KR102145112B1 - Enzyme treated Borage oil comprising high concentration of free gamma-linolenic acid having high alopesia betterment effect, manufacturing method thereof and cosmetic composition comprising the same - Google Patents

Abstract

The present invention enzymatically treats borage oil with a high gamma-linolenic acid content to inhibit 5-alpha-reductase, thereby improving hair loss, that is, high concentration free gamma-linolenic acid, which has excellent hair loss and hair follicle cell recovery effects. It relates to an oil, a method for producing the same, and a cosmetic composition comprising the same, characterized in that it contains free gamma-linolenic acid by enzymatic treatment of borage oil with a lipase enzyme.

Description

Enzyme treated Borage oil comprising high concentration of free gamma-linolenic acid having high alopesia betterment effect, manufacturing method thereof and enzyme treated borage oil containing high concentration free gamma-linolenic acid with high hair loss improvement effect method thereof and cosmetic composition comprising the same}

The present invention relates to an enzyme-treated borage oil containing high concentration free gamma-linolenic acid having a high hair loss improvement effect, a method for preparing the same, and a cosmetic composition comprising the same.In particular, by enzymatic treatment of borage oil having a high gamma-linolenic acid content It relates to an enzyme-treated borage oil containing high concentration free gamma-linolenic acid, which has a high hair loss improvement effect by inhibiting 5 alpha-reductase, that is, a hair loss and hair follicle cell recovery effect, a preparation method thereof, and a cosmetic composition comprising the same.

In general, the number of human hairs is estimated to be about 100,000 to 150,000. It is said that 50 to 100 hairs per day fall out, and when more than 100 hairs per day continue to fall out, hair loss begins. The causes of hair loss are very diverse, and the patterns appear differently from person to person. In particular, men's hair tends to become thinner in their 20s. Among the symptoms of hair loss, male pattern baldness (MPB), which is the most common symptom of hair loss, has a large genetic characteristic, and another major reason is that the hair roots are weakened by an abnormality of the male hormone and the secretion of sebum becomes excessive, causing inflammation in the scalp. The hair will fall out.

The biggest cause (about 90% or more) of the male pattern hair loss is an abnormality of male hormones (androgenic alopecia). Like prostatic hyperplasia, when dihydrotestosterone is excessively synthesized by 5-alpha reductase, dihydrotestosterone binds to androgen receptors around the hair follicle, gradually shrinking and deteriorating the hair follicle, eventually reducing the growth period of the hair. Due to the reduced growth, the root of the hair, that is, the root of the hair, does not develop sufficiently, and at the same time, the pigment accumulation is not sufficient. This weak, immature hair often comes out of the scalp and eventually hair loss begins. Hair follicles that continue to shrink eventually die and permanent hair loss occurs. At this time, treatment other than hair transplantation becomes difficult. Dihydrotestosterone shortens the growth period and lengthens the resting period during the hair growth cycle, thereby providing a factor that makes the size of the hair smaller and smaller as the growth cycle continues. During the process of hair loss, the hair follicles gradually contract, instead the sebaceous glands become larger and the hair becomes thinner and smaller, and usually more and more greasy, leading to a rash or inflammation. The thin and small hair turns into fluffy and becomes invisible to our eyes, and the scalp inside the head begins to look through. The hair follicle dies forever after 20 repetitive processes that fall out and become thinner than before, and no treatment means can recover it again. Testosterone mainly affects the growth of underarm hair and pubic hair, while dihydrotestosterone is known to affect beards and baldness, but it is not clear.

Many drugs are also known for treating enlarged prostate and male pattern hair loss. However, for the fundamental treatment, it is most important to inhibit 5-alpha reductase.

5-alpha reductase synthesis inhibitors include finasteride and dutasteride. Testosterone is converted to dihydrotestosterone by 5-alpha reductase in prostate tissue. Therefore, the size of the prostate can be reduced by taking 5-alpha reductase inhibitors that effectively block the conversion of dihydrotestosterone. It is known that the size of the prostate is reduced to the maximum after 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, 45 times stronger for type 1 enzyme and 2.5 times stronger for type 2 than finasteride. Most of the metabolism is in the liver, so use should be restricted in patients with liver failure. Inhibitors of the synthesis of these 5-alpha reductase enzymes can be said to be a fundamental treatment method for prostatic hyperplasia, but the patient must take 5 mg/day for 4 to 6 months for a long period of time, and the problem of recurrence has been suggested. . As side effects, erectile dysfunction, decreased libido, gynecomastia, and ejaculation disorder have been reported.

