WO2020111402A1 - Enzyme-treated borage oil containing high concentration of free gamma-linolenic acid having high hair loss improvement effect, preparation method thereof, and cosmetic composition containing same - Google Patents

Abstract

The present invention relates to: enzyme-treated borage oil in which borage oil containing a high amount of gamma-linolenic acid is enzyme-treated so that 5α-reductase is inhibited and an excellent hair loss improvement effect, that is, a hair loss and hair follicle cell recovery effect is achieved; a preparation method thereof and a cosmetic composition containing same, wherein the borage oil is enzyme-treated with a lipase enzyme so as to contain free gamma-linolenic acid.

Description

Enzyme-treated borage oil containing high-concentration free gamma-linolenic acid having a high hair loss improvement effect, a method for manufacturing the same, and a cosmetic composition comprising the same

The present invention relates to an enzyme-treated borage oil containing a high concentration of free gamma-linolenic acid having a high hair loss improvement effect, a method for manufacturing the same, and a cosmetic composition comprising the same, in particular, by treating the borage oil having a high content of gamma-linolenic acid 5 Alpha-reducing enzyme to inhibit the high hair loss improvement effect, that is, hair loss and hair follicle cell recovery high-concentration free gamma-linolenic acid-containing enzyme-containing borage oil, relates to a preparation method and a cosmetic composition comprising the same.

The number of human hair is estimated to be approximately 100,000 to 150,000. It is said that 50 to 100 hairs are lost per day, and hair loss has started when more than 100 hairs are lost per day. The causes of hair loss are very diverse, and the pattern varies from person to person. In particular, men's hair tends to become thinner and thinner in their 20s. Male Pattern Baldness (MPB), which is the most common symptom among hair loss symptoms, has a large genetic characteristic, and another major reason is that the hair roots weaken due to abnormal male hormones and excessive sebum secretion causes inflammation of the scalp. Hair falls out.

The most common cause of male pattern hair loss (about 90% or more) is an abnormality of male hormone (androgenic alopecia). As with prostatic hyperplasia, when the dihydrotestosterone is over-synthesized by 5-alpha reductase, the dihydrotestosterone binds to the androgen receptors around the hair follicles, gradually shrinking and degrading the hair follicles, eventually reducing the period of hair growth. Because of the reduced growth period, the roots of the hair, that is, the hair follicles, do not develop sufficiently and at the same time, the pigment accumulation is also insufficient. Hairless immature hair often comes out of the scalp and eventually hair loss begins. The shrinking hair follicles eventually die and permanent hair loss occurs, which makes treatment other than hair transplantation difficult. Dihydrotestosterone provides a factor that makes the size of the hair smaller and smaller as the growth period is shortened and the rest period is shortened during the hair growth cycle. During hair loss, the hair follicles contract more and more, and instead, the sebaceous glands become larger and smaller, making the hair thinner and smaller, and, as a rule, more oily, causing rashes or inflammation. The thin and small hair turns into fluffy and becomes invisible to our eyes, and the scalp in the head begins to be seen. After 20 times of iterative process, which is thinner than before, the hair follicle dies forever and no remedy can restore it. Testosterone is primarily known to affect the growth of underarm hair and pubic hair, while dihydrotestosterone is known to affect beard and baldness, but it is not clear.

Drugs for treating prostatic hyperplasia and male pattern hair loss are also known. However, it is most important to inhibit 5-alpha reductase for fundamental treatment.

5-alpha reductase synthesis inhibitors include finasteride and dutasteride. Testosterone is converted into dihydrotestosterone by 5-alpha reductase in the prostate tissue. Therefore, taking a 5-alpha reductase inhibitor that effectively prevents the conversion of dihydrotestosterone can reduce the size of the prostate. It is known that the size of the prostate is reduced to the maximum after approximately 6 months of use. Finasteride, a competitive inhibitor of 5-alpha reductase developed by Merck, also inhibits type 1, but mainly inhibits type 2 5-alpha reductase, thereby reducing dihydrotestosterone in the prostate and blood. Dutasteride has a longer half-life than finasteride, and is 45 times stronger for type 1 enzymes and 2.5 times stronger for type 2 enzymes. Most of the liver has metabolism, so use should be limited in patients with liver failure. Synthetic inhibitors of these 5-alpha reductases can be said to be a fundamental treatment method for prostatic hyperplasia, but patients who take 5 mg/day for 4 to 6 months for a long period of time can see the effect, and the problem of recurrence has been suggested. . Erectile dysfunction, decreased libido, gynecomastia, and ejaculation disorders have been reported as side effects.

Finasteride is also used to treat male pattern hair loss. Finasteride was originally used to treat prostatic hyperplasia, but the effect of hair loss treatment was also recognized by the US Food and Drug Administration, and the dosage was prescribed at a rate of 1 mg per day and sold as a treatment for hair loss (trade name, Propecia). As mentioned above, this drug also exhibits a hair loss treatment effect through a mechanism for inhibiting 5-alpha reductase present in the scalp. However, at least 3 to 5 months are effective only after taking it and there is a disadvantage of returning to the original state within 12 months if taken and stopped. In addition, since the structure of finasteride itself is similar to sex hormones such as testosterone and progesterone hormones, side effects are often found in women, and particularly, because of the risk of birth defects, the use of mothers is strictly limited. Even factories that produce finasteride prohibit women from participating directly in production.

Minoxidil was also developed for the treatment of hypertension, and it was developed and marketed as a hair growth agent after observing hair loss inhibition in hypertensive patients. Although the mechanism of action of minoxidil is not clear, it can be seen that the blood vessels of the scalp are expanded to easily supply oxygen and nutrients to the hair follicle, thereby promoting hair formation and development. There are few side effects, but in order to achieve the maximum effect, it must be applied for a long period of time for 6 months or more, and the effect disappears when it is discontinued. In addition, it is not an inhibitor of 5-alpha reductase, so it cannot lower the concentration of dihydrotestosterone.

