KR102122614B1 - Composition comprising Tetrathelmis tetrathele extract for improving condition of hair and preventing hair loss - Google Patents

Abstract

The present invention is a novel composition for improving scalp and preventing hair loss, and more specifically, a pharmaceutical composition for improving scalp and preventing hair loss, including an extract of Tetrathelmis tetrathele corresponding to marine microalgae as an active ingredient, health Functional food and cosmetic composition. The present invention suggested the possibility of utilizing the extract of the marine microalgae Tetrathelmis tetrathele ( Tetrathelmis tetrathele ) to prevent hair loss and to improve the scalp. In addition, the extract of the marine microalgae Tetrathelmis tetrathele , which has the function of preventing hair loss and improving the scalp, is a microalgae mainly used as a main food organism of shellfish such as oyster and sea cucumber farming. Because it is cultured in large quantities, it is advantageous for supply and demand of raw materials. In addition, the marine microalgae Tetrathelmis tetrathele ( Tetrathelmis tetrathele ) extract of the present invention can contribute to cost savings because it can be secured at a lower cost than natural products that have the effect of preventing hair loss and improving the scalp.

Description

Composition comprising Tetrathelmis tetrathele extract for improving condition of hair and preventing hair loss}

The present invention is a novel composition for improving scalp and preventing hair loss, and more specifically, a pharmaceutical composition for improving scalp and preventing hair loss, including an extract of Tetrathelmis tetrathele corresponding to marine microalgae as an active ingredient, health Functional food and cosmetic composition.

Recently, hair loss symptoms are one of the biggest concerns caused by the increased interest in external images. Hair loss has usually been considered only as a concern for men in their 40s and 50s, but currently, patients with hair loss in their 20s and 30s are skyrocketing, and female hair loss is also increasing. The symptoms of alopecia are aggravated by many environmental factors such as imbalance of modern people's diet, harmful air, contaminated drinking water, acid rain, and stress. Accordingly, many studies are in progress to prevent and treat hair loss.

Hair loss refers to the absence of hair in areas where hair should normally exist, or refers to a phenomenon in which hair that has stopped growing naturally falls out. More than 50 to 100 hairs are usually lost per day, and the more hairs are removed, the more alopecia is defined (Lee, 2005). The causes of hair loss are very diverse, and the genetic cause and androgen, the male hormone, are thought to be important factors for the development of baldness. It is estimated that some cases of female pattern hair loss occur in the same path as male pattern hair loss, and alopecia areata is thought to be an autoimmune disease. In addition, in the case of resting alopecia, it is caused by severe physical and mental stress such as endocrine diseases, nutritional deficiency, drug use, childbirth, fever, and surgery.This is a temporary hair loss, which causes some of the hair to not fill the growth period and becomes a resting state. Hair loss occurs.

Various attempts have been made to prevent and treat the symptoms of hair loss. There are surgical methods such as scalp flap surgery, hair loss reduction surgery, tissue expansion and autologous hair transplantation, and hair loss prevention and hair growth promoting agents using herbal extracts such as natural products. I am using it. Although the above surgical method can have an excellent effect in a short period of time, there is a problem in that the patient's economic and mental burden for surgery is large. Accordingly, in addition to surgical methods, various external preparations are used to prevent and prevent hair loss, and when using synthetic preparations, side effects may occur. Said side effects include decreased sexual desire, decreased sexual function, such as erectile dysfunction, and women are at risk for birth defects.

Recently, research on using herbal extracts using natural products without side effects as an agent for preventing hair loss and promoting hair growth has been increasing. Conventionally, natural products used for preventing hair loss or promoting hair growth use extracts of various natural products such as Hwangnyeon, Hwangbaek, Golden, Hwangjeong, Hwanggi, Baekji, Wildflowers, Licorice and Ginger. In particular, when used as a hair loss prevention and hair growth promoter using natural products, there is no side effect when used in synthetic preparations, it does not produce an unpleasant odor, and at the same time provides an antioxidant effect and a natural antiseptic effect.

However, the hair loss prevention and scalp improvement agent using natural products is disadvantageous in that it is not economical because the raw material is expensive and the extraction process is expensive.

Therefore, in order to replace the extract for preventing hair loss and scalp improvement, which can be obtained from natural products, the supply and demand of raw materials is relatively easy, and mass production is easy. It is necessary to develop an agent that replaces the synthetic hair used to prevent hair loss and the scalp improving agent derived from expensive natural materials using marine microalgae, which are cultivated in large quantities in enterprises and the like.

Republic of Korea Registered Patent No. 10-0950437

Accordingly, the present inventors have found out that Tetrathelmis tetrathele , a microalgae used mainly as a main food organism of shellfish, has an activity capable of preventing hair loss and promoting hair growth, as in oyster or sea cucumber farming. The present invention was completed.

Accordingly, an object of the present invention is to provide a composition for improving scalp and preventing hair loss, which contains an extract of the microalgae Tetrathelmis tetrathele as an active ingredient.

Another object of the present invention is to provide a health functional food for improving scalp and preventing hair loss, which contains an extract of the microalgae Tetrathelmis tetrathele as an active ingredient.

In addition, another object of the present invention is Tetrathelmis , which is a microalgae tetracelmis. It is to provide a cosmetic composition for improving the scalp and preventing hair loss containing the extract of tetrathele ) as an active ingredient.

In order to achieve the above object, the present invention provides a composition for improving scalp and preventing hair loss, which contains an extract of Tetrathelmis tetrathele , a microalgae as an active ingredient.

In one embodiment of the present invention, the extract may be an extract obtained using water or an organic solvent.