Finasteride is also used to treat male pattern hair loss. Finasteride was originally used to treat an enlarged prostate, but the effect of treating hair loss was also recognized by the US Food and Drug Administration, so it is marketed as a treatment for hair loss (brand name, Propecia), prescribed at a dose of 1 mg a day differently. As described above, this drug also exhibits a hair loss treatment effect through a mechanism that inhibits 5-alpha reductase present in the scalp. However, it takes at least 3 to 5 months to be effective, and if you stop taking it, it returns to its original state within 12 months. In addition, the original finasteride structure itself is similar to sex hormones such as testosterone and progesterone hormones, so side effects are often found in women. In particular, the use of mothers is strictly limited due to the risk of birth defects. Even in factories that produce finasteride, the law prohibits women from directly participating in the production.

Minoxidil was also developed for the treatment of hypertension, and was developed and marketed as a hair growth agent after observing the phenomenon of inhibition of hair loss in hypertensive patients. The mechanism of action of minoxidil is not clear, but it can be seen that it expands the blood vessels of the scalp to easily supply oxygen and nutrients to the hair follicles to promote hair formation and growth. There are almost no side effects, but for maximum effect, it must be applied for a long period of at least 6 months, and the effect disappears when the use is stopped. Also, since it is not an inhibitor of 5-alpha reductase, it cannot lower the concentration of dihydrotestosterone.

Recently, a lot of herbal remedies have emerged in order to compensate for the side effects and disadvantages of conventional drug treatments for the treatment of enlarged prostate and male pattern hair loss. Although herbal therapy shows different possibilities for the treatment of prostatic hyperplasia and male pattern hair loss, no long-term clinical results have been accumulated compared to placebo control to date.

Saw palmetto, which is reported to be used by more than 2 million people in the United States alone, is extracted from the fruit of the saw palmetto, and is a representative natural product-derived functional food most commonly used for prostatic hyperplasia. In the past, Indians have recorded the use of saw palmetto as a treatment for the genitourinary system. The main components of saw palmetto are triglycerides-type fatty acids and phytosterols, which are believed to act as 5-alpha reductase inhibitors to improve prostatic hypertrophy and improve hair loss. In Korea, saw palmetto is marketed as an individually recognized product because it has been recognized for its effectiveness in improving prostatic hyperplasia. Recently, cucurbit seed oil is used as an active ingredient and is being manufactured and sold as a treatment for prostatic hypertrophy. In fact, this seed oil is a European pumpkin seed extract that is imported from European pharmaceutical companies. With its mechanism of action, pharmaceutical companies are explaining that cucurbit seed oil cures urination disorders caused by enlarged prostate by inhibiting blood cholesterol that causes prostate enlargement and preventing changes in male hormones.

Camellia is also called the rose of winter and grows in Shandong Province of China, Taiwan, southern regions of Korea, and Japan. It grows mainly at 300 to 1,100 m above sea level, and is usually 1.5-6 m tall. It blooms from January to March. In Korea, it is grown in Tongyeong and Jeju Island. In particular, camellia oil produced in the Tongyeong region of Korea is imported and sold by a Japanese atopic dermatitis company. Camellia oil is a fatty oil collected from the seeds of Camellia japonica L. and its fauna and flora of Theaceae family. It contains 82-86% of oleic acid because its composition fatty acids are similar to olive oil. It is used in creams and emulsions 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 contained in a lot of camellia oil, lowers the level of LDL cholesterol, which is harmful to the body, while it increases the level of HDL cholesterol, which is a high-density cholesterol, which is good for the body, thereby preventing 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 free oleic acid in nature, but is usually covalently bonded to glycerol in the form of an ester. Oleic acid on the market is almost in the form of oil saponification with K ⁺ or Na ⁺ attached, ethyl ester type through ethanol decomposition (ethanolysis), or manufactured through several stages of industrial process.

Accordingly, the present inventors fermented camellia oil, which is rich in essential unsaturated fatty acids such as oleic acid and linoleic acid, for absorption and use, and oleic acid and linoleic acid free from glycerol skeleton molecules without any other chemical treatment. A fermented natural oil having the effect of preventing and improving prostatic hypertrophy and male pattern hair loss with the ability to inhibit 5-alpha reductase, which is generated by increasing the content of essential fatty acids including, was applied to the Korean Intellectual Property Office to obtain registration number 10-1452320. I have registered a patent.

On the other hand, there are a number of prior technologies related to the effect of improving hair loss.

