KR101177500B1 - Hair growth stimulating composition - Google Patents

Abstract

The present invention relates to a composition for hair growth comprising a compound selected from the group consisting of cypress essential oil extract, fractions thereof, or cuminol, eucarvone, and caramenene isolated therefrom. will be.
Cypress essential oil extract, fractions thereof, or cuminol, eucarvone and caramenene of the present invention are safe for the human body and have excellent effects on hair growth without side effects, thus preventing hair loss. It can be usefully used as a pharmaceutical composition for hair growth or a cosmetic composition for hair growth having activity for improving, improving hair growth.

Description

Hair growth composition {Hair growth stimulating composition}

The present invention relates to a composition for hair growth comprising an essential oil derived from nature as an active ingredient, and a pharmaceutical composition or hair growth cosmetic composition for hair growth using the same as an active ingredient.

Hair grows in hair follicles and hair follicles derived from epithelial cells. In humans, the formation of hair follicles is already done during fetal growth, and after birth, hair follicle formation no longer occurs.

The hair is buried about 3-5mm in the skin and accumulated on the epidermis and dermis. This part is called a blanket. There is dermal papilla, the tissue that controls the hairs on top of the follicles, and there are hair cells that make the hairs just above the papillas, producing new hair and pushing it up while continuing to divide.

 The dermal papilla cells have a period of growth (anagen), degeneration (catagen), and telogen cycles that are actively growing, and when signals are received from adjacent cells after the resting period, they enter the growth phase and regenerate the cells. This results in the formation of new hair. Usually three years of growth, three weeks of retreat, three months of rest, this entire cycle is repeated over three to six years, resulting in an average of 50 to 200 hairs per day. Generally, the term alopecia refers to the increase in the number of hairs that are abnormally dropped due to shortening of the hair growth rate in the growth period and more hairs in the degeneration or rest period.

On the other hand, when the gene expression and activity of cell signaling and related molecules involved in such a normal cycle are affected, hair loss and hair thickness change appear. The dermal papilla cells are derived from mesoderm and are known to be the most similar to the fibroblasts. In addition, versican has been reported as a specific marker of dermal papilla cells in vivo, and it is known that dermal papilla cells expressing versican have hair inducing ability.

Growth of dermal papilla cells is caused by a number of growth factors. Representative growth factors include VEGF (Vascular endothelial growth factor) and KGF (keratinocyte growth factor). These growth factors are important for maintaining the growth of dermal papilla cells.

  In general, baldness or hair loss caused by disorders of the hair formation cycle is more frequently occurring in men than in women. Male type baldness has a familial family history of baldness, and the hair is gradually thinned from the 20s or 30s. Hair loss progresses, and the forehead and hairline borders are pushed backwards. In contrast, female baldness rarely causes baldness like male baldness.

The cause of baldness is not yet clear, but genetic predisposition and androgen, androgens, are considered important factors for the development of baldness, i.e. at a genetically determined point in the lifetime of a person with a family history of baldness, Androgen acts on hair follicles and is believed to cause hair loss. Dihydrotestosterone, a metabolite of testosterone, is known to play an important role in the development of baldness, especially among the metabolites of androgens rather than androgens in the blood. In addition, endocrine disorders, autonomic nervous system abnormalities, allergies, hair root malnutrition, bacterial infection, aging, etc. are suspected to be the cause.

Recently, many people suffer from alopecia areata, a type of baldness caused by environmental factors such as air pollution, mental stress, and excessive use of hair beauty products such as sprays and mousses. to be.

Alopecia like this can be very shocking and severely stressful to the parties, lead to loss of self-confidence, disruption of interpersonal relationships, and serious social problems such as marriage and job hunting. Although it is desirable to treat vulgaris, most of the current baldness treatments on the market are insignificant or ineffective and have not been medically proven.

To date, standard treatments for alopecia generally include the application of minoxidil solution, finasteride medication, and autologous hair transplantation. Minoxidil is a high blood pressure medication developed for the purpose of lowering blood pressure, but it has become more famous as a hair growth agent because of hair growth phenomenon due to side effects of use. When minoxidil solution is used as a hair restorer, it may cause side effects such as debilitating effect due to antihypertensive action, hypotension, tachycardia, etc., and thus it may not be a suitable drug for long-term use. Side effects such as insufficiency or limited effects, and self-hair transplantation is not only expensive for transplanting hairs individually, but also requires a prolonged transplant period, and can only transplant one's own hair. There is a problem that there is.

The present invention, in order to solve the problems of the prior art as described above, provides a composition for hair growth derived from a natural material that is safe to the human body, has no side effects, and shows excellent hair loss treatment effect, also for hair growth using the composition for hair growth as an active ingredient. It is to provide a pharmaceutical composition or cosmetic composition for hair growth.

The present invention provides a composition for hair growth comprising a compound selected from the group consisting of cypress essential oil extract, fractions thereof, or cuminol, eucarvone and caramenene separated therefrom. I would like to.

In addition, the present invention is a pharmaceutical for hair growth comprising a compound selected from the group consisting of cypress essential oil extract, fractions thereof, or cuminol, eucarvone and caramenene separated therefrom. To provide a composition or cosmetic composition for hair growth.

In order to solve the problems of the prior art as described above, the inventors of the invention while testing a material having excellent hair growth effect from various natural substances, using cypress essential oil has a therapeutic effect on hair loss, but is safe for the human body and there is no concern about side effects and conventional It was found that the blendability with the cosmetic ingredients for hair is also excellent and the present invention was completed by identifying what compounds constitute the components of the cypress essential oil exhibiting such hair growth effects.