Recently, herbal remedies have emerged to supplement the side effects and disadvantages of the existing drug therapy for treating prostatic hyperplasia and male pattern hair loss. Herbal therapy has different potential for the treatment of prostatic hyperplasia and male pattern alopecia, but to date, long-term clinical results have not been accumulated compared to the placebo control group.

Saw Palmetto, which is reported to be used by more than 2 million people in the United States alone, is derived from the fruits of the saw palm tree and is the most common natural-derived functional food used for prostatic hyperplasia. In the past, Indians used saw palmetto fruit as a remedy for the genitourinary system. The main components of saw palmetto are triglycerides type fatty acids and phytosterols, which are thought to improve prostate hyperplasia and improve hair loss by acting as a 5-alpha reductase inhibitor. In Korea, saw palmetto is recognized as an effect of improving prostatic hypertrophy, and is marketed as an individually recognized product. Recently, cucurbit seed oil has been used as an active ingredient and has been manufactured and sold as a treatment for prostatic hyperplasia. In fact, this seed oil is imported from European pharmaceutical companies as an extract of pumpkin seeds from Europe. As a mechanism of action, the pharmaceutical company explained that cucurbit seed oil cures blood cholesterol causing prostate hypertrophy and prevents changes in male hormones to treat urination disorders caused by prostate hypertrophy.

Camellia is also called winter rose and grows in China's Shandong Province, Taiwan, South Korea, and Japan. It grows mainly at 300 to 1,100 m above sea level, and is usually about 1.5-6 m tall. It blooms from January to March. It is grown in Tongyeong and Jeju Island in Korea. In particular, camellia oil produced in Tongyeong in Korea is imported and sold by a specialized Japanese atopic dermatitis company. Camellia oil is a fatty oil extracted from the seeds of the Camellia japonica L. of the family Theaceae and its constituent fatty acids similar to olive oil, which contains up to 82-86% of oleic acid. It is used for creams, emulsions, etc. for similar purposes, especially for hair. It does not deteriorate well even at a high temperature of 160 to 220°C. Oleic acid, which is high in camellia oil, lowers the level of LDL cholesterol, a low-density cholesterol that is harmful to the body, while increasing the level of HDL cholesterol, a high-density cholesterol that is good for the body, prevents high blood pressure and heart disease. It is known to be effective in preventing cancer and reducing memory due to aging.

However, it does not exist as a free oleic acid in nature, and is usually covalently attached to glycerol in an ester form. Commercial oleic acid is either a form of K ⁺ or Na ⁺ consisting of saponification of oil, or an ethyl ester type through ethanolysis, or manufactured through several industrial processes.

Thus, the present inventors fermented camellia oils containing abundance of essential unsaturated fatty acids oleic acid and linoleic acid to facilitate absorption and use, and oleic acid and linoleic acid released from glycerol skeleton molecules without any other chemical treatment. A 5-alpha-reductase inhibitory ability generated by increasing the content of essential fatty acids, including fermented natural oil, which has the effect of preventing and improving prostatic hyperplasia and male pattern hair loss, and filed it with the Korean Intellectual Property Office to register No. 10-1452320 Patents have been registered.

Meanwhile, there are a number of prior arts related to the effect of improving hair loss.

For example, Republic of Korea Patent Application Publication No. 10-2009-0076671 (Name of the invention: a method for preparing a composition for preventing hair loss and promoting hair growth) "The present invention is excellent in preventing hair loss, preventing hair loss having a hair growth effect and It relates to a method for preparing a composition for promoting hair growth, alcohol extracts, such as anterior umbilical cord, celestial crown, gangjinhyang, snake beet and coarse, which promote the production of new capillaries among natural products that can improve this in relation to the disappearance and atrophy of hair cells. Contains single or complex compositions of hot water extracts and/or oxidized phospholipids (Oxidized 1-Palmitoyl-2-Arachidonoyl-sn-Glycero-3-Phosphocholine, OxPAPC), and are also commonly used as natural products of sewage, black beans, goji berries, and licorice. It is characterized by having a wool effect that keeps the hair thick and strong by containing an alcohol extract or a hot water extract."

Republic of Korea Patent Publication No. 10-2011-0127447 (Name of the invention: hair loss prevention or hair growth promoting composition) "The present invention relates to a composition for preventing hair loss or hair growth promoting comprising phospholipids as an active ingredient It relates to a composition for preventing hair loss or promoting hair growth, which includes phospholipids extracted from animals as an active ingredient."

In addition, Republic of Korea Patent Publication No. 10-2012-0102111 No. (invention name: reduction of hair loss and / or hair growth and / or promoting method of hair growth) "The present invention delays hair loss or hair growth and / or hair growth It relates to a method of promoting. More specifically, the present invention relates to a method of using the disclosed composition for increasing the rate of hair growth and/or hair growth in mammals."

However, there is still a need for the development of a cosmetic composition having a high hair loss improving effect.

The present invention is an enzyme treatment borage that contains a high concentration of free gamma-linolenic acid excellent in hair loss improvement effect, that is, a high hair loss improvement effect by inhibiting 5 alpha-reductase by enzymatic treatment of borage oil with high gamma-linolenic acid content. It is an object to provide an oil, a method for manufacturing the same, and a cosmetic composition comprising the same.

Enzyme-treated borage oil containing a high concentration of free gamma-linolenic acid having a high hair loss improvement effect according to the present invention includes enzyme-treated borage oil containing free gamma-linolenic acid by enzymatically treating borage oil with a lipase.

According to the present invention, an enzyme treatment comprising a high concentration of free gamma-linolenic acid having a high hair loss improvement effect, that is, a high hair loss improvement effect by inhibiting 5 alpha-reductase by enzymatic treatment of borage oil having a high content of gamma-linolenic acid It has an effect of providing borage oil, a method for manufacturing the same, and a cosmetic composition comprising the same. In particular, the present invention enzymatically treats borage oil with a high content of gamma-linolenic acid with immobilized lipase, dihydrotestosterone (Dihydrotestosterone, which destroys hair follicle cells through high 5 alpha-reductase inhibitory ability including high concentration free gamma-linolenic acid) DHT) has an object to provide an enzyme-treated borage oil that effectively blocks the production, a manufacturing method thereof, and a cosmetic composition comprising the same.