For example, Korean Patent Laid-Open Publication No. 10-2009-0076671 (name of the invention: a method for preparing a composition for preventing hair loss and promoting hair growth) is described as "The present invention has excellent hair loss prevention, hair growth effect, and hair loss prevention and It relates to a method for preparing a composition for promoting hair growth, among natural products that can improve the extinction and atrophy of hair follicle cells, and alcohol extracts such as anterior lobe Eumyanggwak, Sky Tari, Gangjinhyang, Bammu and Jogu, or Contains single or complex compositions of hot water extract or/and oxidized phospholipid (Oxidized 1-Palmitoyl-2-Arachidonoyl-sn-Glycero-3-Phosphocholine, OxPAPC), and also common natural products of sewage, black beans, goji berries, licorice. It is characterized by having a woolen effect that keeps the hair thick and strong by containing alcohol extract or hot water extract."

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

In addition, Republic of Korea Patent Laid-Open Publication No. 10-2012-0102111 (name of the invention: a method of reducing hair loss and/or hair growth and/or promoting hair growth) is “the present invention is a method of delaying hair loss or promoting hair growth and/or hair growth. More specifically, the present invention relates to methods of using the disclosed compositions to increase 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 improvement effect.

The present invention enzymatically treats borage oil with a high gamma-linolenic acid content to inhibit 5-alpha-reductase, thereby improving hair loss, that is, high concentration free gamma-linolenic acid, which has excellent hair loss and hair follicle cell recovery effects. It is an object of the present invention to provide an oil, a method for producing the same, and a cosmetic composition comprising the same.

Enzymatically treated borage oil containing high concentration free gamma-linolenic acid having a high hair loss improving effect according to the present invention includes enzyme-treated borage oil containing free gamma-linolenic acid by enzymatic treatment of borage oil with lipase.

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

1 is an experimental result of inhibition of 5-alpha-reductase activity of free fatty acids, and is a graph showing the results of quantification of DHT generated by 5-alpha-reductase.
Figure 2 is a result of comparing the inhibitory ability of the conventional 5-alpha-reductase inhibitor saw palmetto and finasteride and enzyme-treated vegetable oil, a graph showing the result of quantifying the DHT generated by the 5-alpha-reductase.
FIG. 3 is a graph showing the result of measuring the growth of hair follicle cells by enzyme-treated borage oil, which was divided into a general control group and a control group for 0.1% DMSO, and the cell viability (%) was measured.
4 is a result of a preliminary experiment for measuring the increase in skin absorption ability 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 or glycerin (GC: Glycerin) in a ratio of 1:1 to compare the solubility.
5 is a photograph showing the progress of the experiment by taking a picture using a digital camera (Nikon D90) as a result of an experiment of increasing hair growth in a testosterone-induced hair loss model rat of enzyme-treated borage oil.
6 is a criterion for scoring 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 criterion for scoring in FIG. 6.
8 is a result of measuring the length of hair of 50 strands randomly selected using a dissecting microscope by pulling out part of the hair regenerated on the 27th day after all experiments were completed.
9 is a graph comparing the total weight of regenerated hair.

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

Enzymatically treated borage oil containing high concentration 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 enzymatic treatment of borage oil with lipase. It is characterized.

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 enzyme treatment, and high concentration free gamma-linolenic acid having high inhibitory power against 5 alpha-reductase. It is characterized in that it provides an improved effect compared to the conventional hair loss improvement and treatment composition to obtain a composition containing this. In addition, the enzyme isolated from the strain is immobilized and applied to 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 odor. Provide manufacturing process.

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

Alpha-linolenic acid (ALA: α-linolenic acid) is a type of unsaturated fatty acid, omega 3 fatty acid. It is a precursor that converts into EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) in the body. In the case of EPA and DHA, fish It is contained in a lot, and is contained in a lot of vegetable oils such as flaxseed oil (flexed-seedoil) or walnut oil. In the case of alpha-linolenic acid and linoleic acid, which is an omega 6 fatty acid, it is known that if it is insufficient as an essential fatty acid that is not synthesized in the human body, DHA required for the brain and retina is insufficient, resulting in poor learning ability and visual function. Omega 3 fatty acids protect cells in the body, maintain cell structure, and aid in smooth metabolism. In addition, it has the effect of inhibiting blood film formation and promoting and strengthening bone formation. It is known that lack of omega-3 fatty acids leads to a lack of DHA, which is required for the brain and retina, leading to poor learning and visual function. According to recent research results, clinical studies have reported that alpha-linolenic acid reduced C-reactive protein, a marker of inflammation. Ingestion of alpha-linolenic acid lowers blood cholesterol and reduces the risk of cardiovascular disease. It is known that the effect of protecting the function 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 raw material for soft soap. Also known as 9,12-octadecadienoic acid. As an ester bond with glycerol, it is contained in soybean oil, cottonseed oil, etc. Linoleic acid and linolenic acid are the main components of drying oil, which is easy to oxidize in the air. It is found slightly in animal bodies as fatty acids that make up phospholipids. Some animals cannot synthesize themselves and require them as essential nutrients, but they are also called vitamin F (F in fatty acids) along with linolenic acid. It is used as a raw material for soft soap.

