisting of ethyl ester.

In the present invention, the ethosome composition is vitamin 20 to 70 mg/100ml, dexpanthenol 150 to 250 mg/100ml, ethanol 5 to 15 ml/100ml, peptide 0.1 to 10 mg/100ml, phosphatitylcholine 10 to 1000 It may contain mg/100ml, 10 to 1000 mg/100ml of an amino acid compound.

The vitamin may preferably be contained in an amount of 30 to 60 mg/100ml, and when added in excess of the content, there is a problem that vitamins are precipitated during long-term storage, and when added in a small amount than the content, high skin There is a problem that it is difficult to exhibit a sufficient effect despite the penetration rate.

The dexpanthenol may preferably be contained in an amount of 120 to 220 mg/100ml, but when added in excess of the content, solubilization is difficult and stability is poor, and when added in a small amount than the content, effectiveness There is a problem that the effect exerted by the person is negligible.

The ethanol may preferably be contained in an amount of 7 to 12 mg/100ml, and if it is added in excess of the content, there is a problem that it cannot form an etosomal and becomes dissolved, and is added in a small amount than the content. In this case, there is a problem that the stability of the formulation of the poorly soluble substance is poor because the etosomes are not formed.

On the other hand, the peptide may preferably be contained in an amount of 0.1 to 10 mg/100ml, and if it is added in excess of the content, there is a problem of increasing the manufacturing/production unit cost, and if it is added in a small amount than the above content, In spite of the high skin penetration rate, there is a problem that it is difficult to exhibit sufficient effects.

The phosphatidylcholine may preferably be contained in an amount of 10 to 1000 mg/100ml, and if it is added in an excess of the amount, there is a serious problem with turbidity due to the remaining excess material that does not participate in the formation of ethosomes. When added in a smaller amount, there is a problem in etosomeizing the active substance.

In the case of the amino acid compound, it may be included in an amount of 10 to 1000 mg/100ml, and when added in excess of the content, there is a problem of raising or lowering the pH, and when it is added in a smaller amount than the content, a pH adjuster There is a problem in that it has to be added in excess.

By being added to satisfy the above content, the effective ingredient delivery effect of the ethosome composition has a particularly excellent effect.In particular, in the present invention, the period of rest is shortened by controlling the growth cycle of the hair through effective delivery to the head , It is characterized by having a hair growth, wool effect by sustaining the growth period.

In the present invention, the etosomal contained in the etosomal composition may have an average particle diameter of 90 to 200 nm. Preferably, by having an average particle diameter of 100 to 150 nm, the size of the particles has a uniform effect, and has a more advantageous effect on the delivery of an active ingredient by maintaining a stable etosomal composition.

The etosomal may also have an absolute value of a surface charge of 10 to 100 mV. When it has the above range, the etosomes do not clump together and may be uniformly dispersed in the solution.

The method for preparing an etosomal composition according to the present invention comprises the steps of: (a) solubilizing an amino acid compound in purified water;

(b) solubilizing dexpanthenol in purified water;

(c) solubilizing ethanol, vitamins, phosphatidylcholine, and phenoxyethanol; (d) adding the solution of step (b) to the solution of step (a) and then mixing the solution; (e) adding the solution of step (c) to the solution of step (d) and then mixing the solution; (f) adding a pH adjuster to the solution of step (e) and then mixing; Includes.

Specifically, step (c) may further include one or more selected from the group consisting of surfactants and other wool active ingredients. As the surfactant, for example, a surfactant such as Poloxamer 188, Poloxamer 407, Poloxamer F-127, PEG 60 (hydrogenated cateroil), Polysorbate 80, tween 20, tween 60, and tween 80 may be used.

As the other active ingredients for wool, any active ingredient may be used without any particular limitation, and for example, natural extracts such as melatonin and blueberry extract and types of compounds may be appropriately added as needed.

The pH adjusting agent may include one or more selected from the group consisting of citric acid and salts thereof.

The step (a) may be adding 80 to 120 g of an amino acid compound to 50 to 70 L of purified water. Preferably, it may be to add 90 to 110 g of amino acid compound to 55 to 65L of purified water.

If it is added in excess of the above content, it is not sufficiently dissolved in purified water and there is a problem of precipitation, and if it is added in a small amount than the above content, there is a problem that an excessive amount of a pH adjuster must be used.