In one embodiment of the present invention, the extract is Tetrathelmis Tetrathelmis tetrathele ) microalgal cell extract, a culture of the microalgae, a concentrate of the culture or a dried product of the culture.

In one embodiment of the present invention, the extract may have the effect of improving the scalp and preventing hair loss through the anti-inflammatory activity, the proliferative activity of human dermal papilla cells, and the ability to inhibit 5 alpha-reductase enzyme activity.

In one embodiment of the present invention, the composition is selected from the group consisting of creams, gels, patches, sprays, ointments, warning agents, lotions, linen agents, pasta agents and cataplasmas as external preparations for pharmaceutical use. It may be a formulation.

In addition, the present invention provides a health functional food for improving scalp and preventing hair loss, which contains an extract of the microalgae Tetrathelmis tetrathele as an active ingredient.

In addition, the present invention provides a cosmetic composition for improving scalp and preventing hair loss, which contains an extract of the microalgae Tetrathelmis tetrathele as an active ingredient.

In one embodiment of the present invention, the cosmetic composition may be any one selected from the group consisting of hair tonic, hair nutrient longevity, scalp treatment, hair treatment, hair rinse, hair shampoo and hair lotion.

The present invention suggested the possibility of utilizing the extract of the marine microalgae Tetrathelmis tetrathele ( Tetrathelmis tetrathele ) to prevent hair loss and to improve the scalp.

In addition, the extract of the marine microalgae Tetrathelmis tetrathele , which has the function of preventing hair loss and improving the scalp, is a microalgae mainly used as a main food organism of shellfish such as oyster and sea cucumber farming. Because it is cultured in large quantities, it is advantageous for supply and demand of raw materials.

In addition, the marine microalgae Tetrathelmis tetrathele ( Tetrathelmis tetrathele ) extract of the present invention can contribute to cost savings because it can be secured at a lower cost than natural products that have the effect of preventing and preventing hair loss.

Figure 1 Tetrathelmis This is a micrograph of tetrathele .
Figure 2 Tetrathelmis of the present invention tetrathele This is the result of confirming the NO production amount of RAW 264.7 macrophages of the extract.
3 is Tetrathelmis of the present invention tetrathele It is a graph showing the regeneration efficacy of the extract on HaCaT cells.
4 is Tetrathelmis of the present invention tetrathele It shows the graph of the extract by measuring the regeneration efficacy of the dermal papilla cells.

Hereinafter, the present invention will be described in detail.

All technical terms used in the present invention, unless defined otherwise, are used in a sense as commonly understood by those skilled in the art in the relevant field of the present invention. In addition, although a preferred method or sample is described herein, similar or equivalent ones are included in the scope of the present invention. The contents of all publications described by reference herein are incorporated into the present invention.

The present invention is a new material for improving the scalp, preventing hair loss, and promoting hair growth, Tetrathelmis , a marine microalgae tetracelmis. tetrathele ) It is characterized by providing a new use of the extract.

More specifically, the present invention is Tetrathelmis , which is a microalgae tetracelmis. Provides a composition for improving scalp and preventing hair loss, which contains an extract of tetrathele ) as an active ingredient.

Tetrathelmis tetrathele , an active ingredient contained in the composition for improving scalp and preventing hair loss provided by the present invention, is a microalgae belonging to green algae and is mainly used as a food for shellfish.

The cell size of the microalgae is characterized in that the major axis is 14.4±0.8 and the minor axis is 9.3±1.1.

The present inventors are researching to discover new materials derived from nature other than conventional synthetic compounds as a new scalp improvement and hair loss prevention agent, while Tetrathelmis tetrathele microalgae have the effect of preventing hair loss and promoting hair growth. Found.

That is, according to an embodiment of the present invention, the Tetrathelmis of the present invention tetrathele ) As a result of performing antioxidant and anti-inflammatory activity analysis on the ethanol extract, it showed very low antioxidant activity compared to ascorbic acid used as a conventional antioxidant, while effectively inhibiting the production of nitric oxide, an inflammation-inducing factor, and having anti-inflammatory activity. Appeared (see Figure 2 and Table 6).

In addition, according to another embodiment of the present invention, Tetrathelmis of the present invention tetrathele ) In the case of ethanol extract, it was confirmed that the cell proliferation ability according to the treatment of human papillary cells was excellent (see FIG. 4 and Table 11).

In addition, according to another embodiment of the present invention, Tetrathelmis of the present invention In the case of tetrathele ) ethanol extract, it was confirmed that it effectively inhibits the activity of 5 alpha(α) -reductase , which causes hair loss (see Table 12).

On the other hand, hair loss may be caused by hair loss due to disease, hair loss due to hormonal causes, hair loss due to nutritional imbalance, or hair loss due to stress, and the hair loss caused by the disease may include genetic, autoimmune diseases, burns, chemotherapy, Psoriasis, fungal infections, radiation exposure, or tumors may be the cause, and hair loss due to the hormonal cause may be due to medication, before and after pregnancy, menopause, diabetes, pituitary function abnormalities, parathyroid or thyroid function abnormalities.

In addition, 5α-reductase is an important enzyme that accelerates signaling to AR by converting testosterone to 5α-dihydroxytestosterone (DHT). Propecia) and prostate hyperplasia (benign prostatic hyperplasia, BPH) treatment (trade name; Proscar) is used.

Therefore, it is possible to suppress the activity of the 5 alpha (α)-reductase that causes hair loss, suppress the inflammatory reaction, and in the case of a substance having antioxidant activity, it can prevent hair loss and promote hair growth.

Therefore, Tetrathelmis of the present invention having both of these activities tetrathele ) Microalgae can be used as a new material for preventing hair loss and promoting hair growth.