In the present invention, 'Chamaecyparis obtusa' is an evergreen tree of the genus Cypressaceae, also called cypress. Mainly about 40m in height and 2m in diameter, the branches grow horizontally, the branches are drooped, the bark is reddish brown, and torn vertically. The leaves are egg-shaped ridges, thick in quality, the fruit ripens brown in September to October, the flowers bloom in April, and the male and female run on different branches by monogamy, and the male flowers are yellow. It is native to Japan, and because of its good material, it has been planted in Korea as a planting species in Jeju Island and the south coast.

In the present invention, the term 'essential oil' refers to a volatile substance produced by trees and mainly produced in plants, but usually contained in mammary glands or gland hairs or dissolved in resin. Present in the cavity Most of them are obtained from each part of the seed plant, such as flowers, buds, leaves, branches, stems, and roots, and the composition varies depending on the type of plant. Some of the essential oils are capable of sterilizing bacteria, viruses, molds, etc., and provide various functions such as anti-inflammatory, insect repellent, and deodorant. In particular, essential oils, which are volatile components present in trees, have various physiological activities as secondary metabolites of plants, and essential oils extracted from conifers such as pine, pine, cypress, cypress, etc. are effective in lowering blood cholesterol levels. It has been reported.

'Baekbaek essential oil' has been traditionally used for urinary tract infections, and recent studies have shown that it has a strong antibacterial effect, which reduces the effect of atopic itch, in addition to killing bacteria in wounds, promoting blood circulation, and increasing immunity. It is known to have a function of protecting skin and softening the skin.

In the present invention, 'vascular endothelial growth factor (VEGF)' is an important growth factor that forms nitric oxide and prostacyclin, affects cell survival and proliferation and increases blood and production. . VEGF is also involved in cancer and inflammatory responses associated with angiogenesis. The hair growth cycle consists of hair growth, decline, rest and hair loss. In particular, rodent hair growth stage VEGF induces capillary proliferation of hair follicles, and these findings suggest that capillary vessels have an important effect on the hair growth cycle. VEGF gene expression has also been found around human hair follicles. Significant increases in VEGF gene expression are observed in hair follicle hair follicles, while in the resting and hair loss periods the gene expression decreases. VEGF thus acts as an autocrine growth factor for hair follicle cells. VEGF overexpression in hair follicle cells increases the formation of blood vessels around the hair follicle, leading to hair loss and hair growth. Minoxidil, a well-known hair loss prevention, is known to prevent hair loss by increasing the expression of VEGF in artificial hair follicle cells.

In the present invention, 'keratinocyte growth factor (KGF)' is a member of the fibroblast growth factor (FGF) family and is also known as fibroblast growth factor-7 (FGF-7). Members of this family are characterized by their binding to heparin, which plays an important role in mediating the interaction between growth factors and their receptors (Schlessiger et al., Cell 83: 357-360, 1995). KGF was first isolated in 1989. It is unique that KGF is produced by cells of mesenchymal origin but acts first on cells of epithelial origin. KGF stimulates proliferation and differentiation of epithelial cells (Aaronson et al., Annals of the New York Academy of Sciences 638, 62-77, 1991).

In the present invention, 'Cuminol' is known to have antibacterial and antifungal, antioxidant, and anticancer effects, and 'eucarvone' is known as a substance that increases skin penetration, and 'Calamene' ) 'Has been shown to increase the functional maturation and differentiation of human monocyte-derived dendritic cells in the laboratory, and to have an effect on cancer and to inhibit gastric ulcer and anti-inflammatory activity.

The present invention provides a composition for hair growth comprising a cypress essential oil extract as an active ingredient.

The extracting method of the cypress essential oil of the present invention may extract essential oil using a plant essential oil extraction method commonly used in the art, but preferably steam distillation, solvent extraction, leaching, compression, supercritical gas extraction, etc. This can be used, more preferably can be extracted according to the water vapor distillation method (water vapor distillation method).

In one example of the present invention, the leaves, stems, roots, pulp, flowers, fruits, seeds or bark of the cypress, preferably the leaves are shredded so that water, preferably distilled water is 1: 2 to 15 (leaves of cypress: Distilled water), preferably in a ratio of 1: 3 to 10, more preferably in a ratio of 1: 4 to 7, and heated, preferably in accordance with the steam distillation method at 70 to 130 ℃, more preferably 90 to 110 ℃ Can be extracted.

In addition, the present invention provides a composition for hair growth comprising a fraction of the extract of the cypress essential oil as an active ingredient.

The active fraction of the cypress essential oil extract obtained by the steam distillation may be separated by performing a conventional fractionation and purification process in the art, and preferably silica gel column chromatography may be used. The extraction solvent used in the fractionation may be an organic solvent, preferably hexane, chloroform, acetone, ethyl acetate, butanol, ethanol (including alcohol), methanol, lower alcohol or a mixed solvent thereof, more preferably hexane (hexane ), Ethyl acetate or a mixed solvent thereof. Hereinafter, the fractions of the extract of cypress essential oil obtained by extracting hexane and ethyl acetate will be referred to as 'CH-HE' in the present invention.