1 is a graph showing the results of quantifying DHT produced by 5 alpha-reductase as an experimental result of 5 alpha-reductase activity inhibition of free fatty acids.

Figure 2 is a graph showing the results of quantifying the DHT produced by the 5 alpha-reductase as a result of comparing the inhibitory ability of the existing 5 alpha-reductase inhibitors saw palmetto and finasteride with enzyme-treated vegetable oil.

FIG. 3 is a graph showing the results of measuring the cell viability (%) by dividing the enzyme-treated borage oil into hair follicle cell growth promoting experiments, divided into a general control group and a control group for 0.1% DMSO.

4 is a result of a prior experiment for measuring the increase in skin absorption of enzyme-treated borage oil, borage oil, enzyme-treated borage oil and enzyme-treated borage oil and butylene glycol (BG: 1,3-Butylene glycol) ), Propanediol (Propandiol) or glycerin (GC: Glycerin) is a picture showing the results of a comparison of solubility by mixing in a 1:1 ratio.

FIG. 5 is a test result of an increase in hair growth of testosterone-induced hair loss model rats of enzyme-treated borage oil, and is a photograph showing the progress of the experiment by taking a picture using a digital camera (Nikon D90).

FIG. 6 is a criterion for scoring a visual evaluation of hair growth related to FIG. 5.

7 is a graph showing a result of scoring the progress of FIG. 6 as a reference for scoring of FIG. 6.

8 is a result of measuring the length of 50 randomly selected hairs using a dissecting microscope by extracting a part of the hair regenerated on the 27th day after all the experiments were finished with tweezers.

9 is a graph comparing the total weight of the regenerated hair.

The present invention provides enzyme-treated borage oil containing high-concentration free gamma-linolenic acid with high hair loss improvement, characterized in that it contains free gamma-linolenic acid by enzymatically treating borage oil with a lipase enzyme. do.

In addition, the present invention, in the best form, in a method for producing enzyme-treated borage oil by enzymatic treatment of borage oil, (1) Borage oil preheated to a temperature within the range of 10 to 40°C to resin Enzyme treatment step of enzymatic treatment under conditions of contact with the immobilized lipase enzyme for 10 to 100 hours; And (2) a fractionation step in which the enzyme treatment obtained by completing the enzyme treatment is mixed with 1,3-butylene glycol, and then dissolved to recover a layer containing fatty acids; It provides a method for producing enzyme-treated borage oil characterized in that it comprises a.

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

Enzyme-treated borage oil containing a high concentration of free gamma-linolenic acid having a high hair loss improvement effect according to the present invention comprises enzymatically treated borage oil containing free gamma-linolenic acid by enzymatically treating borage oil with a lipase It is characterized by.

That is, according to the present invention, the content of free essential unsaturated fatty acids such as gamma-linolenic acid, alpha-linolenic acid, linoleic acid, oleic acid, etc. is increased through enzymatic treatment, and high concentration of free gamma-linolenic acid with high inhibitory ability against 5 alpha-reductase It is characterized in that it obtains the composition included, thereby providing an improved effect compared to the existing composition for improving hair loss and treatment. In addition, the enzyme separated from the strain is immobilized and applied to the enzyme treatment (fermentation) to shorten the production time, simplify the manufacturing process through simplification of purification after enzyme treatment, and minimize the disadvantages of the existing fermentation method by preventing fermentation. Provide a manufacturing process.

Gamma-linolenic acid (GLA) is a type of omega 6 fatty acid that is an unsaturated fatty acid. It is usually found in vegetable oils, and is found in evening primrose oil, blackcurrant seed oil, and borage oil in nature. It is a substance essential for in vivo synthesis of prostaglandin, which is effective in lowering the level of cholesterol in the blood, and is also known to be effective in lowering blood sugar, anti-inflammatory, anti-cancer, weight-loss, osteoporosis, rheumatoid arthritis, menopause syndrome, and menstrual syndrome.

Alpha-linolenic acid (ALA) is a type of omega 3 fatty acid that is an unsaturated fatty acid. It is a precursor that is converted into ePA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) in the body. In the case of EPA and DHA, fish It is found in a lot, and it is also found in vegetable oils such as flaxseed oil and walnut oil. In the case of alpha-linolenic acid and linoleic acid, which is an omega 6 fatty acid, it is known that when it lacks as essential fatty acid that is not synthesized by the human body, it lacks DHA required for the brain and retina, thereby reducing learning and visual functions. Omega 3 fatty acids protect cells in the human body, maintain cell structure and aid in smooth metabolism. In addition, it suppresses the film formation of blood and promotes and strengthens bone formation. It is known that a lack of omega 3 fatty acids leads to a lack of DHA for the brain and retina, leading to a decrease in learning and visual function. According to the recently researched results, clinical research results have been reported that alpha-linolenic acid reduced the C-reactive protein, which is a marker of inflammation, and ingestion of alpha-linolenic acid lowers cholesterol in the blood and reduces the risk of cardiovascular disease. It is known that an effect that a function is protected can be obtained.

Linoleic acid (LA) is an unsaturated fatty acid with two double bonds, insoluble in water, soluble in ether and alcohol, and used as a source of soft soap. Also called 9,12-octadecadienoic acid. It is contained in soybean oil, cottonseed oil, etc. among vegetable oils by ester bonding with glycerol. Dry oil, which tends to be oxidized in the air, is composed mainly of linoleic acid and linolenic acid. It is found slightly in the animal body as a fatty acid constituting phospholipids. Some animals cannot synthesize on their own and require it as an essential nutrient. It is also called vitamin F (F of fatty acids) along with linolenic acid. It is used as a raw material for soft soap.