The composition for preventing hair loss and promoting hair growth provided by the present invention is Tetrathelmis as an active ingredient tetrathele ) microalgae may include an extract of itself, a culture of the microalgae, a concentrate of the culture or a dried product of the culture.

In addition, Tetrathelmis The tetrathele ) extract is cultured using the above-mentioned microalgae using a medium, and then cultured Tetrathelmis Tetrathelmis After the tetrathele ) strain is collected by centrifugation or the like, it may be an extract extracted by adding an organic solvent to the strain, and lower alcohol having 1 to 4 carbon atoms such as water, methanol, ethanol, butanol including purified water or a mixture thereof. The extract may be obtained using a solvent selected from a solvent, and an extract may be obtained using a mixed solvent of water and ethanol, more preferably 50 to 100% ethanol, and even more preferably 70% ethanol.

Tetrathelmis of the present invention The composition containing tetrathele ) extract as an active ingredient may be used as a pharmaceutical composition for preventing hair loss, improving scalp and promoting hair growth, and a cosmetic composition.

The cosmetic composition according to the present invention has an active ingredient of tetracelmis tetracel ( Tetrathelmis tetrathele ) extract, as well as ingredients commonly used in cosmetic compositions, and may include, for example, antioxidants, stabilizers, solubilizers, conventional adjuvants such as vitamins, pigments and flavors, and carriers.

In addition, the composition of the present invention, in addition to the above-mentioned tetracelmis tetracele ( Tetrathelmis tetrathele ) extract, can be used by mixing a hair or hair growth agent that has been conventionally used to the extent that it does not impair the action of preventing hair loss (hair loss) and improving hair. have.

In addition, the cosmetic composition of the present invention can be prepared in any formulation conventionally prepared in the art, for example, hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair rinse, Hair treatment, hair cream, hair nutrition cream, hair moisture cream, hair massage cream, hair wax, hair aerosol, hair pack, hair nutrition pack, hair soap, hair cleansing foam, hair oil, hair drying agent, hair preservative treatment agent, hair dye, It can be manufactured in various forms such as wave agent for hair, hair decolorant, hair gel, hair glaze, hair dresser, hair lacquer, hair moisturizer, hair mousse, and hair spray. Particularly, hair tonic, hair nutrition, scalp treatment, hair Treatment, hair rinse, hair shampoo, and can be prepared in any one formulation selected from the group consisting of hair lotion, but is not limited thereto.

According to a preferred embodiment of the present invention, the Tetrathelmis of the present invention The content of tetrathele ) extract is 0.00001-30% by weight relative to the total weight of the composition, preferably 0.5-20%, more preferably 1.0-10% by weight. If the content of the extract is less than 0.00001% by weight, the effect of preventing hair loss and improving hair growth by the extract is greatly reduced, and if it exceeds 30% by weight, it may cause skin irritation, and there is a problem in formulation.

In addition, the composition of the present invention may be a pharmaceutical composition comprising tetraselmis tetracele ( Tetrathelmis tetrathele ) extract as an active ingredient.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant, excipient, disintegrant, sweetener, binder, coating agent, expander, lubricant, A lubricant or flavoring agent may be used.

The pharmaceutical composition may be preferably formulated into a pharmaceutical composition for administration, including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectables. For example, for formulation in the form of tablets or capsules, the active ingredient can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and colorants can also be included in the mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, trachocanth or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include, as sterile and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components can be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents can be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA by appropriate methods in the art.

In one embodiment of the present invention, the extract of the present invention may be included in 0.00001 to 30% by weight relative to the total weight of the composition.

The pharmaceutical composition for external application for skin of the present invention is an external preparation for skin having the effect of preventing hair loss, promoting hair growth, and improving the scalp. Cream, gel, patch, spray, ointment, warning agent, lotion agent, linement agent, pasta agent, or cataplasma It can be prepared and used as a pharmaceutical composition in the form of an external preparation for skin, but is not limited thereto.

The composition of the present invention may also be a food composition, which is an active ingredient tetracelmis tetrasele ( Tetrathelmis In addition to containing the tetrathele ) extract, it can contain various flavoring agents or natural carbohydrates, etc., as additional ingredients, as in conventional food compositions.

Examples of the natural carbohydrates described above include monosaccharides, such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, etc.; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-mentioned flavoring agents may advantageously use natural flavoring agents (taumatine), stevia extracts (eg rebaudioside A, glycyrrhizine, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).

The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplement foods. There is this.

In addition, the food composition is a variety of nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and neutralizing agents (cheese, chocolate) in addition to the active ingredient Tetrathelmis tetrathele extract Etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonic acid used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain natural fruit juice and fruit juice for the production of fruit juice beverages and vegetable beverages.

Tetrathelmis , an active ingredient of the present invention Since tetrathele ) extract is a natural substance and has little toxicity and side effects, it can be used safely even for long periods of time for the purpose of preventing hair loss, promoting hair growth, and improving the scalp.

The present invention is also Tetrathelmis tetrathele ) Provides health functional food for preventing hair loss, improving scalp and preventing hair loss, which contains extract as an active ingredient.

The health functional food of the present invention may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing hair loss, promoting hair growth and improving scalp.

In the present invention, the health functional food refers to food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to the Act No. 6727 on the Health Functional Food Act, and it regulates nutrients with respect to the structure and function of the human body. Or it means taking it for the purpose of obtaining a useful effect for health purposes such as physiological action.

The health functional food of the present invention may include a normal food additive, and whether or not it is suitable as a food additive is related to the item according to the general rules and general test methods of food additives approved by the Food and Drug Administration, unless otherwise specified. Judging by standards and standards.