As an example of the present invention in which the active fraction is separated using the solvent, an appropriate blending ratio of hexane and ethyl acetate is 5 to 35: 1 (v / v), preferably 15 to 25: 1 (v /) based on the volume. v) fractions (hereinafter referred to as 'CH-HE-1') can be obtained using a solvent mixed with v. Hexane when the fraction (CH-HE-1) obtained is fractionated once more The appropriate blending ratio of ethyl acetate and ethyl acetate may be fractionated using a solvent mixed at a volume of 3 to 15: 1 (v / v), preferably 5 to 13: 1 (v / v) (hereinafter referred to as' CH -HE-2 '). When the amount of the extraction solvent is too small, sufficient extraction is not performed. When the extraction solvent is too large, the amount of solvent used may be excessively excessive compared to the extraction solvent efficiency.

As an example of the present invention in which the active fraction is separated using the solvent, an appropriate blending ratio of hexane and ethyl acetate is CH-HE-1-6 using a solvent mixed at a volume of 20: 1 (v / v) based on volume. To CH-HE-1-7, and when the fraction CH-HE-1-7 is fractionated once more, the appropriate mixing ratio of hexane and ethyl acetate is 10: 1 (v / v based on volume). CH-HE-2-A to CH-HE-2-G can be obtained using the solvent mixed with

Extracts obtained according to the above methods CH-HE-1-6, CH-HE-1-7, CH-HE-2-A, CH-HE-2-B, CH-HE-2-C, CH-HE -2-D, CH-HE-2-E, CH-HE-2-F and CH-HE-2-G significantly increase VEGF gene expression as evidenced by the examples below to increase hair growth effects. Preferably, CH-HE-2-A, CH-HE-2-B, CH-HE-2-D, CH-HE-2-E and CH-HE-2-F were 8 compared to the control group. More than double the VEGF gene expression increased hair growth effect, more preferably CH-HE-2-D and CH-HE-2-E showed a hair growth effect by increasing the expression of KGF gene as well as VEGF gene expression. . Therefore, the fraction can be used as an effective active fraction in the composition of the present invention, the extract and fraction inhibits the expression of growth factors expressed during hair growth other than VEGF (Vascular endothelial growth factor) and KGF (keratinocyte growth factor) or As can be increased, which growth factors are expressed does not limit the scope of patent rights of the present invention.

In the present invention, for example, in the case of using DMSO used as a solvent for the protein extract, 0.005% to 0.05%, preferably 0.01% may be added in consideration of the solubility and toxicity of the protein and its fractions.

The present invention provides a composition for hair growth comprising a compound selected from the group consisting of cuminol, eucarvone, and caramenene as an active ingredient.

In this case, the cuminol (Cuminol), eucarvone (eucarvone) or caramenene (calamenene) may be separated from the fraction of the extract of the cypress essential oil, but may be purchased commercially, the present invention is limited by the acquisition route It doesn't work.

As an example of a method of separating the compound from the active fraction of the essential oil extract as described above, CH-HE-2-D and CH-HE-2-E, which is an excellent fraction of the fraction (CH-HE-2), is selected. By gas chromatography and mass spectrometry (GC / MS analysis), each chemical structure can be derived by comparison with standard library data.

In addition, Cuminol, eucarvone or / and caramenene compounds may be synthesized through the synthesis and fractionation of conventional substituents (Herbert O. House: Modern Synthetic Reactions, 2nd ED). , The Benj amin / Cummings Publishing Co., 1972).

 Cuminol, eucarvone or / and caramenene compounds of the invention can be prepared with salts and solvates of such compounds according to methods conventional in the art.

 As the salt of the compound, acid addition salts formed by free acid are useful. Acid addition salts can be prepared by conventional methods, for example by dissolving the compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and acid or alcohol (eg, glycol monomethylether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

Salts of the compounds of the invention include salts of acidic or basic groups which may be present in the compounds of the invention, unless otherwise indicated. For example, salts include sodium, calcium and potassium salts of the hydroxy group; other salts of the amino group include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, Tartrate, lactate, mandelate, methanesulfonate (mesylate), and p-toluenesulfonate (tosylate) salts include, but are not limited to, salts known in the art and may be prepared by methods or processes for preparing salts known in the art. have.

 The present invention is to provide a pharmaceutical composition for hair growth comprising an extract of cypress essential oil, a fraction thereof, or a compound separated therefrom as an active ingredient.

The cypress essential oil extract exhibits excellent hair growth effect with little side effects or toxicity, and thus may be useful for promoting hair growth or preventing hair loss in humans and animals with alopecia.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, dogs, livestock, humans, or the like by various routes such as oral or parenteral, and can be administered by injection such as oral, rectal or intravenous, intramuscular, subcutaneous, and the like. . Preferably it is applied in the form of topical application during parenteral administration, more preferably by application.

Compositions containing the extracts of cypress essential oil according to the present invention are conventional agents in the pharmaceutical field, for example tablets, capsules, troches, solutions, suspensions, according to conventional pharmaceutical methods when used for hair growth promotion and hair loss prevention. Injectable preparations, ointments, creams, lotions, etc., in the form of oral preparations such as preparations, injectable solutions or suspensions, or ready-to-use injectable dry powders which can be prepared by injection with distilled water for injection. It can be formulated into various formulations such as topical formulations such as liquids and sprays.

Carriers that can be used for the formulation of the compositions of the invention are customary in the pharmaceutical art, for example in the case of oral preparations binders, suspending agents, disintegrating agents, excipients, solubilizers, dispersants, stabilizers, Pigments, flavoring agents, and the like. In the case of injections, preservatives, analgesics, solubilizers, stabilizers, and the like, and topical administration preparations include bases, excipients, lubricants, preservatives, and propellants. Pharmaceutical preparations thus prepared can be administered orally or topically. The composition of the present invention is preferably formulated into topical formulations, such as ointments, lotions, sprays, and the like, and is directly applied to the hair loss site.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, .