Oleic acid (OA) is a major component of fatty acids in olive oil and is an omega-9 unsaturated fatty acid. It plays a role in lowering blood pressure in olive oil. According to the type of fatty acid, which is a component of triglycerides, it is classified into saturated fatty acids and unsaturated fatty acids, and unsaturated fatty acids are classified into monounsaturated fatty acids and polyunsaturated fatty acids according to the number of carbon double bonds. Oleic acid is a monounsaturated fatty acid that has only one double bond between carbon atoms. It is a fatty acid that is widely found in plants and animals, as well as vegetable oils such as olive oil and canola oil, as well as animal fats such as cattle and pigs. It is hardly soluble in water, but soluble in ethanol, benzene, and chloroform. When platinum black, nickel, or the like is reduced with hydrogen as a catalyst, stearic acid, which is a saturated fatty acid without a double bond, is formed by a hydrogenation reaction. Pure is a colorless, odorless liquid, but is oxidized in the air to turn yellow or brown and has a rotting odor. Serum cholesterol concentration is lowered and high density cholesterol (HDL-cholesterol) concentration is not lowered, which is especially beneficial for hyperlipidemia patients. It is the most fatty acid contained in breast milk and helps the baby grow and develop. In animals, glycerin and esters are formed and stored in the subcutaneous fat or liver, and used as a raw material for soap or as a waterproofing agent for cloth.

Borage oil is Borage (Scientific name: Borago officinalis ) As a plant-derived oil, borage oil is known to be rich in gamma-linolenic acid and is known to be effective in lowering blood sugar, anti-inflammatory, weight loss, osteoporosis, and reducing menopausal and menstrual syndrome in women. It is reported to give.

According to the present invention, as used to enzymatically treat the borage oil, the lipase enzyme is enzymatically processed in a batch manner using an immobilized enzyme immobilized on a resin, preferably a lipase derived from the microorganism of the genus Lysopus, and centrifuged. Separating and filtering the enzyme residue and resin by separation, mixing the obtained enzyme treatment solution with 1,3-butylene glycol, and then separating the layer containing the fatty acid and the enzyme-free borage oil. By fractionating the oily layer, the layer containing the fatty acid can be subsequently processed to be used as a raw material for the cosmetic composition, and the oily layer containing unenzymatic borage oil can be recovered and introduced again into the enzyme treatment process. In the present invention, 1,3-butylene glycol is particularly used to recover the layer containing the fatty acid from the enzyme treatment, and 1,3-butylene glycol is widely used as a cosmetic raw material, particularly for the enzyme treatment of oil. Not only can it dissolve the fatty acid and glycerol produced by it, it can be used as a solvent for various raw materials for general cosmetics, so a layer containing fatty acids recovered with 1,3-butylene glycol can be used as a cosmetic ingredient as it is. When applying to cosmetics, there is no need to use an emulsifier with skin irritation.

The lipase is inoculated with the microorganism of the genus Rizopus in an amount of 10% by volume relative to the medium volume, and cultured under aerobic conditions, pH 5.5 to 6.5, culture conditions of 25 to 35°C, and 800 to 1 to 10°C The supernatant obtained by centrifugation at 1200 rpm may be precipitated and concentrated by ammonium sulfate, and then may be a dialysate obtained by dialysis, which is used directly as a microorganism by using a culture concentrate of microorganisms of the genus Rizopus In contrast, it was confirmed by the present inventors that the amount of production of unsaturated essential fatty acids can be greatly increased and fermentation odor can be eliminated.

At this time, the medium may be 2 to 10% by weight of potato starch, 0.5 to 3% by weight of dextrose and PDB (Potato Dextrose Broth) containing water as the remaining amount. Cultivation using the PDB may be preferably performed at a pH of 6.0±0.2 and aerobic conditions, and the PDB preferably further comprises 0.1 to 10% by weight of olive oil based on the total weight of the PDB. have.

Particularly, it was confirmed by the present inventors that the fermentation culture time in the culture conditions of the culture concentrate of the genus Rhizopus is the best when it is cultured within the range of 80 to 150 hours.

Rhizopus sp . ) Microorganisms are the first genus of fungi of Mucorales , and the stolon spreads and stretches to the side, where it touches the medium, i.e., one to several from the node. Sporangia and rhizoids are common. Sporangia large, usually black. The main column (columella) is hemispherical, mycelium occurs in large quantities, and it is elongated to fill the lid of the petri dish. Meteorically, the two sperm bags contact fusion to form spores. It is present in soil, fruits, etc., and rarely has pathogenicity in humans, animals, and certain species of plants. It has a strong starch saccharification power and a very small amount, but it also has alcohol fermentation power, proteolytic power, and organic acid generation ability such as lactic acid and fumaric acid. For example, it is used for brewing Chinese liquor or for the production of rice wine in Southeast Asia. R. delemar and R. japonicus are important amylophiles for alcohol production. R. nigricans ( R. nigricans ) is a fruit of peaches, strawberries, grains, and bread, and is a source of sweet potato soft disease, but it is also known to have strong fumaric acid production capacity.

The culture concentrate of the microorganism of genus Rizopus includes lipase, and the lipase means an enzyme that breaks down oil into glycerol and fatty acids.

The enzyme treatment of the borage oil is 10 to 80 hours, preferably 15 to 50 hours, and more preferably 20 to 80 hours, with lipase enzyme immobilizing borage oil preheated to a temperature within the range of 10 to 40°C. It can be carried out by enzymatic reaction under the reaction conditions for 30 hours to contact, and the microorganism of the genus Lysopus can be preferably Rhizopus oryzae .

In addition, the method of manufacturing an enzyme-treated borage oil according to the present invention, in a method for producing an enzyme-treated borage oil by enzymatic treatment of borage oil, (1) preheated to a temperature within the range of 10 to 40°C Enzyme treatment step of enzymatic reaction in the enzyme treatment conditions to contact the lipase enzyme immobilized on borage oil for 10 to 80 hours; And (2) a fractionation step in which the enzymatic treatment obtained in the enzymatic treatment step is mixed with 1,3-butylene glycol, followed by layer separation to recover the fraction containing fatty acid.