Examples of the food additives receivable include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamonic acid; Natural additives such as decolorant, licorice extract, crystalline cellulose, high pigment, and guar gum; And mixed preparations such as L-sodium glutamate, noodle-added alkalis, preservatives, and tar colorants.

For example, in the form of tablets, the health functional food is granulated by mixing the mixture of the extract, which is the active ingredient of the present invention with excipients, binders, disintegrating agents, and other additives, in a conventional manner, and then adding a lubricant, etc. Alternatively, the mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain a mating agent or the like as necessary.

Hard capsules of capsules in the form of health functional foods can be prepared by filling a mixture of the above-mentioned extract, which is the active ingredient of the present invention, with additives such as excipients, etc. in a conventional hard capsule, and the soft capsules are tetracelmis tetracel ( Tetrathelmis tetrathele ) can be prepared by filling a capsule base such as gelatin with a mixture of extracts mixed with additives such as excipients. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

The health functional food in the form of a ring can be prepared by molding a mixture of the extract, excipient, binder, disintegrant, etc., which is the active ingredient of the present invention, by molding in a conventionally known method, and peeling it with white sugar or other skin repellent if necessary. Alternatively, the surface may be coated with a material such as starch or talc.

The granular form of the health functional food can be prepared in the form of a mixture of the above-mentioned extract, excipient, binder, disintegrant, etc., which is the active ingredient of the present invention, in a granular form by a conventionally known method, and if necessary, flavoring agents, mating agents, etc. It may contain.

The health functional food may be beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

In addition, the present invention provides a method for preventing or treating hair loss, which comprises administering an extract of Tetrathelmis tetrathele to a mammal.

The term "mammal" as used herein refers to a mammal that is the subject of treatment, observation or experiment, preferably human.

The term “therapeutically effective amount” as used herein refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, physician or other clinician, which And an amount that induces relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and frequency of administration of the active ingredients of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, the type of disease, the severity of the disease, the content of active ingredients and other ingredients in the composition, the type of formulation, and the patient's age, weight, general health status , Gender and diet, time of administration, route of administration and composition, secretion rate, duration of treatment, and drugs to be used simultaneously. In the treatment method of the present invention, in the case of an adult, Tetrathelmis of the present invention When the tetrathele ) extract is administered once to several times a day, it is preferable to administer it at a dose of 1 mg/kg to 250 mg/kg.

In the treatment method of the present invention, a composition comprising the tetracelemis tetrathele extract of the present invention as an active ingredient is oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, It can be administered in a conventional manner via intraocular or intradermal routes.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

< Example 1>

Marine microalgae Tetrathelmis tetrathele Characteristics and culture

<1-1> Microalgae Tetrathelmis tetrathele Characteristics

Microalgae Tetrathelmis of the present invention The properties of tetrathele are shown in Table 1 below, and FIG. 1 shows a micrograph of the microalgae.

Tetrathelmis Characteristics of tetrathele Species Source of strain Cell size(mean±sd,um) Major axis Minor axis characteristic Tetrathelmis tetrathele TT KMCC P-2 14.4±0.8 9.3±1.1 Oyster feed, green algae

<1-2> Composition of medium

Microalgae Tetrathelmis of the present invention The culture of tetrathele was cultured under the following conditions (see Table 1 below) in a static culture, and the medium was used as a Conwy medium, and the composition is shown in Table 3 below.

Tetrathelmis Culture conditions of tetrathele Culture conditions Political culture
Temperature 23±1℃
Illuminance 400 Watt metal halide lamp
Culture tank round acrylic water tank

Composition of the medium f/2 medium Solution A- Chemicals Stock solution Quantity Molar concentration in final medium Sodium nitrate
(NaNO3) 1.76M 5 mL 8.83×10 ^-4 M Sodium phosphate monobasic (NaH2PO4) 0.06M 5 mL 3.63×10 ^-5 M Sodium silicate
(Na2SiO39H2O) 0.49M 5 mL 1.07×10 ^-4 M f/2 trace metal solution see recipe below 2.5 mL - f/2 vitamin solution see recipe below 1.25 mL - Solution B- Trace metal solution Stock solution Quantity Iron(III) chloride hexahydrate (FeCl36H2O) 10mM 3.15 g 1×10 ^-5 M Na2EDTA2H2O 10mM 4.36 g 1×10 ^-5 M Copper(II) sulfate pentahydrate (CuSO45H2O) 40mM 1 mL 4×10 ^-8 M Sodium molybdate dihydrate(Na2MoO42H2O) 30mM 1 mL 3×10 ^-8 M Zinc sulfate heptahydrate(ZnSO47H2O 80mM 1 mL 8×10 ^-8 M Cobalt(II) chloride hexahydrate(CoCl26H2O 40mM 1 mL 5×10 ^-8 M Manganese(II) chloride tetrahydrate(MnCl24H2O 0.9M 1 mL 9×10 ^-7 M Make final volume up to 1 L with DW and autoclave Solution C-Vitamin solution Stock solution Quantity Vitamin B1 (Thiamin) 0.3mM 1 mL 1×10 ^-10 M Vitamin B12 (cyanocobalamine) 0.73μM 10 mL 2×10 ^-9 M Biotin 0.82mM 200 mg 3×10 ^-7 M 100 ml. Dissolve in distilled water and store in the refrigerator

Each solution is mixed with 1L, sterilized with autoclave, and kept refrigerated before use.

<1-3> Microalgae Tetrathelmis tetrathele Recovery and extract extraction

Tetrathelmis cultured with the culture medium presented above To prepare the tetrathele extract, centrifugation at 3,000 rpm for 15 minutes was performed by separating and recovering the microalgae, and the supernatant was concentrated and used as a microalgal culture. In order to extract the active ingredients of the microalgae, 70% ethanol solution was added to the collected microalgae and concentrated microalgae culture by adding an amount corresponding to 5 times on a wet basis. The extract was extracted while shaking at 40° C. for 3 hours, filtered, and the filtrate was concentrated and used as a sample.