On the other hand, the step (b) may be adding 100 to 300 g of dexpanthenol to 25 to 35L of purified water, and preferably, 150 to 250g of dexpanthenol to 27 to 32L of purified water.

In this case, if it is added in excess than the above content, it is not sufficiently dissolved in purified water or the solubilization time is long, there is a problem of precipitation, and if it is added in a small amount than the above content, the effect is insignificant even if the etosomal composition is applied. have.

The ethosome composition according to the present invention can be used as a composition for promoting hair growth and/or wool. As described above, by effectively permeating the active ingredient having the effect of improving skin aging and preventing aging into the scalp using the etosome composition. , Prevents telogen hair loss by inducing the aging improvement effect of the scalp, and induces hair growth and/or wool effects by sustaining the growth period.

Likewise, it provides a composition for preventing telogen hair loss capable of preventing telogen hair loss by using the etosomal composition according to the present invention.

The composition according to the present invention can be used to induce hair growth, wool, or to improve or prevent hair loss.

On the other hand, the present invention can provide a composition for promoting hair growth and/or wool and a composition for preventing telogen hair loss, which is optimized according to the user's gender.

The above-described etosomal composition may be used without distinction between a female and a male, but by configuring to further include oleanolic acid and/or apigenin for males, a composition particularly suitable for male users can be provided.

The oleanolic acid is a triterpenoid saponin, which is a pentacyclic triterpenoid. By using this, it is possible to provide a composition particularly suitable for men.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily implement the present invention, but this is only an example, and the scope of the present invention is based on the following content. Is not limited.

[Production Example]

Examples and comparative examples were prepared in the following order with reference to the component tables of Tables 1 to 3.

1.Injection/solubilization of arginine in purified water

2. Purified water, NAC, panthenol stirring/solubilization

3. Mixing after adding the mixture of 2 to the mixture of 1

4. Ethanol, vitamin E acetate, poloxamer F-127, phosphatidylcholine, phenoxyethanol, oleanolic acid, apigenin stirring/solubilization

5. Mixing after adding the mixture of 4 to the mixture of 3

6. Purified water, citric acid, sodium citrate agitation/solubilization (pH=7.0)

7. Mix after adding the mixture of 6 to the mixture of 5

[Experimental Example 1: Ethosome size and surface charge measurement]

1) Equipment: Marlvern Nano ZS

2) Experimental method: The test material was diluted with a 0.2% solution using distilled water and then measured, and measured using a Disposable cell.

For Examples 1, 5, 10 and 14, the results of measuring the size and surface charge of the etosomes are shown in FIGS. 2 to 5.

In the case of ethosomes, it may be difficult to have a particle size distribution in the 100 nm range due to poorly soluble substances and suspending agents, but in the case of 1, 5, 10, and 14 of the present invention, uniform particle size distribution as shown in the figure below. It can be seen that and have a relatively high surface charge.

In the case of the surface charge value, it is known that the stability in the solution can be maintained when the absolute value is 20 mV or more. As a result of the experiment, it was confirmed that it had a value of -40 to -20 mV, and from this, it was confirmed that the formulation has excellent long term stability.

[Experimental Example 2: Stability Test]

1) Experiment method

-In the same experimental method as in Experimental Example 1, changes in particle size and surface charge were tested for 0 days, 1 day, 1 week, 4 weeks, 8 weeks, 12 weeks, and 24 weeks.

-Each sample was stored at 37°C and 70% humidity, and the samples were collected at a predetermined time, diluted and measured.

The stability measurement results for 1 to 5 and 10 to 14 of Examples to which ZnO was not added are shown in Table 4 below. The particle size distribution of the examples prepared according to the manufacturing process was a formulation having a very even particle size distribution of about 100 nm to 160 nm based on the Z-average size, and in the case of Zeta potential, Examples 3 and 14 had the lowest charge. Results with values were obtained, suggesting that the formulation has excellent long-term storage stability. In the case of Examples 1 and 10, there was no change in the particle size distribution as time passed, but it was confirmed that the surface charge value increased, which means that the stability of the etosomes increased after 1 to 2 days or more than the initial stage.

[Experimental Example 3: Skin penetration test]

1) Equipment: Biotek's Epoch 2

2) Analysis wavelength: 260 nm

3) Test kit: Pion's skin pmapa test kit is used.

4) Experimental method: A sample was injected into the donor (pH=5.4) using a 32°C incubator, and the amount of the sample penetrated toward the aceeptor (pH=7.4) was measured using a microplate reader.