Oleic acid (OA) is the main 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 that 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 poly unsaturated fatty acids according to the number of carbon double bonds. Oleic acid belongs to monovalent unsaturated fatty acids that have only one double bond between carbon atoms. It is a fatty acid widely present in animals and plants, contained in vegetable oils such as olive oil and canola oil as well as fats and oils of animals such as cattle and pigs. Almost insoluble in water, but soluble in ethanol, benzene, and chloroform. When platinum black, nickel, etc. are used as a catalyst to reduce it to hydrogen, a saturated fatty acid stearic acid without double bonds is produced by hydrogenation. Pure is a colorless, odorless liquid, but it is oxidized in the air and turns yellow or brown, with a rancid odor. It is particularly beneficial for hyperlipidemia patients as it lowers the serum cholesterol concentration and does not lower the concentration of high-density cholesterol (HDL-cholesterol). It is the fatty acid that is most often contained in breast milk and helps the baby's growth and development. In animals, it forms esters with glycerin and is stored in the subcutaneous fat or liver, and is used as a raw material for soap or as a waterproofing agent for cloth.

Borage oil is borage (scientific name: Borago officinalis ) As an oil derived from a plant, 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, and osteoporosis, and by reducing menopausal syndrome and menstrual syndrome in women. It is reported as giving.

According to the present invention, it is used to enzymatically treat the borage oil, and the lipase enzyme is particularly enzymatically treated in batch using an immobilized enzyme in which a lipase derived from a microorganism of the genus Rhizopus is immobilized on a resin, and then centrifuged. The enzyme residue and the resin were separated and filtered by separation, and the obtained enzyme treatment solution was mixed with 1,3-butylene glycol, and then layer-separated to contain a fatty acid-containing fraction and unenzyme-free borage oil. The oily layer is fractionated and the layer containing fatty acids is subsequently processed to be used as a raw material for a cosmetic composition, and the oily layer containing unenzyme-treated borage oil can be recovered and introduced back into the enzyme treatment process. In the present invention, in particular, 1,3-butylene glycol is used to recover the fatty acid-containing layer from the enzyme treated product, and 1,3-butylene glycol is widely used as a cosmetic raw material, especially for enzymatic treatment of oil. Not only can it dissolve fatty acids and glycerol produced by it, but it can also be used as a solvent for various raw materials of general cosmetics, and therefore, a fractionated layer containing fatty acids recovered as 1,3-butylene glycol can be used as a raw material for cosmetics. When applying to cosmetics, it is not necessary to separately use emulsifiers with skin irritation that are commonly used.

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

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

In particular, it was confirmed by the present inventors that the fermentation culture time under the culture conditions of the culture concentrate of the genus Rhizopus has the best lipase activity when cultured within the range of 80 to 150 hours.

Rhizopus sp . ) Microorganisms are a genus of fungi of the order Mucorales , where the stolon spreads to the side and elongates, and this is one to several from the point where it contacts the medium, that is, from the node. Sporangial cyst disease and rhizoids develop. Sporangia large, usually black. The main axis (columella) is hemispherical, and the hyphae occurs in large quantities, and also elongates to fill the lid of the petri dish. Sexually, the two gametes are fused to form spores. It is present in soil and fruits, and in rare cases, some have pathogenicity to humans, animals, and certain species of plants. There are many kinds of fermentation industry because it has strong starch sugar heating power and very small amount, but it has alcohol fermentation power and protein decomposition power, and also has strong ability to produce organic acids such as lactic acid and fumaric acid. For example, it is used for the brewing of Chinese liquor or for the production of rice wine in Southeast Asia. R. delemar and R. japonicus are important as amylogens for alcohol production. R. nigricans ( R. nigricans ) occurs well in fruits such as peaches and strawberries, grains, and bread. It is also known to have a strong ability to produce fumaric acid, although it can cause sweet potato rot.

The culture concentrate of the microorganism of the genus Rhizopus includes lipase, and the lipase refers to an enzyme that decomposes oil into glycerol and fatty acids.

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

In addition, in the method for producing enzyme-treated borage oil according to the present invention, in the method for producing enzyme-treated borage oil by enzymatic treatment of borage oil, (1) preheated to a temperature within the range of 10 to 40°C. An enzyme treatment step of enzymatic reaction in enzyme treatment conditions in which borage oil is brought into contact with the lipase enzyme immobilized on the resin for 10 to 80 hours; And (2) a fractionation step of mixing the enzyme-treated product obtained in the enzyme treatment step with 1,3-butylene glycol, and then separating the layer to recover a fractional layer containing fatty acids.