The application amount of the composition according to the present invention is appropriately selected depending on the type and severity of the alopecia, the condition of the patient, etc., but generally 0.0001 to 10000 mg (as dry matter) of the extract of the white sperm oil per kg body weight per day in adults, Preferably it can be administered in an amount of 0.001 to 300mg (as a dry matter), it can be administered once to several times a day, and in the case of external preparations once a day in an amount of 1.0 to 6.0 ml per day on an adult basis Apply five times and continue for at least one month. However, the dosage does not limit the scope of the present invention.

In addition, the present invention is to provide a cosmetic composition for hair growth comprising a extract of cypress essential oil, fractions thereof, or a compound separated therefrom as an active ingredient.

The components included in the cosmetic composition of the present invention include components conventionally used in cosmetic compositions in addition to the cypress essential oil as an active ingredient, and include, for example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings, and Carrier. In addition, the cosmetic composition may further include a skin absorption promoting substance to improve its effect.

 In the composition of the present invention, the cypress essential oil extract is preferably contained in 0.0001 to 99% by weight, in particular 0.001 to 90% by weight.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, the composition of the present invention further contains a cosmetically acceptable carrier, such as creams, lotions, tonics, sprays, It may be prepared in conventional formulations such as aerosols, oils, solutions, suspensions, gels, ointments, emulsions, waxes, pastes, cosmetic liquids, hair cosmetics and the like. The compositions of the present invention can be prepared using any of the devices and methods commonly used or well known in the pharmaceutical or cosmetic manufacturing arts (e.g., Remington's Pharmaceutical Science, latest edition).

The composition of the present invention usually contains a base, and other ingredients generally used as cosmetic raw materials may be appropriately blended as necessary. The base of the composition of the present invention may be used as long as it is commonly used, examples thereof include purified water, mineral water, ethanol, glycerin, squalene, 1,3-propylene glycol, 1,3-butylene glycol, castor oil, Tsubaki oil and liquid petrolatum. Examples of other components to be incorporated into the composition of the present invention include, but are not limited to, surfactants, emulsifiers, thickeners, preservatives, antioxidants, fragrances. The surfactant can be a cationic surfactant, anionic surfactant or nonionic surfactant. Examples of cationic surfactants include alkali salts of higher fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid and oleic acid (eg, sodium salts, potassium salts, ammonium salts, triethanolamine salts); Alkyl sulfate ester salts such as sodium lauryl sulfate and lauryl sulfate triethanolamine; And alkyl ether sulfate ester salts such as polyoxyethylene lauryl ether sulfate and polyoxyethylene lauryl ether sulfate triethanolamine. Examples of the nonionic surfactant include polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether and polyoxyethylene oleyl ether; And alkanolamides such as palm oil fatty acid diethanolamide and lauric acid diethanolamide. Examples of anionic surfactants may include stearyltrimethyl ammonium chloride, cetyltrimethyl ammonium chloride and stearylbis (diethylalcohol) hydroxyethyl ammonium chloride.

 Examples of emulsifiers include cetanol, stearyl alcohol and behenyl alcohol. Examples of thickeners may include sodium alginate, methyl cellulose, carboxymethyl cellulose, polyvinyl alcohol and polyvinyl pyrrolidone.

 Examples of preservatives include ethyl parahydroxybenzoate, butyl parahydroxybenzoate and benzalkonium chloride. Examples of antioxidants may include butyl hydroxytoluene, propyl gallate and butyl hydroxy anisole. The fragrance may be a parfum mainly used for general purposes, examples may include citrus, lavender, floral.

 The composition of the present invention, in addition to the above components from monosaccharides such as glucose, xylose, mannose, arabinose and the like, disaccharides such as maltose, sucrose, cellobiose, trehalose, etc. It may further comprise one or more, the amount thereof is preferably 0.01 to 5% by weight of the total composition. In addition, the composition of the present invention may include hair growth promoting auxiliary components commonly used, for example pepper tink, vitamin E (tocopherol), vitamin B6 (pyridoxine), cysteine, pantothenic acid, salicylic acid, tocopherol acetate, l-menthol, grapefruit seed extract, and the like, but are not limited to these. The amount thereof is preferably 0.0001 to 10% by weight of the total composition.

 There is no particular limitation on the application of the composition of the present invention, the composition of the present invention can be applied to all animals, including humans as well as animals, for example, breeding animals or pets. The compositions of the present invention can be used by methods such as hair or scalp, direct application or spreading on the skin. Subjects to which the compositions of the present invention are applied include hair follicles and hair follicles of the head, hair and eyelashes and cilia, beards, armpits, pubic hair, and all parts of the hair follicles and hair follicles throughout the body. Accordingly, the present invention provides a hair tonic, hair lotion, hair cream, hair spray, hair mousse, hair gel, hair conditioner, hair shampoo, hair rinse, hair pack, hair treatment, eyebrow hair restorer, eyelash hair restorer or eyelash nutrient, or pet It can be used as a shampoo and pet rinse.

 The amount and frequency of use according to the present invention should be appropriately determined in consideration of sex, age, hair loss, hair condition, and the like.