The lipase enzyme, in particular, is enzymatically processed in a batch manner using an immobilized enzyme immobilized on a resin, preferably a lipase derived from the genus Lyzopus, after which the enzyme residue containing the resin is separated and filtered and obtained. After mixing the enzyme treatment with 1,3-butylene glycol, the layer is separated to separate the layer containing the fatty acid and the oily layer containing the enzyme-untreated borage oil, and the layer containing the fatty acid is subsequently processed to make a cosmetic. Used as a raw material for the composition, the oily layer containing unenzymatic borage oil can be recovered and introduced again into the enzyme treatment process.

The lipase enzyme used for enzyme treatment, in particular, the lipase enzyme derived from the microorganism of the genus Lyzopus, the medium and culture conditions and culture time used for the preparation of the enzyme are the same and/or similar to those described above, and repeated descriptions will be avoided. , The microorganism of the genus Rhizopus may preferably be Rhizopus oryzae .

The enzyme-treated borage oil according to the present invention has a characteristic that the content of free essential unsaturated fatty acids is significantly higher than before the enzyme treatment by enzymatic treatment by the above-described method.

The free essential unsaturated fatty acids include gamma-linolenic acid (GLA), alpha-linolenic acid (ALA), linoleic acid (LA) and oleic acid (OA).

Gamma-linolenic acid is an unsaturated fatty acid component found only in women's milk and some plants, and is a necessary precursor for synthesizing prostaglandins known as active substances in the body. When the body lacks gamma-linolenic acid, the prostaglandins E1 and E2, which are bioactive substances, are not synthesized, so the essential amino acids and other components necessary for the normal functioning of the body are not well formed, resulting in abnormal immune systems.

The free essential unsaturated fatty acid means a form in which fatty acids are not bound to glycerol in the form of glycerides. As the content of free essential unsaturated fatty acids increases, stickiness decreases and skin absorption improves.

According to the present invention, a composition for improving hair loss containing the above-mentioned enzyme-treated borage oil as an active ingredient may be provided, but it is understood by those skilled in the art that the present invention is not intended to be limited to these. .

The composition for improving hair loss may be prepared in various formulations such as cream, lotion, emulsion, lotion, essence, mist, gel, pack, mask pack, oil, colorant, soap, body wash, shampoo, rinse, bathing agent, scrub agent, etc. .

The composition for improving hair loss may further include at least one additive selected from the group consisting of purified water, oil, surfactant, moisturizer, stabilizer, alcohol, emulsifying agent, chelating agent, coloring agent, preservative, and fragrance.

Preferred examples and comparative examples of the present invention will be described below.

The following examples are intended to illustrate the invention and should not be understood as limiting the scope of the invention.

Preparation Example 1. Purification of immobilized enzyme required for enzyme treatment

In order to obtain a lipase for enzymatic treatment of vegetable oils such as borage oil, first, microorganisms of the genus Lysopus and microorganisms of the genus Candida, Rhizopus oryzae , Candida lugosa ( Candida rugosa ), Pseudomonas fluorescence and Pseudozima sp . ) Were cultured. The composition of the medium used for culturing Rizopus oriose was prepared with 2% olive oil, 4% potato starch, and 1% dextrose, and cultured at a temperature of 28°C and pH 6.0 for 120 hours. The composition of the medium used for Candida Lugosa culture was 2% olive oil, 0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% dextrose, pH Prepared at 7.0, and incubated at 30°C for 90 hours. To cultivate Pseudomonas fluorescens, 0.5% peptone, 0.5% yeast extract, and 0.8% salt were added to a nutrient medium (Nutrient broth) to prepare a pH of 7.0, and cultured at 28°C for 32 hours. The medium used for cultivation in Pseudojima is 0.01% glucose, 0.1% olive oil, 0.003% yeast extract, 0.003% malt extract, 0.003% peptone, 0.003% soybean flour, 0.02% ammonium sulfate, 0.001% potassium phosphate, 0.005% magnesium sulfate , 0.001% calcium chloride and 0.0001% sodium chloride were used to prepare pH 7.0 and incubated at 37°C for 24 hours. Thereafter, each strain culture solution was centrifuged at 8000 rpm to precipitate microbial cells to recover only the supernatant, and then slowly added 70% ammonium sulfate at 4°C to precipitate the coenzyme mixture. Thereafter, the supernatant was removed by centrifugation for 30 minutes at 4°C and 12000 rpm, and the precipitate was dissolved in 50 mM phosphate buffer solution (pH 7.0) and dialyzed at 4°C using a dialysis membrane to use it as a lipase concentrate. Did.

Adsorptive resins suitable for enzyme immobilization include Amberlite XAD-7, anion exchange resins, porous glass, porous chitosan beads, polypropylene powder, CaCO ₃ A lamp is used, and Amberlite XAD-7 resin (Sigma-aldrich), which is relatively stable to double heat and physical impact, is used. The Amberlite XAD-7 resin was washed with ethanol and phosphate buffer solution (pH 7.0), stirred at 37°C for 1 hour and filtered through a filter. Pierce ^TM Protein is quantified using the method provided by the manufacturer using the Bicinchoninic acid (BCA) Protein Assay kit (Thermoscientific), and the enzyme concentrate is used to adjust the total protein amount to 10 mg/ml using 0.01 M Tris-HCl buffer solution (pH 8.6). Did. After the enzyme concentrate was added to the filtered Amberlite XAD-7 resin, the reaction was performed for 10 hours in a 200° C. 200 rpm shake incubator, and the reaction was centrifuged at 3000 rpm for 5 minutes to separate the Amberlite XAD-7 resin layer. Then, it was washed three times with 0.01 M Tris-HCl buffer solution (pH8.6). After drying it completely with a vacuum desiccator for several hours, it was used as an immobilization enzyme.