In particular, the lipase enzyme is subjected to an enzyme treatment in a batch manner using an immobilized enzyme in which a lipase derived from a microorganism of the genus Rhizopus is preferably immobilized on a resin, and then the enzyme residue including the resin is separated and filtered by centrifugation, and the obtained After mixing the enzyme-treated product with 1,3-butylene glycol, the layers are separated to separate the fatty acid-containing layer and the oily layer containing unenzyme-treated borage oil, and the fatty acid-containing layer is subsequently processed to make cosmetic products. The oily layer, which is used as a raw material for the composition and contains unenzyme-treated borage oil, can be recovered and introduced into the enzyme treatment process again.

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

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

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 present only in female breast milk and some plants, and is an essential precursor for synthesizing prostaglandins, known as active substances in the body. When gamma-linolenic acid is insufficient in the body, prostaglandin E1 and E2, which are bioactive substances, are not synthesized. Therefore, components necessary for the normal functioning of the body, including essential amino acids, are not well formed, causing abnormalities in the immune system.

The free essential unsaturated fatty acid refers to 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 is reduced and skin absorption is improved.

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

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, color cosmetic, soap, body wash, shampoo, conditioner, bath 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, heavy agent, chelating agent, colorant, preservative, and fragrance.

In the following, preferred embodiments and comparative examples of the present invention will be described.

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

Manufacturing example 1. Purification of immobilized enzymes required for enzyme treatment

In order to obtain lipase for enzymatic treatment of vegetable oils such as borage oil, Rhizopus orrize ( Rhizopus) as a microorganism of the genus Rhizopus and a microorganism of the genus Candida. oryzae ), Candida lugosa ( Candida rugosa ), Pseudomonas fluorescence , and Pseudozima sp . ) Were each cultured. The composition of the medium used for the cultivation of Rhizopus orris was prepared with 2% olive oil, 4% potato starch, and 1% dextrose, and cultured at 28° C. and pH 6.0 for 120 hours. The composition of the medium used in the culture of Candida lugosa is 2% olive oil, 0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% dextrose, and pH. Prepared at 7.0, and incubated for 90 hours at 30 ℃. Pseudomonas fluorescein culture was prepared by adding 0.5% peptone, 0.5% yeast extract, and 0.8% salt to nutrient broth to pH 7.0, and cultured at 28°C for 32 hours. The medium used for the culture of Pseudomonas genus was 0.01% glucose, 0.1% olive oil, 0.003% yeast extract, 0.003% malt extract, 0.003% peptone, 0.003% soy 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 a pH of 7.0, and cultured at 37°C for 24 hours. Thereafter, the culture solution of each strain was centrifuged at 8000 rpm to precipitate the microbial cells to recover only the supernatant, and then 70% ammonium sulfate was slowly added thereto at 4° C. to precipitate the coenzyme mixture. After that, centrifugation at 4℃ and 12000 rpm for 30 minutes to remove the supernatant, dissolve the precipitate in 50 mM phosphate buffer (pH 7.0), and dialyzate it at 4℃ using a dialysis membrane to use it as a lipase concentrate. I did.

Adsorptive resins suitable for enzyme immobilization include Amberlite XAD-7, anion exchange resins, porous glass, porous chitosan beads, polypropylene powder, and CaCO ₃ Is used, and Amberlite XAD-7 resin (Sigma-aldrich), which is relatively stable against heat and physical impact, was used. Amberlite XAD-7 resin was washed with ethanol and phosphate buffer (pH 7.0), stirred at 37° C. for 1 hour, and filtered through a filter. Pierce ^TM Quantify protein by the method provided by the manufacturer using Bicinchoninic acid (BCA) Protein Assay kit (Thermoscientific), and use an enzyme concentrate with a total protein amount of 10 mg/ml using 0.01 M Tris-HCl buffer solution (pH 8.6). I did. After the enzyme concentrate was added to the filtered Amberlite XAD-7 resin, reacted for 10 hours in a shaking incubator at 37°C at 200 rpm, and the reaction product was centrifuged at 3000 rpm for 5 minutes to separate the Amberlite XAD-7 resin layer. Then, it was washed 3 times with 0.01 M Tris-HCl buffer solution (pH8.6). This was completely dried for several hours with a vacuum desiccator, and then 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. 3 g of the immobilized enzyme was diluted in 57 g of a sodium phosphate buffer (50 mM) pH 7.0 to prepare an enzyme reaction mixture. That is, in the enzyme treatment, the immobilized enzyme reaction mixture in which the immobilized enzyme was diluted 5% in a phosphate buffer solution and vegetable oil (borage oil or camellia oil) were mixed in a ratio of 4:6, and then 60 at 300 rpm and 37°C. It was run for hours. The vegetable oil that has been treated with the immobilized enzyme is centrifuged at 10,000 rpm for 10 minutes to separate the lipase-immobilized resin, and then passed through a filter membrane having a pore size of 0.22 µm to treat the immobilized enzyme. Oil and enzyme-treated camellia oil were obtained, respectively.