As described above, the composition comprising the extract of cypress essential oil, fractions thereof, or a compound separated therefrom as an active ingredient can be used safely without cytotoxicity and skin side effects, so it is useful as a hair growth composition or as an additive of an existing hair growth agent. It can be used, and the composition can be usefully used as a pharmaceutical composition for hair growth or cosmetic composition for hair growth having hair loss prevention, improvement and hair growth promoting activity as an active ingredient.

FIG. 1 shows chemical structures of cuminol, eucarvone and caramenene.
Figure 2 is to examine the effect of cypress essential oil on the hair growth effect in the mouse. The hair of the mouse is pushed and the hair grows every 10 days. The picture above does not apply the extract of the essential oil, and the picture below shows the extract of the extract of essential oil.
Figure 3A is a 24 hours treatment of various concentrations of DMSO to HaCaT cells to confirm the survival rate in DMSO and Figure 3B confirms the highest solubility and toxicity of the cypress essential oil and its fractions. In this case, Fraction 1 to Fraction 7 means CH-HE-1-1 to CH-HE-1-7.
Figure 4 is to examine the growth factor expression in HaCaT cells by applying the primary fractions (CH-HE-1-1 to CH-HE-1-7) of the extract of the white sperm oil.
Figure 5 is applied HaCaT by applying the primary fractions (CH-HE-1-6, CH-HE-1-7) and secondary fractions (CH-HE-2-A to CH-HE-2-G) of the extract Growth factor expression was examined in cells.

Hereinafter, the present invention will be described in more detail with reference to Examples, Experimental Examples, and Production Examples. However, the following Examples, Experimental Examples and Preparation Examples are for exemplifying the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples, and Preparation Examples or the scope of the present invention is limited.

Example

Example 1 Extraction Method of Cypress Oil

The cypress essential oil of the present invention was extracted according to the water vapor distillation method. The leaves of cypress and distilled water were mixed in a ratio of 1: 5, placed in a round flask, and placed in a heating mantle. Then, the heating mantle was heated to a temperature of 100 ° C. As the temperature rises, volatile components and water vapor are mixed and evaporated from the heated leaves and distilled water of the cypress and condensed in a condenser connected to a round flask, which is divided into a water layer and a volatile component layer. It was used as the test of the present Example just as a white powder essential oil.

Example 2 Fraction of Cypress Essential Oil

The cypress essential oil extract was fractionated by silica gel column chromatography. Essential oil extract (240.40 g) was added to a silica gel column (1529.5 cm, # 1.07734.9025, Merck, Darmstadt, Germany) and eluted with hexane and ethyl acetate (20: 1, v / v) at 20 ml / min. The final 527 fractions were collected using Erlenmeyer flasks (# A0205, Dong-Sung Science, Bucheon, Korea,) at a volume of 100 ml. Finally, the residue was eluted with ethyl acetate to prepare fraction 528 (CH-HE). Thin layer chromatography (TLC, Thin Layer Chromatography, plate # 1.05715.0001, Merck) using 50% sulfuric acid and 50% hexane: ethyl acetate (8: 1, v / v) for each fraction (CH-HE) ) And the fractions were divided into seven groups according to the components. After concentrating with a concentrator, the first fraction CH-HE-1-1 to CH-HE-1-7 was obtained.

The primary fraction CH-HE-1-7 was further fractionated using silica gel column chromatography. CH-HE-1-7 fraction (5 ml) was added to a silica gel column (4.531 cm) and eluted with hexane and ethyl acetate (10: 1, v / v) at 2 ml per minute. 250 ml of fractions were collected in Erlenmeyer flasks, and a total of 43 fractions (CH-HE-2) were obtained. The silica gel column residue was re-eluted with 1 litter of ethyl acetate to obtain the 44th fraction. Each fraction was visualized by TLC using 50% sulfurinc acid and 50% hexane: ethyl acetate (8: 1, v / v), and then grouped into seven groups (A to G). Divided. After concentration with a concentrator, secondary fractions CH-HE-2-A to CH-HE-2-G are prepared.

Example 3 Active Compounds from Cypress Extract

The active fraction of the essential oil was subjected to gas chromatography and mass spectrometry (GC / MS analysis). GC (#HP 6890, Hewlett Packard, Santa Clara, USA) and DB-5 MS column (30 mx 0.25 mm id, 0.25 um film thickness, J & W Scientific, Folsom, USA) were used and the insertion and detection temperatures were respectively 250 ° C and 280 ° C. Helium was used as the carrier gas at a rate of 1 ml per minute. After holding the initial oven temperature at 70 ° C. for 10 minutes, the temperature was increased by 5 ° C. per minute to reach a final 280 ° C. After holding the initial oven temperature at 70 ° C. for 10 minutes, the temperature was increased by 5 ° C. per minute to reach a final 280 ° C. MS (#HP 5973N, Hewlett Packard) was used in EI mode (electron energy, 70 eV; scan range, 35-600 amu; source temperature, 230 ° C). Each chemical structure was derived by comparison with standard library data.