< Experimental Example 1>

Marine microalgae Tetrathelmis tetrathele Antioxidant activity for scalp improvement, hair loss prevention and treatment effect

It is known that the formation of radicals inducing diseases of the scalp and hair is the cause of hair loss. Therefore, when inhibiting the production of radicals and inhibiting the activity of enzymes causing oxidation, hair loss can be prevented and treated.

Accordingly, the present inventors obtained Tetrathelmis obtained in Example 1 above. In order to confirm that the tetrathele extract has antioxidant activity, DPPH radical scavenging activity was evaluated by Park et al. (2005, Park, EY, Murakami, H., Mori, O. and Matsumura, Y. 2005. Effects of protein and peptide addition on lipid oxidation in powder model system.J. Agric.Food Chem. 53, 137-144.

Specifically, 500 μL of the sample solution was added to a test tube, and 500 μL of ethanol and 250 μL of 0.5 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH)/ethanol solution were added and left at room temperature at 25° C. for 30 minutes. Then, absorbance was measured at 517 nm. As a control, DMSO, a sample solvent of 500 μL, was used instead of the sample, and 750 μL ethanol was used instead of DPPH and distilled water for the blank test. Antiradical activity was calculated as% of DPPH decolorization according to the following formula.

Tetrathelmis The tetrathele extract was tested and compared with ascorbic acid, which is known as a powerful antioxidant. DPPH radical scavenging activity of ascorbic acid showed a scavenging capacity of about 50% at 0.04 mg/mL. On the other hand, the radical scavenging activity of Tetrathelmis tetrathele extract, which is the present material, was 7.8 mg/mL, which was only about 1/200 of that of ascorbic acid, and the culture medium showed little activity in the measured concentration range (see Table 4).

Tetrathelmis used in the present invention DPPH (EC ₅₀ ) measurement result of tetrathele extract sample EC ₅₀ , mg/mL Ascorbic acid (positive control group) 0.04 Tetrathelmis tetrathele ethanol extract 7.8 Tetrathelmis tetrathele culture medium extract -

< Experimental Example 2>

Marine microalgae Tetrathelmis tetrathele Anti-inflammatory activity for scalp improvement, hair loss prevention and treatment effect

In addition to radical formation, inflammation is known to cause inflammation of the scalp and hair. Therefore, when suppressing inflammation, the present inventors confirmed whether the microalgal extract of the present invention has anti-inflammatory activity because it can prevent and treat hair loss.

<2-1> RAW Cytotoxicity measurement for 264.7 cells

We are Tetrathelmis Prior to measuring the anti-inflammatory effect of the tetrathele extract, the cytotoxicity of the microalgal extract was confirmed for RAW 264.7 cells pre-sold by the Korea Cell Line Bank (KCLB, Seoul, Korea).

That is, RAW 264.7 cells in a cell culture flask were cultured using Dulbecco's modified Eagle's Medium (DMEM) medium containing 10% FBS, and then dispensed into 96-well plates at a concentration of 1×10 ⁵ cells/well, 24 The cells were cultured in a CO ₂ incubator adjusted to a temperature of 37° C., 95% humidity and CO _2, 5% for a period of time, and then, when replaced with a new medium, the final concentration of the microalgal extract of the present invention was 0.1, 1 Cells were treated with 10, 100 and 200 μg/mL, respectively, and then cultured for 24 hours. To measure the viability of the cell line, MTS[3-(4,5-dimethyl-thiazol-2-yl)-5-carboxy-methoxyphenyl2-(4-sulfophenyl)-2H-tetrazolium] reagent was used. After treatment for an hour, the absorbance was measured at 490 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA). The survival rate of the cells was expressed as the absorbance of the sample treatment group compared to the control group without treatment.

As a result of the analysis, as shown in Table 5, the toxicity to the Tetrathelmis tetrathele extract of the present invention was not toxic until a concentration section of 100 ug/mL, but showed a rather high toxicity at a concentration of 200 ug/mL. Therefore, through the above results, the present inventors found that the extract of the present invention did not induce cytotoxicity until the concentration section of 100 ug/mL with respect to the cells, and thus the anti-inflammatory effect of the extract was the section excluding the concentration of 200 ug/mL It was measured at.

Tetrathelmis used in the present invention Toxicity of tetrathele extract to RAW264.7 Sample concentration (ug/mL) Cell viability,% Control 100.0±5.5 Tetrathelmis tetrathele ethanol extract 0.1 101.2±2.2 One 98.8±3.4 10 109.9±6.6 100 133.2±1.7* 200 34.0±8.2*

*p<0.5, compared with control

<2-2> Nitrogen Oxide Inhibition Activity Analysis

We are Tetrathelmis To measure the anti-inflammatory effect of tetrathele extract, a RAW 264.7 cell line was dispensed into 96 wells, and then cultured until the cells reached about 80% of the bottom. Treated as much as possible. Thereafter, the Tetrathelmis tetrathele extract of the present invention was treated to have concentrations of 0.1, 1, 10, and 100 μg/mL, respectively, and after 24 hours of incubation, the amount of nitrogen oxide (NO) produced was analyzed with Griess reagent. At this time, the control group used only the group treated with lipopolysaccharide (LPS) and the group not treated with anything.