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

HPLC was performed to analyze the free fatty acid content of the enzyme-treated vegetable oil in Example 1. Standards 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 in the analysis are HPLC grade isopropanol (DUKSAN) and acetonitrile (Acetonitrile, DAEJUNG), and samples and standards are dissolved in isopropanol (DUKSAN), and then 0.22 µm membrane filter (PVDF Syringe Filter, 13 mm, FUTECS Co., Ltd.), and the free fatty acid was analyzed using a C18 column (250 x 4.6 mm, Gemini 5 μPhenomenex). A 10 µl sample was injected at a flow rate of 1.0 ㎖/min, and measured by ultraviolet rays having a wavelength of 210 nm. As the mobile phase, 0.005% trifluoroacetic acid (Trifluoroacetic acid, Millipore Co.) in acetonitrile: 0.005% trifluoroacetic acid in water started with 80:20 (v/v) as the gradient condition of the mobile phase. Then, for 45 minutes, fatty acids were analyzed by a gradient method.

The results of analyzing the content of free fatty acids in the enzyme-treated vegetable oil are shown in Table 1 below.

As a result of the analysis of the content of free fatty acids, it was found that when lipase was immobilized through Rhizopus orrase, it was superior to the total content of free fatty acids compared to the lipase immobilized with other strains. In addition, the ratio of gamma-linolenic acid, linoleic acid, and oleic acid of camellia oil and borage oil was different depending on the enzyme isolated strain, and the content and ratio of gamma-linoleic acid were also the best when using lipase from Rhizopus oris. . Therefore, in a later experiment, enzyme treatment of borage oil was performed through lipase of Rhizopus oryzae, and in order to compare with vegetable oils with a low gamma-linolenic acid content, enzymes with lipase of R. Treated camellia oil was prepared, and its content is indicated in Table 1.

Experimental example 2. Enzyme treatment of vegetable oil 5 alpha -Inhibition of reductase activity

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

Since the negative control is the amount of DHT inherently present in the prostate of SD rats, after subtracting this, the amount of newly generated DHT is calculated to determine the half maximal inhibitory concentration (IC ₅₀ ) of the amount of DHT production in the positive control. 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 of linoleic acid, indicating that the 5-alpha-reductase activity inhibition rate was the best. Therefore, the ratio and content of gamma-linolenic acid in the enzyme-treated vegetable oil 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 Rhizopus oris It was judged to be the best, so I decided to verify this.

Next, the inhibitory ability of the conventional 5-alpha-reductase inhibitors saw palmetto and finasteride and 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 as above. A 1, 2.5, 5, 10, 50, 100 ppm and finasteride were 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 carried out at 37°C for 1 hour. Likewise, DHT generated by 5-alpha-reductase according to the experimental method provided by the manufacturer using DHT (Dihydrotestosterone) ELISA Kit (MyBioSource). Was quantified. The results are shown in Fig. 2, and the IC ₅₀ value is calculated and shown in Table 3.

Finasteride is reported in the ranges of 300 to 500 nM and 3 to 10 nM, respectively, with IC ₅₀ values of type 1 and type 2 5-alpha-reductase, and when converted to ppm, about 0.12 to 0.18 ppm and 0.02 to 0.04 ppm Becomes. Compared with the results of FIGS. 1 and 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 is all suppressed in type 2 5 alpha-reductase, It appears to be mainly similar to the IC ₅₀ value for type 1 5-alpha-reductase. The difference from the values reported by previous researchers appears to be an error that appeared because the experiment was performed using prostate homogenate rather than purely purified enzyme. It was confirmed that the 5-alpha-reductase inhibition rate of camellia oil and borage oil was increased by enzyme treatment, and moreover, the 5-alpha-reductase inhibition rate of enzyme-treated borage oil, which was rich in the ratio and content of gamma-linolenic acid, was saw palmetto. Compared to enzyme-treated camellia oil, which contains most of and oleic acid, it showed superior effect.