Constituents of the Secondary Fraction of CHB-HE-2-D from GC / MS Analysis RT Area (%) CAS No. MW Library Quality 3.86 1.17 123-42-2 116.16 Hydroxy-4-methyl-2-pentanone 83 17.11 0.59 4621-04-09 142.24 4- (1-methylethyl) -Cyclohexanol 83 18.54 0.4 122-00-9 134.18 1- (4-methylphenyl) -Ethanone 97 18.67 5.59 768-91-2 150.26 1-Methyladamantane 83 20.13 0.28 106-22-9 156.27 Citronellol 97 20.95 0.62 106-24-1 154.25 Geraniol 93 22.41 5.51 536-60-7 150.22 Cuminol 98 22.94 8.23 503-93-5 150.22 Eucarvone 91 23.54 0.73 22539-72-6 152.23 p-Mentha-1,4-dien-7-ol 90 24.73 0.51 5580-33-6 149.19 3,4-Dimethylbenzamide 89 29 0.24 483-77-2 202.34 (-)-Calamenene 96 29.53 0.41 21391-99-1 200.32 Calacorene 93 29.68 0.41 639-99-6 222.37 Elemol 93 32.12 0.3 4630-07-3 204.35 (+)-Valencene 93 32.21 0.91 87-85-4 162.27 Hexamethylbenzene 95 46.65 0.26 103-23-1 370.57 Di (2-ethylhexyl) adipate 93 48.95 0.21 27554-26-3 390.56 Diisooctyl phthalate 91

Constituents of the Secondary Fraction of CHB-HE-2-E from GC / MS Analysis RT Area (%) CAS No. MW Library Quality 10.88 0.43 99-87-6 134.22 Cymene 95 18.99 0.31 1197-01-9 150.22 Cymene-8-ol 91 22.36 0.48 536-60-7 150.22 Cuminol 98 22.59 4.1 30434-64-1 110.15 3,4-dimethyl-2-Cyclopenten-1-one 87 22.88 2.09 503-93-5 150.22 Eucarvone 90 29.01 1.02 483-77-2 202.34 (-)-Calamenene 98 30.06 0.46 30364-38-6 170.27 1,1,6-Trimethyl-1,2-dihydronaphthalene 86 32.74 0.59 483-78-3 198.3 Cadalene 99

Constitution of CH-HE-2-D and CH-HE-2-E, which are fractions showing the hair growth effect found by the following examples among the secondary fractions (CH-HE-2-A to CH-HE-2-G) The results of analysis of min by GC / MS are shown in Table 1 and Table 2, and the secondary fraction CH-HE-2-D consists of 17 substances, and the main substances were 1-methyladamantane (5.59%), cuminol ( 5.51%), eucarvone (8.23%) (Table 1). Secondary fraction CH-HE-2-E consists of 10 substances. The major components are 3,4-dimethyl-2-cyclopenten-1-one (4.1%), eucarvone (2.09%) and (-)-calamene (1.02%) (Table 2). Common compounds of these two secondary fractions were found to be cuminol, eucarvone and caramenene. The chemical formulas of these compounds can be found in FIG.

Example 4 Data Analysis

All data were analyzed using a nonparametric one-way analysis-of-variance method using the Kruskal Wallis test (followed by Dunnett's test for multiple comparisons to vehicle). All data values were statistically significant when P <0.05 using SPSS for Windows Edition (SPSS Inco, Chicago, IL, USA).

Experimental Example

Experimental Example 1 Hair Growth Effect by Cypress Essential Oil

Six-week-old C57BL / 6 mice were taken from Coatech (Pyeongtaek-si, Gyeonggi-do), followed by a period of approximately one week of gestation, and general health conditions were used to study only healthy animals. The hair follicles of the 7 week old mice are at rest. After removing the back hair of the mouse using a hair removal agent and an animal clipper, 1% Cypress essential oil is dissolved in 40% ethanol and used as a test drug. Experimental and control (40% ethanol) 0.2ml each day for 30 days percutaneous application was evaluated for each hair growth effect. Gross hair growth effects were photographed by anesthetizing Ketamine with Xylazine after 0, 10, 20 and 30 days from the start of drug treatment. Visually evaluate the score of the area of hair growth.

As shown in FIG. 2, the difference between the treatment group and the untreated group was clearly revealed from the 10th day of the drug treatment, and the extract of the extract was excellent in the hair growth effect. At this time, the weight of the treated group and the control group was constant during the application period of the protein, and no abnormal reaction could be observed, and it was confirmed that the difference in hair growth was due to the protein.

Example 2 Survival Test of HaCaT Cells by Cyclones and Fractions thereof

 Human keratinocytes (HaCaT, Dermatology College of Medicine, Chungnam National University) were penicillin and streptomycin (Gibco / Invitrogen) and 10% fetal bovine serum (DMEM, Gibco / Invitrogen, Co., Carlsbad, CA, USA) medium in Dulbecco's Modified Eagle Media. FBS; Gibco / Invitorgen) is added and cultured.

First, the survival rate of HaCaT cells by DMSO used as a solvent for protein conversion is examined, and the survival rate of the treated group is indicated by considering the cell survival rate of the untreated group in DMSO considered as the optimal concentration as 100%.

Cytotoxicity test was performed using Cell Counting Kit-8 (CCK-8, Dojindo Lab., Tokyo, Japan). One day prior to drug treatment, 2500 cells are mixed in DMEM (containing 10% FBS) medium and incubated in 96-well plates. After 24 hours, replace the plate with 100ul medium containing cypress essential oil and its fractions. After another 24 hours of incubation, 10ul of CCK-8 was added. After incubation in a CO ₂ incubator for 3 hours, the absorbance is measured at a wavelength of 450 nm / 600 nm in a microplate reader.

As shown in Figure 3A, as a result of 24 hours of treatment with various concentrations of DMSO HaCaT cells, a strong reduction in survival was observed between DMSO 3.1 and 6.5%. As a result, the final concentration of DMSO was chosen as 2%.