Analysis using the Griess reagent was first added to 1% sulfanilamide in 5% nitric acid solution, 0.1% naphthylethylene-diamine di-hydrochloride was added and mixed, and 50 μL of the supernatant of the cultured cells was taken from a 96-well plate. , 50 µL of Griess reagent was added to the mixture, and reacted at room temperature for 15 minutes. Thereafter, absorbance was measured at 540 nm using a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA). The change in the amount of nitrogen oxide produced was expressed as the amount of nitrogen oxide produced in the sample treatment group based on the amount of nitrogen oxide produced in the control group without treatment.

As a result of analysis, as shown in FIG. 2 and Table 6, as a result, when compared with the group treated with only 1 ug/mL of LPS, Tetrathelmis In the case of tetrathele ethanol extract, a production effect of 8.5% was observed at a concentration of 0.1 ug/ml, and a high NO production of 59.8% was suppressed at a concentration of 100 ug/ml. Also, as the concentration increased, the production of NO decreased concentration-dependently, resulting in Tetrathelmis tetrathele It was confirmed that the extract was effective in inhibiting NO production. However, in the case of the culture solution, it was confirmed that there is no efficacy to suppress the production of NO.

Tetrathelmis used in the present invention NO production amount of RAW264.7 of tetrathele extract Sample concentration (ug/mL) NO production,% Control 19.6±4.8 LPS ** 100.0±1.4 Tetrathelmis tetrathele ethanol extract + LPS 0.1 91.5±3.2* One 89.2±9.7 10 88.9±8.8* 100 40.2±5.9* Tetrathelmis tetrathele culture medium extract 0.1 101.6±2.5 One 107.2±1.9 10 126.4±3.1* 100 122.3±4.8*

*p<0.5, compared with control, ** LPS: lipopolysaccharide

<2-3> PGE2 Production inhibition activity analysis

Prostaglandin (PGE) is an immunomodulatory substance known to have an activity that induces an inflammatory response. Therefore, when the prostaglandin production or activity is inhibited, the inflammatory reaction can be suppressed, thereby preventing hair loss caused by inflammation. Therefore, the present inventors have Tetrathelmis to make sure they produce PGE ₂ inhibitory effect of the extract tetrathele Prostaglandin E ₂ EIA Kit; PGE ₂ by the extract of the present invention using a (Cayman Chemical Company Ann Arbor, Michigan , USA) The production inhibitory effect was analyzed. At this time, the measurement method was quantified according to the manufacturer's analysis method. First, after treating Tetrathelmis tetrathele extract with RAW 264.7 cells at a concentration of 0.01, 0.1, 1, 10, 100, 200 ug/mL with 0.1% LPS for 24 hours, , After loading the cell culture supernatant into goat anti-mouse coated 96-well plate by 50 uL and adding 50 uL of primary antibody solution and 50 uL of PGE ₂ conjugate thereto, reacted at 4° C. for 18 hours, After washing 5 times with a washing solution, Ellman's reagent was treated with 200 uL and reacted while stirring for 60 minutes, and absorbance was measured at 405-420 nm.

As a result of the measurement, as shown in Table 7 below, when only LPS 1 ug/mL was compared with the treated group, Tetrathelmis It was confirmed that tetrathele ethanol extract had no significant inhibition of production.

Tetrathelmis used in the present invention PGE2 production amount for RAW264.7 of tetrathele extract Sample concentration (ug/mL) NO production,% Control 13.2±64.64.8 LPS** 100.0±2.7 Tetrathelmis tetrathele ethanol extract + LPS 0.1 - One 97.6±0.6 10 94.1±0.5 100 94.1±0.2

*LPS: lipopolysaccharide

< Experimental Example 3>

Marine microalgae Tetrathelmis tetrathele For human epidermal cells for scalp improvement, hair loss prevention and treatment effect Proliferative capacity Measure

In order to prevent the hair from falling out, the health of the scalp supporting the hair is most important. The purpose of this study was to determine the effect on the health of the scalp by measuring the regenerative efficacy of the scalp cells forming the scalp.

<3-1> HaCaT Cytotoxicity measurement for cells

We are Tetrathelmis The cytotoxicity of the sample against epidermal cells was confirmed before confirming the cell's regenerative efficacy of the tetrathele extract.

HaCaT cells corresponding to human skin epidermal cells were cultured with DMEM (Gibco) medium containing 10% calf serum (FBS; Gibco), and medium was changed once every 2-3 days during the culture. HaCaT cells were dispensed in a 96 well plate to be 5×10 ⁴ cells/well, and stabilized by incubating in a CO ₂ incubator controlled at 37° C., humidity 95%, and CO ₂ 5% for 24 hours. Samples were prepared in a new medium so that the final concentrations were 0.01, 0.1, 1, 10, and 50 μg/mL, treated with cells, and cultured for 24 hours. The viability of the cell line was measured at 490 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA) using MTS reagent. The survival rate of the cells was expressed as the absorbance of the sample treatment group compared to the control group without treatment.

As a result, as shown in Table 8 below, it was confirmed that cytotoxicity was not observed in all concentration sections.

Tetrathelmis used in the present invention Assessment of toxicity of tetrathele extract to HaCaT cells Sample concentration (ug/mL) Cell viability,% Control 100.0± 4.5 Tetrathelmis tetrathele ethanol extract 0.01 122.4± 4.8 0.1 112.8± 1.5 One 110.7± 4.9 10 105.7± 6.2 50 105.1±11.7 Tetrathelmis tetrathele culture medium extract 0.01 116.8±11.5 0.1 115.4± 2.8 One 115.7± 1.7 10 111.4± 3.4 50 104.0±3.9

<3-2> HaCaT Regeneration efficacy on cells

The cell regeneration efficacy of the sample was confirmed using HaCaT cell, a human skin epidermal cell. HaCaT cells were dispensed into 96 well plates at 1×10 ⁴ cells/well, and cultured in a CO ₂ incubator controlled at 37° C., humidity 95%, and CO ₂ 5% for 24 hours. Samples were prepared in a new medium so that the final concentrations were 0.01, 0.1, 1, 10, and 50 µg/mL, treated with cell lines, and cultured for 1, 3, and 5 days to measure cell proliferation capacity. The cell proliferation ability was performed in the same way as the cytotoxicity test, and absorbance was measured at 490 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA) using MTS reagent. The survival rate of the cells was expressed as the absorbance of the sample treatment group compared to the control group without treatment.