Experimental example 3. Enzymatic treatment of borage oil Hair follicle Promote growth

Cell growth ability of borage oil and enzyme-treated borage oil was measured using human dermal papilla cells. Cell culture was performed at 37° C. and 5% CO ₂ using DMEM (Dulbecco Modified Eagle Medium) medium added with 10% Fetal bovine serum (FBS). The viability of cells was analyzed by MTT (3-4,5-dimethyl thiazol-2-yl-2,5-diphenyl tetrazolium bromide, Sigma) measurement. Dermal papilla cells were dispensed at 1 x 10 ⁴ cells/ml per well of a 96 well plate, and cultured at 37°C and 5% CO ₂ for 24 hours. After 24 hours, the medium used for the culture was removed, and borage oil and enzyme-treated borage oil were dissolved in DMSO, and then treated to 1, 10, 100, and 1000 ppm, respectively, at 37℃ and 5% CO ₂ . And incubated for 24 hours. Thereafter, 20 µl of 0.2% MTT solution was added to each well, and reacted in a 37°C, 5% CO ₂ incubator for 3 hours. After the reaction, all the supernatant was removed, and 150 µl of DMSO was added to dissolve all the formazan generated at room temperature for 10 minutes, and the absorbance was measured at 570 nm using an ELISA reader. In order to examine the effect on DMSO, the experiment was divided into a general control group and a control group for 0.1% DMSO, and the cell viability (%) was 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, when the enzyme-treated borage oil was treated with more than 100 ppm, it rather promoted the growth of dermal papilla cells, which is the enzyme-treated borage oil. This means that it is also helpful in the recovery of scalp cells.

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

One of the drawbacks as a formulation for application of vegetable oils is low skin absorption. To improve this, 1:1 in borage oil, enzyme-treated borage oil, enzyme-treated borage oil, butylene glycol (BG: 1,3-Butylene glycol), propanediol or glycerin (GC: Glycerin). It was mixed in proportions and compared to experiment. Among them, butylene glycol was the only solvent capable of sufficiently dissolving the enzyme-treated borage oil, and as shown in FIG. 4, propanediol and glycerin exhibited distinct delamination and were excluded from the skin absorption test. In order to find out 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 black mouse skin tissue (C57BL/60, Samtaco, Inc.), and black mouse skin tissue (C57BL/60) with an area of about 2 x 2 cm was fixed between the donor and the receptor phase. 6 ml of each sample 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 time and skin area was compared and shown in Table 4. As a result, the skin absorption power of borage oil itself increased by 1.46 times by enzyme treatment, and moreover, when the enzyme-treated borage oil was dissolved in butylene glycol, skin absorption capacity increased by 2.63 times, resulting in better skin absorption. I could confirm.

Experimental example 5. Enzyme-treated borage oil increases hair growth in testosterone-induced hair loss model mice

Seven-week-old female C57BL/6 mice were distributed from Samtaco Co., Ltd. and used in the experiment after a 1-week acclimation period. During the experiment period, the experimental animals were reared in a mouse cage for isolation, and the environmental conditions of the breeding room were kept at room temperature 25 ±3℃, relative humidity 50 ±5%, and the lighting cycle was maintained for 12 hours each night and day. Tap water and solid feed for experimental animals (Samtaco) were allowed to be consumed freely. The mouse hair was firstly removed using a cutting length Adjustable Pro Hair Clipper (Joas, JC-5000), and then NiClean hair removal cream (Ildong Pharmaceutical) was treated for 1 minute, and washed three times with purified water to remove. The shaved mice were reared by dividing into 2 control groups and 5 experimental groups, each of 5 mice, and are shown in Table 5. Testosterone and each sample were applied every day for 27 days during 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 a hair loss state. One hour after testosterone was sufficiently absorbed into the skin, 150 µl of each sample was applied. A sample in which borage oil, enzyme-treated borage oil, and enzyme-treated camellia oil were diluted 10% in olive oil known to have no effect on hair loss improvement, and enzyme-treated borage oil was dissolved in butylene glycol in a 1:1 ratio (Enzyme-treated borage oil (BG)) was diluted to 20% in olive oil and used. Each concentration is shown in Table 5 below. To compare the effects of conventional hair loss drugs and enzyme-treated vegetable oils, 0.5% testosterone was applied and 150 µl of 5% minoxidil was applied one hour later. The positive control was applied with 50% ethanol, and olive oil was applied after one hour, and the negative control was applied with 0.5% testosterone and olive oil was applied after one hour. After the experiment was progressed, all mice were anesthetized by inhalation with ethyl ether on the 0th, 7th, 12th, 17th, 22nd, and 27th, and then photographed using a digital camera (Nikon D90) to proceed with the experiment. The process is shown in Figure 5. Visual evaluation of hair growth was scored based on the items indicated in FIG. 6, and the results are shown in a graph in FIG. 7. On the 27th day after all experiments were completed, part of the regenerated hair was pulled with tweezers and the result of measuring the length of 50 strands randomly selected using a dissecting microscope is shown in FIG.8. The average of the weight of the graph was compared in FIG. 9. As a result of FIGS. 5 and 7, it was shown that the hair loss improvement effect appeared evenly from the 12th day of the experiment due to the high absorption capacity of the enzyme-treated borage oil (BG) and the fastest improvement. It showed the closest hair growth power. The single effect of enzyme-treated borage oil also showed superiority close to that of minoxidil, an existing hair loss treatment. The length of the regenerated hairs of FIG. 8 grew close to the control group on average, but the total weight of the regenerated hairs of FIG. 9 showed a difference according to the regeneration area of the hairs. It was able to confirm the excellence.