As shown in FIG. 3B, considering the highest solubility and toxicity of the cypress essential oil and its fractions, it was found that it is optimal to add the cypress essential oil and its fractions in DMSO at 0.01%. This study was conducted to determine the optimal treatment concentration of cypress essential oil and its fractions.

Experimental Example 3 Effects of Cypress Oil and Its Fraction on the Growth Factor Expression

Experimental Example 3-1 Cell Culture

Human keratinocytes (HaCaT, Department of Dermatology, College of Medicine, Chungnam National University) were penicillin and streptomycin (Gibco / Invitrogen) and 10% fetal bovine serum (DMEM, Gibco / Invitrogen, Co., Carlsbad, CA, USA) medium in Dulbecco's Modified Eagle Media. FBS; Gibco / Invitorgen) is added and cultured. In the hair growth functional gene test of cypress essential oil and its fractions, 0.3 X 10 ⁶ cells are cultured in 6-well plates. After drug treatment, the effect of each drug after 24 is analyzed using real-time PCR.

Experimental Example 3-2: Relative mRNA Quantitation by Real-time PCR

 To examine the molecular mechanism of the hair growth effect of cypress essential oil, growth factor expression in HaCaT cells was assayed using real-time PCR.

Gene expression of the five growth factors (VEGF1, TGFβ1, KGF, IGF, HGF) was examined in untreated HaCaT cells of the E. coli essential oil extract and the expression of the growth factor after application of the E. coli essential oil extract or fractions thereof.

Intracellular total RNA was prepared using TRIzol reagent (Invitrogen, Co.,) and the concentration was measured using a 260 nm absorbance meter. RT-PCR uses existing experimental methods. Briefly, 1 ug of total RNA was synthesized using a first strand complementary DNA (M-MLV reverse transcriptase (Invitrogen, Co.) and random primer (9-mer, TaKaRa Bio., Inc, Otsu, Shiga, Japan)). cDNA). Genes of interest include vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGFβ1), keratinocyte growth factor (KGF), insulin-like factor-1 (IGF-1), hepatocyte growth factor (HGF) and control genes. GAPDH was used. Real-time PCR consists of 10 ul of TaqMan Universal PCR Master Mix (Applied Biosystems, Foster, CA) and 1 ul of 20X Assays-on-DemandTM Gene Expression Assay Mix (Applied Biosystems, (VEGF; Hs00173626_m1, TGFβ: Hs00171257_m1, KGF: Hs00173565_m1) , IGF-1: Hs00153126_m1, HGF: Hs00300159_m1, GAPDH: Hs00266705_g1), and 5ul cDNA were mixed and reacted with a total of 20ul. Perform 10 min pre-incubation at ° C. Perform PCR for a total of 40 cycles, each cycle was performed at 95 ° C. for 15 seconds and at 60 ° C. The relative target gene expression was determined by GAPDH expression. After leveling, relative percentages were used All real-time analyzes were performed using RQ software (Applied Biosystems).

As a result, it was found that VEGF and KGF genes are expressed in HaCaT cells. As shown in Figure 4, after treatment of the protein and its fractions, the expression of growth factors in HaCaT cells were examined, the fractions of CH-HE-1-6 and CH-HE-1 than those measured whole whole protein The expression of VEGF gene was significantly increased at -7, but not changed in other fractions and protein. As shown in FIG. 5, the primary protein fraction CH-HE-1-7 showing the best results was separated into seven secondary fractions (CH-HE-2-A to CH-HE-2-G). Later, when treated with HaCaT cells, CH-HE-2-A to CH-HE-2-G increased the expression of VGEF gene of CH-HE-1-7 or higher, and among them, secondary CH-HE-2. -A, CH-HE-2-B, CH-HE-2-D, CH-HE-2-E, CH-HE-2-F increased VGEF gene expression by at least 8-fold compared to the control. Secondary fractions CH-HE-2-D and CH-HE-2-E also significantly increased KGF gene as well as VEGF. GC / MS analysis confirmed the composition of this secondary fraction, the results are shown in Example 3 and FIG.

Formulation example

Hereinafter, an example of preparation of a cosmetic composition for hair growth comprising an extract of a cypress essential oil of the present invention, a fraction thereof, or a compound separated therefrom as an active ingredient will be described, but the present invention is not intended to be limited thereto. to be.

Formulation Example 1. Softening Longevity (Content: Weight%)

 Cypress Essential Oil Extract (or Fractions thereof) 0.01

 Glycerin 3.0

 Butylene Glycol 2.0

 Propylene Glycol 2.0

 Carboxy Vinyl Polymer 0.1

 Ethanol 10.0

 Triethanolamine 0.1

 Preservatives, trace pigments, trace fragrances, trace amount of purified water

Total 100.0

 Formulation Example 2 Nutrients (Content: Weight%)

 Cypress Essential Oil Extract (or Fractions thereof) 0.01

 Beeswax 4.0

 Polysorbate 60 1.5

 Sorbitan Sesquioleate 0.5

 Liquid Paraffin 5.0

 Squalane 5.0

 Caprylic / Capric Triglycerides 5.0

 Glycerin 3.0

 Butylene Glycol 3.0

 Propylene Glycol 3.0

 Carboxy Vinyl Polymer 0.1

Triethanolamine 0.2

Preservatives, trace pigments, trace fragrances, trace amount of purified water

Total 100.0

Formulation Example 3 Nutrition Cream (Content: Weight%)