Example 1. Preparation of enzyme-treated vegetable oil

Vegetable oil was enzymatically treated using the immobilized enzyme prepared in Preparation Example 1. The enzyme reaction mixture was prepared by diluting 3 g of immobilized enzyme in 57 g of a phosphate buffer solution of pH 7.0 (Sodium phosphate buffer, 50 mM). That is, in the enzymatic treatment, the immobilized enzyme reaction mixture diluted with 5% of the immobilized enzyme in a phosphate buffer solution and vegetable oil (borage oil or camellia oil) are mixed at a ratio of 4:6, and then 60 at 300 rpm and 37°C. It was run for an hour. After the immobilized enzyme treatment, the vegetable oil is centrifuged at 10,000 rpm for 10 minutes to separate the lipase-immobilized resin, and again, pass through a filtration membrane having a pore size of 0.22 μm to see the enzyme treatment with the immobilized enzyme. Oil and enzyme-treated camellia oil were obtained, respectively.

Experimental Example 1. Fatty acid content of enzyme-treated vegetable oil

In Example 1, HPLC was performed to analyze the free fatty acid content of the enzymatically treated vegetable oil. Standard products used for this are alpha-linolenic acid (ALA: α-Linolenic acid, Sigma-aldrich), gamma-linolenic acid (GLA: γ-Linolenic acid, Sigma-aldrich), linoleic acid (LA: Linoleic acid, Sigma-aldrich) and oleic acid. (OA: Oleic acid, Sigm-aldrich). Solvents used for analysis are HPLC grade isopropanol (DUKSAN) and acetonitrile (Acetonitrile, DAEJUNG), samples and standards are dissolved in isopropanol (Isopropanol, DUKSAN), and then 0.22 μm membrane filter (membrane filter; PVDF Syringe Filter, 13 mm, FUTECS Co., Ltd.), and analyzed free fatty acids using a C18 column (250 x 4.6 mm, Gemini 5 μPhenomenex). A sample of 10 µl was injected at a flow rate of 1.0 ml/min, and measured with ultraviolet light having a wavelength of 210 nm. As a mobile phase, 0.005% trifluoroacetic acid in acetonitrile (Trifluoroacetic acid, Millipore Co.): 0.005% trifluoroacetic acid in water starts at 80: 20 (v/v) as a gradient condition of the mobile phase Thus, fatty acids were analyzed by a gradient analysis for 45 minutes.

Table 1 shows the results of analyzing the free fatty acid content of the enzyme-treated vegetable oil.

As a result of analyzing the content of free fatty acids, when the lipase was immobilized through lysopus oryase, it was found to be superior to the total content of free fatty acids compared to immobilizing the lipase with other strains. In addition, the ratio of gamma-linolenic acid, linoleic acid, and oleic acid of camellia oil and borage oil was different according to the enzyme-separated strain, and the content and ratio of gamma-linoleic acid was also the best when lyase of Rizopus orioli was used. . Therefore, in the subsequent experiments, enzyme treatment of borage oil was performed through lipase of lysopus oriose, and further, in order to compare with vegetable oils with a low content of gamma-linolenic acid, lyase of lysopus oriose lipase in the same way Processed camellia oil was prepared and its contents are listed in Table 1.

<Table 1>

Experimental Example 2. Inhibition of 5 alpha-reductase activity of enzyme-treated vegetable oil

In order to confirm the efficiency of each free fatty acid, the 5 alpha-reductase inhibition rate of gamma-linolenic acid, linoleic acid, and oleic acid was first tested. After receiving the Sprague Dawley Rat (SD rat) from Samtaco Co., Ltd., after abdominal surgery, the prostate was removed. The prostate was weighed and washed twice with 1x Phosphate-buffered saline (PBS) buffer. The washed prostate was crushed with surgical scissors with the same weight of 0.25 M sucrose solution. Thereafter, the pulverized prostate solution was transferred to a homogenizer by a glass homogenizer, homogenized, and centrifuged at 4°C for 20 minutes at 1000 g to recover the prostate supernatant. Mix 0.1 M potassium phosphate buffer (pH 6.8) with a mass of 3 times the weight of the prostate supernatant, and testosterone at a final concentration of 20 μM, 200 μM β-nicotinamide adenine dinucleotide phosphate (NADPH: Nicotinamide adenine dinucleotide phosphate) was added to prepare a reaction mixture. Each fatty acid dissolved in DMSO was treated with a concentration of 1, 5, 10, 15, and 25 ppm in a reaction mixture using the prostate homogenate. At this time, the positive control group only added DMSO to the reaction mixture, and a test mixture without testosterone and β-NADPH added to the negative control group was separately prepared and stored at 4°C to prevent reaction. The reaction of the 5 alpha-reductase of each reaction mixture was carried out at 37°C for 1 hour, and then using DHT (Dihydrotestosterone) ELISA Kit (MyBioSource), DHT produced by the 5 alpha-reductase according to the manufacturer's test method Was quantified. The results are shown in FIG. 1.

Since the negative control is the amount of DHT inherently present in the prostate of the SD rat, after subtracting it, the amount of newly generated DHT is calculated to calculate the half maximal inhibitory concentration (IC ₅₀ ) of the positive control DHT production. It was calculated and shown in Table 2.

As a result, gamma-linolenic acid exhibited an IC ₅₀ of about 7.89 times that of oleic acid and about 6.14 times that of linoleic acid, indicating that the activity inhibition rate of 5 alpha-reductase was the best. Therefore, the ratio and content of gamma-linolenic acid among the enzymatically treated vegetable oils are excellent, and the inhibitory efficiency of the 5 alpha-reductase of the enzyme-treated borage oil obtained by enzymatic treatment with lipase isolated and immobilized from lysopus oriose. It was judged to be the best and decided to verify it.