As a result, as shown in Table 9 and Figure 3, Tetrathelmis In the case of tetrathele ethanol extract, the cells were proliferated for 3 days to a concentration of 10 ug/mL, but the proliferative capacity was decreased on the 6th day. But Tetrathelmis In the case of tetrathele culture, it was confirmed that the cells proliferated in all concentration sections during all incubation periods of 6 days. All test groups incubated on the 1st, 3rd, and 6th days showed excellent proliferation efficacy, and especially at 10 ug/ml, it showed a high proliferative activity more than 2 times after 3 days incubation, so that certain components of the culture medium proliferated It is judged to promote.

Tetrathelmis used in the present invention Regeneration efficacy of tetrathele extract on HaCaT cells Sample concentration (ug/mL) Cell growth,% 1 day 3 day 6 day Control 100.0±7.1 100.0±9.3 100.0±8.9 Tetrathelmis tetrathele ethanol extract 0.01 113.6±11.3 120.7±2.7* 101.6±9.8 0.1 138.9±7.7* 122.5±5.2* 101.0±4.0 One 128.4±18.2 123.3±3.8* 111.0±8.0 10 129.2±15.9* 130.4±8.2* 118.1±7.7* 50 110.5±5.9 99.2±9.0 2.4±18.7 Tetrathelmis tetrathele culture medium extract 0.01 125.5±0.6* 129.9±12.2* 129.5±5.8* 0.1 121.6±2.1* 135.6±12.8* 147.1±2.6* One 115.7±8.1* 155.6±9.5* 172.4±3.8* 10 123.5±8.9* 211.0±13.5* 202.0±6.0* 50 122.4±7.8* 161.6±8.0* 212.9±8.3*

*p<0.5, compared with control

< Experimental Example 4>

Marine microalgae Tetrathelmis tetrathele Human for improvement of scalp, prevention of hair loss and treatment effect Breast nipples For cells Proliferative capacity Measure

Hair growth takes place in the human papilla cells (HDFPC, human follicle dermal papilla cells), receiving nutrients from the papilla.

The proliferation ability of the papillary cells was measured to confirm the ability to induce hair growth of the sample.

<4-1> Human Breast nipples Cytotoxicity measurement for cells

We are Tetrathelmis Cytotoxicity of the sample to the cell was confirmed before confirming the regenerative effect of tetrathele extract on human papillary cells.

The dermal papilla cells were tested by purchasing primary cells (C-12071) derived from human follicle dermal papilla from Promocell GmbH (Heidelberg, Germany). As a medium for cell culture, follical dermal papilla cell growth medium (C-26501) was purchased and used by the same company that purchased the cell, and cultured with a medium containing 10% calf serum (FBS; Gibco). These cells were dispensed into a 96-well plate at 2000 cells/well, and cultured by processing samples at each concentration after 24 hours. The survival rate of the cells was measured by treating the MTS reagent of Promega Corporation for 1 hour in the same manner as the above method, and absorbance was measured at 490 nm with a microplate reader (Perkin Elmer 1420 Multilabel Counter VICTORTM, USA). The survival rate of the cells was expressed as the absorbance of the sample treatment group compared to the control group.

As a result, as shown in Table 10 below, Tetrathelmis Tetrathele cells and cultures showed no toxicity at all concentrations.

Tetrathelmis used in the present invention Toxicity of tetrathele extract to dermal papilla cells Sample concentration (ug/mL) Cell viability,% Control 100.0±10.5 Tetrathelmis tetrathele ethanol extract 0.01 124.6±13.4 0.1 110.9±15.4 One 110.1± 9.7 10 101.2± 9.6 50 107.8± 9.3 Tetrathelmis tetrathele culture medium extract 0.01 105.6± 8.2 0.1 123.6± 7.5 One 101.4± 8.5 10 106.6± 2.0 50 117.1±12.6

*p<0.5, compared with control

<3-2> To the papillary cells For regenerative efficacy

Human dermal papilla cells were cultured in a cell culture flask with folicle dermal papilla cell growth medium containing 10% FBS. HDFPC cells were dispensed into 96 well plates at a concentration of 0.5×104 cells/well, and cultured in a CO ₂ incubator controlled at 37° C., humidity 95%, and CO ₂ 5% for 24 hours. Samples were prepared in a new medium so that the final concentrations were 1, 10, 100 and 200 μg/mL, treated with cells, and cultured for 1, 3 and 5 days. All cell line viability was measured by the same method.

As a result, as shown in Table 11 and Figure 4, it was possible to confirm the proliferation capacity in all concentration sections. Tetrathelmis The tetrathele ethanol extract was able to confirm the cell proliferation capacity at a temperature of 10 ug /ml or more when cultured per day, and the cell proliferation capacity increased significantly as the culture day increased. In particular, when cultured at a high concentration of 200 ug/ml for 5 days, it showed a large proliferation capacity of 41.5%. But Tetrathelmis In the case of tetrathele culture, a significant increase rate of cell proliferation capacity could not be confirmed.