5) Experiment time: The experiment time was measured using a microplate or HPLC for the amount of target material that moved toward the acceptor for 6 hours based on 6 hours. It was calculated using the following formula.

6) Calculation: The penetration rate was calculated using the formula below, and a calculation method using ascorbic acid was included as a representative example.

Penetration (%) = [(Ascorbic acid amount in acceptor)/(Ascorbic acid amount in donor)] * 100

7) Composition of Experimental Group: An experimental group in which a substance to be measured was added to a buffer solution provided by Pion was selected as a control group, and the experimental group was compared and analyzed using the formulation corresponding to the example.

The experimental results measured using the above test method are shown in FIGS. 6 and 7. As a result, the penetration rate of the control group was calculated as 100%, and the relative penetration rate was shown. 6 is an experiment result of confirming the skin penetration rate for dexpantenol, which is a hydrophilic substance, and even though it is a water-soluble substance, it was confirmed that the penetration rate increased due to the high penetration rate of the etosomal when using the ethosomal formulation.

[Experimental Example 4-1: Wool Effect Test]

1) Test group composition

2) Hair removal: To evaluate the hair growth efficacy of the test substance, 42-day-old male C57bl/6 mice showing a pink color of the back skin were used. The back hair was carefully removed using an Animal clipper (Panasonic ER1431p, Japan) and a stabilization period of 1 day was given. After 28 days, a test material of 200 μL/head was applied to the hair removal area using a pipette. As a positive control, 2% minoxidil (ROGAINE SOLUTION 2%) was applied in the same amount.

3) Test method

3.1) Photographing and observation of changes in the hair growth cycle

Visual observation was conducted every day after the application of the test substance was started to calculate the period of introduction of the growth phase, which is represented by a change in skin color (from light pink to blue), for each individual, and the period of time when the hair begins to be observed, and periodic photographing was performed. The degree of hair growth of each group was visually determined for each animal by the scoring system. According to the degree of hair growth, 0% was 0, 1-25% was 1, 26-50% was 2, and 51-75% was 3 points and 76~100% were divided into 4 points.

3.2) Histological examination

For histological observation, the skin tissue was removed and fixed in 10% neutral formalin for 48 hours or more while attached to filter paper. After fixation was completed, the tissue was cut from the head to the tail to a thickness of 5 to 10 mm, and then subjected to general tissue treatment of alcohol, xylene, and paraffin. After embedding with Parisin, a 4 μm thin section was made using a rotary thinner and stained with hematoxylin and eosin (H&E). While observing with an optical microscope, the thickness of the skin tissue (dermis + epidermis) was measured, and the ratio of growing and resting hair follicles among 100 hair follicles per animal was calculated.

4) result

4.1) Photographing and observation of changes in the hair growth cycle

The C57BL/6 mice used in this experiment have the advantage of being able to determine whether or not the hair follicle has been introduced to the growth stage simply by observing the change in skin color with the naked eye because the melanin production cycle and the period of introduction of the hair follicle growth phase coincide. Therefore, in this experiment, in order to confirm the hair growth promoting effect of the test substance, every day immediately after application of the test substance was started, the time when the skin color change and hair were first observed was measured.

9 is a photograph of the skin tissue of the experimental group in which the hair was H&E dyed after applying the test substance of Experimental Example 4-1, and FIG. 10 is a photograph of the regrowth state of the hair after applying the test substance to the hair during the experiment period of Experimental Example 4-1. to be.

It is known that the skin of C57BL/6 mice begins to change from gray to black as it enters the growth stage 5 (anagen 5), followed by regrowth of hair. In this experiment, compared to the positive control, Rogaine group (24.00 ± 7.38), the 5, 7, 11, and 12 groups showed a faster transition rate to the growth phase. In particular, groups 6 and 7 showed a rapid growth phase transition rate of 7.62 days and 4.87 days on average compared to each other, showing the most pronounced hair growth promoting effect. In the observation result of hair regrowth period, compared to the Rogaine group (26.00 ± 7.37), the 6 and 11 groups showed an average of 8.12 days and 4.12 days faster effect.

These results were confirmed to show a similar trend in the results expressed by converting the number of animals introduced in the growth phase to a percentage. In groups 6 (100%), 11 (75%), and 12 (75%) groups, a higher percentage of animals introduced into the growth phase than in the Rogaine group (60%) were observed.