<Table 2>

Next, the inhibitory capacity of the existing 5 alpha-reductase inhibitors saw palmetto and finasteride and the enzyme-treated vegetable oil was compared. The final concentration by diluting saw palmetto, borage oil, enzyme-treated borage oil, camellia oil and enzyme-treated camellia oil in dimethyl sulfoxide (DMSO) in the reaction mixture through the prostate homogenate prepared by the above method 1, 2.5, 5, 10, 50, 100 ppm, and finasteride was also diluted in DMSO and added to the reaction mixture so that the final concentration was 0.1, 0.25, 0.5, 1, 10 ppm. The 5 alpha-reductase reaction of each reaction mixture was performed at 37° C. for 1 hour, and similarly, DHT produced by 5 alpha-reductase according to an experimental method provided by the manufacturer using a DHT (Dihydrotestosterone) ELISA Kit (MyBioSource). Was quantified. The results are shown in Fig. 2, and the IC ₅₀ values are calculated and shown in Table 3.

Finasteride IC ₅₀ values of type 1 and type 2 5 alpha-reductases have been reported in the range of 300 to 500 nM and 3 to 10 nM, respectively. When converted to ppm, about 0.12 to 0.18 ppm and 0.02 to 0.04 ppm It becomes. When compared with the results of FIG. 1 and Table 2, in this experiment, 0.37 ppm, which is slightly higher than the IC ₅₀ value of type 1 5 alpha-reductase, was recorded, and this value was inhibited for all type 2 5 alpha-reductase, It seems to be similar to the IC ₅₀ value for mainly type 1 5 alpha-reductase. What appears to be somewhat different from the values reported by previous researchers seems to be an error that appears because the experiment was performed using a prostate homogenate rather than a purely purified enzyme. It was confirmed that the 5 alpha-reductase inhibition rate of camellia oil and borage oil was increased by enzymatic treatment. Moreover, the 5 alpha-reductase inhibition rate of enzyme-treated borage oil rich in the ratio and content of gamma-linolenic acid was saw palmetto. And oleic acid showed excellent effect compared to enzyme-treated camellia oil.

<Table 3>

Experimental Example 3. Enzyme-treated borage oil promotes hair follicle cell growth

Cell growth capacity of borage oil and enzyme-treated borage oil was measured using human dermal papilla cells. The cell culture was cultured at 37° C., 5% CO ₂ using Dulbecco Modified Eagle Medium (DMEM) medium with 10% FBS (Fetal bovine serum) added. Cell viability was analyzed by MTT (3-4,5-dimethyl thiazol-2-yl-2,5-diphenyl tetrazolium bromide, Sigma). The dermal papilla cells were dispensed at 1 x 10 ⁴ cells/ml per well of a 96-well plate, and cultured for 24 hours at 37°C and 5% CO ₂ . After 24 hours, the medium used for the culture was removed, and the borage oil and the enzyme-treated borage oil were dissolved in DMSO and treated at concentrations of 1, 10, 100, and 1000 ppm, respectively, at 37°C and 5% CO ₂ to 5 Cultured for 24 hours and 24 hours. Thereafter, 20 µl of a 0.2% MTT solution per each well was added and reacted for 3 hours in a 37°C, 5% CO ₂ incubator. After the reaction, all the supernatant was removed and 150 μl of DMSO was added to dissolve all of the formazan produced at room temperature for 10 minutes, and absorbance was measured at 570 nm using an ELISA reader. To investigate the effect on DMSO, the experiment was divided into a control group and a control group for 0.1% DMSO, and the cell survival rate (%) was used in Equation 1, and the results are shown in FIG. 3. As a result, it was confirmed that borage oil did not significantly affect the growth of cells. In contrast, enzyme-treated borage oil promoted the growth of dermal papilla cells when treated at 100 ppm or more, which is an enzyme-treated borage oil. This means that it also helps to recover the scalp cells.

<Equation 1>

Cell growth rate (%)=absorbance of sample treatment group/absorbance of control group×100

Experimental Example 4. Enzyme-treated borage oil increases skin absorption

One of the drawbacks of vegetable oils as a formulation for application is low skin absorption. To improve this, borage oil, enzyme-treated borage oil and enzyme-treated borage oil and butylene glycol (BG: 1,3-Butylene glycol), propanediol (Propandiol) or glycerin (GC: Glycerin) 1:1 The ratio was mixed for comparison experiment. Among these, butylene glycol was the only solvent capable of sufficiently dissolving the enzyme-treated borage oil, and as shown in FIG. 4, propanediol and glycerin had distinct layer separation and were excluded from the skin absorption test. In order to investigate the skin absorption capacity, a skin absorption capacity experiment was conducted using the Franz diffusion cell method. In the skin absorption test, the absorption power was confirmed using the black mouse skin tissue (C57BL/60, Samtaco), and about 2 x 2 cm wide black mouse skin tissue (C57BL/60) was fixed between the donor and receptor phases. Each sample of 6 ml was administered to the prepared Franz diffusion cell, and the temperature was maintained at about 32.5°C using a constant temperature water bath. Each sample was allowed to be absorbed for 24 hours, and after 24 hours, the receptor phase was recovered and its weight was measured, and the weight of each sample absorbed per hour and skin area was compared and shown in Table 4. As a result, the skin absorbing power of borage oil itself increased by 1.46 times by the enzyme treatment, and when the enzyme-treated borage oil was dissolved in butylene glycol, the skin absorbing power of 2.63 times increased to have better skin absorbing power. I could confirm.