Experimental example 6. Simple clinical trial of enzyme-treated borage oil for hair loss improvement

In order to test the direct hair loss improvement effect of enzyme-treated borage oil, a simplified clinical trial was conducted on 18 men who showed mild alopecia from 30 to 58 years old (awareness that hair loss progresses and averages more than 50 hairs per day). The test was carried out. Divide 6 people into 3 groups, apply 5 ml of borage oil, enzyme-treated borage oil, and enzyme-treated borage oil (BG) to the hair loss area once a day before bed, and wash their hair the next morning. Proceeded. It was conducted for a total of 8 weeks, and the evaluation of the effect was scored based on Table 6 using the average number of hair loss hairs, visual evaluation, and individual findings of the test subject after washing the hair. The comprehensive test results are shown in Table 7. The improvement effect compared to the placebo group, borage oil, was calculated for the enzyme-treated borage oil and the enzyme-treated borage oil (BG), respectively, and sequentially indicated in the remarks column. As a result of a simplified clinical trial, the enzyme-treated borage oil showed an overall improvement of 33%, and in the case of the enzyme-treated borage oil (BG), a total improvement of 41% was confirmed.

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

(No drawing sign)

Claims (9)

delete delete delete delete A composition for improving hair loss comprising borage oil containing free gamma-linolenic acid as an active ingredient.
(1) an enzyme treatment step of enzymatic treatment under conditions of contacting a lipase enzyme derived from a microorganism of the genus Rhizopus immobilized on an adsorptive resin with borage oil preheated to a temperature within the range of 10 to 40°C for 10 to 100 hours; And
(2) a fractionation step of mixing the enzyme-treated product obtained by completing the enzymatic treatment with 1,3-butylene glycol and dissolving it to recover a fraction containing fatty acids;
Composition for improving hair loss, characterized in that prepared, including The borage oil is a skin external preparation for improving hair loss containing 0.001% to 90% of the total weight of the skin external preparation of borage oil containing free gamma-linolenic acid.
(1) an enzyme treatment step of enzymatic treatment under conditions of contacting a lipase enzyme derived from a microorganism of the genus Rhizopus immobilized on an adsorptive resin with borage oil preheated to a temperature within the range of 10 to 40°C for 10 to 100 hours; And
(2) a fractionation step of mixing the enzyme-treated product obtained by completing the enzymatic treatment with 1,3-butylene glycol and dissolving it to recover a fraction containing fatty acids;
Skin external preparation for improving hair loss, characterized in that prepared including The external preparation for skin for improving hair loss according to claim 6, wherein the external preparation for skin is in the form of an ointment, patch, gel, cream, mist or spray. It is a cosmetic composition for improving hair loss that contains borage oil containing free gamma-linolenic acid in an amount of 0.001% to 90% based on the total weight of the cosmetic composition.
(1) an enzyme treatment step of enzymatic treatment under conditions of contacting a lipase enzyme derived from a microorganism of the genus Rhizopus immobilized on an adsorptive resin with borage oil preheated to a temperature within the range of 10 to 40°C for 10 to 100 hours; And
(2) a fractionation step of mixing the enzyme-treated product obtained by completing the enzymatic treatment with 1,3-butylene glycol and dissolving it to recover a fraction containing fatty acids;
Cosmetic composition for improving hair loss, characterized in that prepared, including The method of claim 8, wherein the cosmetic composition is a formulation of lotion, emulsion, lotion, cream, serum, essence, emulsion, cosmetic ointment, spray, gel, pack, cleanser, soap, shampoo, rinse, bath agent, or detergent. Cosmetic composition for improving hair loss.

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