4.2) Comparison of hair regrowth area

In order to compare the hair regrowth area, the difference between the test groups was compared by scoring the regrowth area by visual observation every day. As a result, groups 6 (3.00 + 0.46 points) and 12 groups (2.13 + 0.61 points) showed higher scores than the Rogaine (1.80 + 0.92 points) group. In addition, groups 7 (1.63 + 0.56) and 11 (1.75 + 0.31) showed similar scores to those of the Rogaine group.

4.3) Histological examination

According to the growth cycle of the hair follicle, many changes appear in the skin tissue. In the growing season, the skin color changes to black, and in addition to the change in skin color, the thickness is accompanied by a change. When the hair follicle grows, in particular, the thickness of the subcutis layer increases, and the thickness of the epidermis layer also increases. Therefore, histological changes were observed to confirm the hair growth promoting effect of the test substance. On the 28th day of application of the test substance, skin tissue was collected and H&E stained, and then the proportion of hair follicles in the growth phase and the change in the thickness of the skin tissue were measured. The rate of growth of hair follicles was calculated after checking a total of 100 hair follicles for each animal.

As a result, 4.9 ± 3.3% of growing hair follicles were observed in the negative group, whereas 45.5 ± 21.6% of the growth phase hair follicles were observed in the Rogaine group. In the test chemical group, the proportion of hair follicles during growth was higher in groups 6 (61.4 + 14.1%) and 11 (48.9 + 13.1%) than in the Rogaine group.

Fig. 9 shows the results of measuring the thickness of the skin tissue. In the negative group, it was 264.8 ± 17.3 μm, whereas in the Rogaine group, it was 476.4 ± 95.3 μm, indicating a distinct increase in skin thickness. In the test substance group, the skin thickness was higher than that of the group 6 (569.9 ± 54.5 μm) Rogaine group.

[Experimental Example 4-2: Wool Effect Test]

1) Experiment method

1.1) Photographing and observation of changes in the hair growth cycle

Visual observation was conducted every day after the application of the test substance was started to calculate the introduction of the growth phase, which is represented by a change in skin color (from light pink to blue), for each group, and the hair growth period at which the hair starts to be observed, and periodic photographing was performed. In addition, the degree of hair growth of each group was visually determined for each animal by the scoring system, and according to the degree of hair growth, 0% was 0 points, 1-25% was 1 point, 26-50% was 2 points, and 51-75%. Was divided into 3 points, and 76~100% was divided into 4 points.

1.2) Histological examination of skin tissue

For histological observation, skin tissues were removed, fixed in 10% neutral formalin, washed with running water after 24 hours, dehydrated with alcohol, xylene, and paraffin, and embedded in paraffin after transparence and infiltration. The embedded tissue was made into a 4 μm thin section, stained with hematoxylin and eosin (H&E), and observed with an optical microscope.

1.3) Measuring the duration of the growth period

The duration of the growth phase of the test substance was measured. As for the measurement method, the period when pigmentation such as skin color appears from pink to gray or black is the period of introduction of the growth period, and after hair growth occurs, the change in skin color is observed once a day, and the period when the first skin color change area changes back to pink is terminated. The difference (day) was compared with the time period.

In order to compare the hair regrowth area, the difference between the test groups was compared by scoring the regrowth area by visual observation every day. As a result, all test substance applied groups showed higher scores compared to the negative control group.

11 is a photograph of a skin tissue after H&E staining of Experimental Example 4-2, and FIG. 12 is a photograph of a regrowth state of hair during the experimental period of Experimental Example 4-2.

For comparison with Experimental Example 4-1, Example 1 was placed in Experimental Group 3 to conduct an experiment. As a result, groups 4, 5, 8 and 12 showed particularly high scores. In groups 8 and 12, which showed a rapid growth phase, hairs began to be observed on the 24th and 21st days, respectively, and in the test group, which was applied with the positive control material, Hyundai Pharmaceuticals, Minoxil, hairs began to be observed on the 23rd day. As a result of the evaluation conducted up to the 32nd day after application, the average score of group 4 was 3.1 ± 0.7 points, and group 12 scored 2.5 ± 0.7 points or higher, which was higher than Hyundai Pharmaceutical's Mainoxil 2.8 ± 1.3 points.

In order to more objectively present these results, the regrowth state of the hair was recorded as a photograph at regular intervals after application of the test substance, and the photographic results are shown in FIG. 10. In the 4 group, hair was uniformly observed without any deviation, and it was confirmed that hair was observed in a uniform and wide area in the group to which the test material of group 12 (Example 18) was applied than in the positive control group Myoxyl group.