Tetrathelmis used in the present invention Regeneration efficacy of tetrathele extract on dermal papilla cells Sample concentration (ug/mL) Cell proliferation,% 1 day 3 day 5 day Control 100.0±2.3 100.0±3.9 100.0±2.8 Tetrathelmis tetrathele ethanol extract 0.1 104.0±4.4 110.4±2.0* 122.2±9.2* One 103.4±3.0 107.1±2.3* 132.9±12.6* 10 106.6±1.6* 108.5±4.9* 132.9±4.6* 100 117.6±2.1* 125.3±4.8* 129.5±3.6* 200 135.7±7.3* 141.9±2.8* 141.5±3.3*

< Experimental Example 5>

Tetrathelmis tetrathele Alpha for improving scalp and preventing hair loss Lyduck Tase activity Inhibitory ability analysis

One of the causes of hair loss is 5α-dihydrotestosterone (dihydrotestosterone). 5α-dihydrotestosterone is a substance in which testosterone, a male hormone, is produced by an enzyme called 5α-reductase in a blanket. It is now known that excessive production of this substance causes hair loss. In this study, the activity of inhibiting the activity of 5α-reductase was investigated using the liver tissue of rats containing a large amount of 5α-reductase.

That is, since the activity inhibitory activity of 5 alpha reductase inhibits the production of dehydrotestosterone, testosterone and NADPH are added to the liver tissue homogenate containing a large amount of this enzyme, and the removal rate of NADPH is measured by absorbance to measure 5 alpha. The activity inhibitory ability was measured by measuring the activity inhibitory ability of reductase. To this end, first, SD rat liver is firstly centrifuged by adding 4 times of 48 uM EDTA2Na-5.02 mM MgCl26H2O-131.5 mM Surose-2.57 mM 2-Mercaptoethanol-50 mM NaCl-1mM Tris-HCl (pH 7.0) and homogenizing. (10,000×g at 0° C. for 10 min). After taking the supernatant, the second centrifugation was performed once again (30,000×g at 0° C. for 1 hr). The supernatant from which the fat layer was removed was measured for protein by the BCA method, and the protein concentration was adjusted to 5.0 mg/mL and stored at 70°C for use in this study.

5α-reductase activity inhibitory ability was measured by modifying the method of Wu JH et al. (2013). That is, after adding 1 uL of sample, 20 uL of 33 uM Testosterone 20 uL 1 mM Tris HCl (pH 7.0) 139 uL, 33 uM NADPH to 20 uL of rat microsomal suspension, and mixing it, 0 to 10 at a wavelength of 340 nm, which is the NADPH measurement wavelength. The absorbance of the minutes was measured every minute to calculate the rate of reduction of NADPH. The activity inhibitory ability of 5 alpha reductase of the microalgae extract was measured as a ratio to the rate of NADPH reduction of the sample containing the solvent instead of the sample.

As a result, as shown in Table 12 below, Tetrathelmis The tetrathele ethanol extract showed inhibitory activity in all concentration sections as the concentration increased. In particular, in the case of Tetrathelmis tetrathele ethanol extract, a high inhibitory activity of 57.0% was found at a concentration of 10 ug/mL. When comparing the activity with finasteride used as a drug for preventing hair loss, it showed an activity inhibitory ability of 50% at a concentration of 92.4 ng/ml. Tetrathelmis tetrathele ethanol extract showed an activity inhibitory activity of about 23% at a concentration of 100 ng/ml, showing half the activity of finasteride. In addition, Tetrathelmis tetrathele ethanol extract showed an inhibitory activity of 57% at a concentration of 10000 ng/ml, showing a very high inhibitory activity when compared to a single component finasteride.

Tetrathelmis used in the present invention Inhibition of 5α-reductase activity of tetrathele extract Sample(ng/mL) Inhibition,% Finasteride IC ₅₀ 92.4 ng/mL Tetrathelmis tetrathele ethanol extract One 6.6±14.7 10 18.4±13.0 100 23.0± 6.2 500 35.3±18.2 1000 45.3±14.8 10000 57.0±45.9

So far, the present invention has been focused on the preferred embodiments and experimental examples. Those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

TT ethanol ext: Tetrathelmis tetrathele Ethanol extract
TT culture media ext: Tetrathelmis tetrathele Culture medium extract

Claims (8)

A pharmaceutical composition for improving scalp and preventing hair loss, which contains an extract of the microalgae Tetrathelmis tetrathele as an active ingredient. According to claim 1,
The extract is a pharmaceutical composition for improving scalp and preventing hair loss, characterized in that the extract is extracted using water or an organic solvent. According to claim 1,
The extract is Tetrathelmis tetrathele ) A pharmaceutical composition for improving scalp and preventing hair loss, comprising a microalgal cell extract, a culture of the microalgae, a concentrate of the culture, or a dried product of the culture. According to claim 1,
The extract is a pharmaceutical composition for improving scalp and preventing hair loss, characterized in that it has anti-inflammatory activity, proliferation activity of human dermal papilla cells and inhibitory activity of 5 alpha-reductase enzyme. According to claim 1,
The pharmaceutical composition is an external skin preparation for cream, gel, patch, spray, ointment, warning agent, lotion, linen agent, pasta agent, and cataplasma, which is a formulation selected from the group consisting of scalp improvement and hair loss prevention. Pharmaceutical composition. A health functional food for improving scalp and preventing hair loss, which contains an extract of microalgae Tetrathelmis tetrathele as an active ingredient. A cosmetic composition for improving the scalp and preventing hair loss, which contains an extract of the microalgae Tetrathelmis tetrathele as an active ingredient. The method of claim 7,
The cosmetic composition is a cosmetic composition for improving scalp and preventing hair loss, characterized in that it is any one selected from the group consisting of hair tonic, hair nutrient longevity, scalp treatment, hair treatment, hair rinse, hair shampoo and hair lotion .

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