 Cypress Essential Oil Extract (or Fractions thereof) 0.01

 Beeswax 10.0

 Polysorbate 60 1.5

 Sorbitan Sesquioleate 0.5

 Liquid Paraffin 10.0

 Squalane 5.0

 Caprylic / Capric Triglycerides 5.0

 Triethanolamine 0.2

 Preservatives, trace pigments, trace fragrances, trace amount of purified water

Total 100.0

Formulation Example 4 Preparation of Hair Tonic (Content: Weight%)

 Cypress Essential Oil Extract (or Fractions thereof) 10.0

 Resorcinol 0.1

 Menthol 0.05

 Panthenol 0.2

 Salicylic Acid 0.1

 Tocopheryl Acetate 0.1

 Pyridoxine hydrochloride 0.1

 Castor Oil 5.0

 Pigment amount

 Spices

 Ethanol

 Purified water balance

Total 100.0%

Formulation Example 5 Preparation of Hair Conditioner (Content: Weight%)

 Mixture of Cuminol, eucarvone and caramenene 0.5

 Cetanol 3.5

 Self-emulsifying glycerin monostearate 1.5

 Propylene Glycol 2.5

 Stearylmethylbenzyl Ammonium Chloride (25%) 7.0

 Methyl Paraoxybenzoate 0.3

 Pigment amount

 Spices

Purified water balance

Total 100.0%

Formulation Example 6 Preparation of Hair Lotion (Content: Weight%)

Cypress Essential Oil Extract (or Fractions thereof) 5.0

Resorcinol 2.0

Menthol 2.0

Panthenol 0.5

Pyroxtonolamine 0.1

Fragrance, pigment 0.5%

Purified water balance

Total 100.0%

Formulation Example 7 Preparation of Hair Soap (Content: Weight%)

Cypress Essential Oil Extract (or Fractions thereof) 0.1

Titanium Dioxide 0.2

Polyethylene Glycol 0.8

 Glycerin 0.5

Ethylenediaminetetraacetic Acid 0.05

Sodium 1.0

Pigment amount

Soap flavor

Cosmetic soap base (moisture 13, parts by weight)

Total 100.0

Hereinafter, a preparation example of a hair growth pharmaceutical extract of the present invention or a fraction thereof, or a compound separated therefrom as an active ingredient will be described. However, the present invention is not intended to be limited thereto, but is intended to be described in detail only. to be.

Formulation Example 1 Preparation of Ointment

Cypress Essential Oil Extract (or Fractions thereof) 3g

Despanthenol 1.5g

1.0 g stearic acid

5.0g of liquid paraffin

4.0 g of light solder

Cetanol 3.0 g

Propylene Glycol 13.0g

1.5 g triethanolamine

Dibutylhydroxytoluene 0.025g

Ethylbenzoate 0.0225 g

0.015 g of propylbenzoate

0.1g polysorbate

65.0 g of purified water

The ointment was prepared according to the conventional method for preparing the ointment using the above components.

The ointment prepared by the above method is applied twice a day, 0.5 to 1 g at a time continuously for 3 to 6 months. The total daily weight should not exceed 2 g. After application to the affected area, it is preferable to massage gently to promote absorption.

Formulation Example 2 Lawson

1.0 g of cypress essential oil extract (or fractions thereof)

Dexpanthenol 1.5g

Glycerin 0.6g

Hydroxypropylcellulose 0.085 g

Sword 0.0255g

Purified water quantity

Using the above ingredients, the lotion was prepared according to the conventional method for preparing the lotion.

The lotion agent prepared in the above manner is applied twice a day, 0.5 to 1 ml at a time continuously for 3 to 6 months. The total daily amount should not exceed 2 ml.

Formulation Example 3 Spray

3.0 g of essential oil extract (or fractions thereof)

Dexpanthenol 1.5g

Propylene Glycol 20.0g

Ethanol 65ml

Purified water quantity

Using the above ingredients, a spray was prepared according to a conventional method for preparing a spray.

Spray the spray agent prepared by the above method 4 times or 8 times in one time (approximately 0.12ml injection), and when the amount of remaining liquid in the container is not good, it is difficult to spray the chemical liquid 21 times or 42 times in the hair loss site with a brush. Apply After application, it is desirable to massage gently to promote absorption.

Claims (17)

A pharmaceutical composition for hair growth comprising at least one compound selected from the group consisting of cuminol, eucarvone and caramenene as an active ingredient. The method of claim 1,
The compound is a hair growth pharmaceutical composition, characterized in that obtained by extraction and separation from cypress essential oil. delete delete delete delete delete delete delete The pharmaceutical composition for hair growth according to claim 1 or 2, wherein the composition is formulated as a topical formulation. The method of claim 10,
The formulation is a pharmaceutical composition for hair growth, characterized in that the ointment, lotion or spray. delete Hair growth cosmetic composition containing as an active ingredient at least one compound selected from the group consisting of cuminol, eucarvone and caramenene. The method of claim 13,
The compound is a cosmetic composition for hair growth, characterized in that obtained by extracting and separating from cypress essential oil.
delete delete The method according to claim 13 or 14,
The composition includes hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nourishment cream, hair moisturizer cream, hair massage cream, hair wax, hair aerosol, Hair Pack, Hair Nutrition Pack, Hair Soap, Hair Cleansing Foam, Hair Oil, Hair Dryer, Hair Preservative, Hair Dye, Hair Wave, Hair Bleach, Hair Gel, Hair Glaze, Hair Dresser, Hair Lacquer, Hair Moisturizer, Hair Cosmetic composition for hair growth, characterized in that the formulation selected from the group consisting of mousse or hairspray.

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