[Experimental Example 5: Growth period sustained / resting period inhibition effect test]

1) Test group composition

2) Test method

Carefully remove the hairs of the C57BL/6 mouse on the back of a 42-day-old male C57BL/6 mouse with a pink skin color using an Animal clipper (Panasonic ER1431p, Japan), and then apply a depilatory cream to remove all fine hair. Removed. After 5 days of hair removal, dexamethasone was applied 5 times every other day from the point when the skin color began to change to light black (introduction of the growth phase) to induce an artificial regression. In the test group, the test substance and dexamethasone were applied together from the 4th day after hair removal to confirm the artificial degenerative induction inhibition/growth sustaining effect.

3) Evaluation items

3.1) Photographing and observation of changes in the hair growth cycle

In order to determine the effect of promoting the transition to the growth phase, visual observation was conducted every day after application of the test substance, and the growth phase represented by the change in skin color (from pale pink to blue) for each group was calculated, and periodic photographing was performed. In addition, in order to confirm the degenerative memory suppression effect, a photo was taken at a specific time point to observe the change in skin color.

3.2) Histological examination of skin tissue

For histological observation, skin tissues were removed, fixed in 10% neutral formalin, washed with running water after 24 hours, dehydrated with alcohol, xylene, and paraffin, and embedded in paraffin after transparence and infiltration. The embedded tissue was made into a 4 μm thin section, stained with hematoxylin and eosin (H&E), and observed with an optical microscope to measure the ratio of the growing and resting hair follicles and the change in the thickness of the skin tissue.

The purpose of this experiment is an experiment conducted in order to confirm the effect on the sustaining/resting effect of the Example.

13 is a graph showing the thickness of the skin tissue in the growth period sustained / resting period inhibition effect test, Figure 14 is a graph showing the growth period hair follicle ratio in the growth period sustained / resting period inhibition effect test.

FIG. 15 is a photograph showing the difference between each group on the 19th day after the start of the test in the growth period sustaining/resting period inhibition effect test, and FIG. 16 is a graph scoring the growth period maintenance state of the growing period sustaining/resting period suppression effect experiment.

To confirm this, an animal model was used to induce rapid resting phase introduction by applying Dexamethasone, a steroid-based drug. In order to compare the hair regrowth area, the regrowth area was scored while observing with the naked eye every day, and the differences between the test groups were compared, and the results are shown in FIG. 15. As a result, it was confirmed that the test substance applied group showed a faster wool effect than the drug-treated group. In particular, it was confirmed that the proportion of hair follicles during growth was very high.

In order to more objectively present these results, the photographic results after application of the test substance are shown in the drawings. When comparing with the naked eye through photographs of the experimental group treated with the example and the non-treated group, it was confirmed that the test substance application group in which the skin color was discolored had a long-lasting entry into the growth phase.

Claims (17)

delete delete delete delete delete delete delete Contains vitamins and dexpanthen enclosed etosomes,
The etosomal is a method for preparing a composition for promoting hair growth and/or hair comprising ethanol, a peptide, phosphatidyl choline, and an amino acid compound,
(a) solubilizing an amino acid compound in purified water;
(b) solubilizing dexpanthenol in purified water;
(c) solubilizing ethanol, vitamins, phosphatidylcholine, and phenoxyethanol;
(d) adding the solution of step (b) to the solution of step (a) and then mixing the solution;
(e) adding the solution of step (c) to the solution of step (d) and then mixing the solution;
(f) adding a pH adjuster to the solution of step (e) and then mixing; A method for producing a composition for promoting hair growth and/or wool comprising a.
The method of claim 8,
The step (c) further comprises at least one selected from the group consisting of surfactants, other wool active ingredients, and triterpenoid saponins.
The method of claim 8,
The method for producing a composition for promoting hair growth and/or wool, wherein the pH adjusting agent comprises at least one selected from the group consisting of citric acid and salts thereof.
The method of claim 8,
The step (a) is a method for producing a composition for promoting hair growth and/or wool, characterized in that 80 to 120 g of an amino acid compound is added to 50 to 70L of purified water.
The method of claim 8,
The step (b) is a method for producing a composition for promoting hair growth and/or wool, characterized in that 100 to 300 g of dexpanthenol is added to 25 to 35L of purified water.
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