<Table 4>

Experimental Example 5. Enzyme-treated borage oil testosterone-induced hair loss model in rats Hair growth increase

Female C57BL/6 mice, 7 weeks of age, were pre-sale from Samtaco Co., Ltd. and were used for the experiment after a 1 week acclimation period. During the experimental period, the experimental animals were kept in a mouse cage for isolation, and the environmental conditions of the breeding room were maintained at room temperature of 25 ±3℃, relative humidity of 50 ±5%, and the illumination cycle was maintained for 12 hours and nights. During this time, tap water and solid feed for experimental animals (Samtaco) were allowed to be consumed freely. The mouse hair was first removed using a device of a Cutting Length Adjustable Pro Hair Clipper (Joas, JC-5000), and then treated with Niclean hair removal cream (Ildong Pharmaceutical) for 1 minute and washed 3 times with purified water. The epilated mice were bred into 2 control groups and 5 experimental groups of 5 animals, and are shown in Table 5. Testosterone and each sample were applied daily for 27 days after the experiment. Testosterone was dissolved in 50% ethanol at a concentration of 0.5%, and 150 µl was applied to the back of the mouse to induce hair loss. One hour after testosterone was sufficiently absorbed into the skin, 150 μl of each sample was applied. Borage oil, enzyme-treated borage oil, and enzyme-treated camellia oil diluted to 10% in olive oil, which is known to have no effect on hair loss improvement, and a sample obtained by dissolving enzyme-treated borage oil in butylene glycol in a 1:1 ratio. (Enzyme-treated borage oil (BG)) was used after being diluted to 20% in olive oil. Each concentration is indicated in Table 5 below. To compare the effects of the existing hair loss drug and enzyme-treated vegetable oil, 0.5% testosterone was applied and 150 µl of 5% minoxidil was applied an hour later. 50% ethanol was applied to the positive control group, and olive oil was applied after an hour, and 0.5% testosterone was applied to the negative control group, and olive oil was applied similarly after an hour. After the experiment was conducted, all mice were inhaled anesthesia with ethyl ether on 0, 7, 12, 17, 22, and 27, and then photographed using a digital camera (Nikon D90) to proceed with the experiment. The process is shown in FIG. 5. The visual evaluation of hair growth was scored based on the items shown in FIG. 6, and the results were shown graphically in FIG. 7. Fig. 8 shows the result of measuring the length of 50 randomly selected hairs using a dissecting microscope by pulling out a part of the regenerated hair on the 27th day after all the experiments were finished, and regenerating the hair per mouse by re-hairing the regenerated hair. The average of the weights was compared graphically in FIG. 9. As a result of FIG. 5 and FIG. 7, the effect of improving the hair loss due to the high absorbency of the enzyme-treated borage oil (BG) appeared evenly from the 12th day of the experiment, showing the fastest improvement, and finally on the 27th day of the experiment, the control group It showed the closest hair growth power. The sole effect of the enzyme-treated borage oil was also superior to that of the existing hair loss treatment, minoxidil. The length of the regenerated hair in FIG. 8 grew close to the control group on average, but the total weight of the regenerated hair in FIG. 9 was different according to the regeneration area of the hair, and at this time, the enzyme-treated borage oil (BG) was the most. Excellent was confirmed.

<Table 5>

Experimental Example 6. Simple clinical trial of enzyme-treated borage oil to improve hair loss

To test the effect of enzymatically treated borage oil on the improvement of direct hair loss, a simple clinical trial was conducted in 18 men with mild alopecia (aware of the fact that more than 50 hairs are lost per day on average) from 30 to 58 years of age. The test was run. Divide the group into 3 groups of 6 people and apply 5 ml of borage oil, enzyme-treated borage oil, and enzyme-treated borage oil (BG) once a day to the hair loss area before bedtime and then wash your hair the next morning. Proceeded. The evaluation was performed for a total of 8 weeks. After the hair was washed, the average hair loss number, visual evaluation, and personal opinions of the subjects were evaluated and scored based on Table 6. The overall test results are shown in Table 7. The improvement effect compared to placebo group borage oil was calculated for enzyme-treated borage oil and enzyme-treated borage oil (BG), respectively, and was sequentially indicated in the remarks column. As a result of the simple clinical trial, the enzyme-treated borage oil showed a comprehensive improvement of 33%, and in the case of the enzyme-treated borage oil (BG), an overall improvement effect of 41% was confirmed.

<Table 6>

<Table 7>

In the above, the present invention has been described in detail only with respect to the described embodiments, but it is apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and it is natural that such modifications and modifications belong to the appended claims.

Claims (9)

1. Enzyme-treated borage oil containing high-concentration free-gamma-linolenic acid with high hair loss improvement, characterized in that the borage oil is enzymatically treated with a lipase enzyme and contains free gamma-linolenic acid.
2. According to claim 1, Enzyme-treated borage oil containing a high concentration of free gamma-linolenic acid with a high hair loss improvement effect, characterized in that the lipase enzyme used for enzymatic treatment of borage oil is a lipase enzyme derived from the genus Lyzopus.
3. According to claim 2, Enzyme-treated borage oil comprising a high concentration of free gamma-linolenic acid with a high hair loss improvement effect, characterized in that the lipase enzyme is an immobilized enzyme immobilized on a resin.
4. According to claim 1, After dissolving the enzyme-treated borage oil by mixing with 1,3-butylene glycol, high-concentration free gamma-linolenic acid having a high hair loss improvement effect, characterized in that it is recovered as a layer containing fatty acids. Contains enzyme-treated borage oil.
5. In the method for producing an enzyme-treated borage oil by enzymatic treatment of borage oil,

   (1) Enzyme treatment step of enzymatic treatment under conditions of contacting the lipase enzyme immobilized on the resin with preheated borage oil at a temperature within the range of 10 to 40° C. for 10 to 100 hours; And

   (2) a fractionation step in which the enzyme treatment obtained by completing the enzymatic treatment is mixed with 1,3-butylene glycol, and then dissolved to recover a layer containing fatty acids;

   Method for producing enzyme-treated borage oil comprising a.
6. A skin external preparation for improving hair loss, comprising the enzyme-treated borage oil according to any one of claims 1 to 4 at 0.001% to 90% of the total weight of the external preparation for skin.
7. The method of claim 6, wherein the external preparation for skin is a formulation for ointment, patch, gel, cream, mist or spray.
8. A cosmetic composition for improving hair loss, comprising the enzyme-treated borage oil according to any one of claims 1 to 4 in an amount of 0.001% to 90% based on the total weight of the cosmetic composition.
9. The cosmetic composition of claim 8, wherein the cosmetic composition is a lotion, emulsion, lotion, cream, serum, essence, emulsion, cosmetic ointment, spray, gel, pack, cleanser, soap, shampoo, rinse, bath, or detergent formulation. Cosmetic composition for improving hair loss.

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