KR102353372B1 - Composition for preventing hair loss or promoting hair growth comprising extract pupae of male bees - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing hair loss or promoting hair growth, comprising an extract of the pupa of larvae.
Since the composition containing the pupa extract is recognized for its efficacy in inhibiting and preventing hair loss and promoting hair growth, it can be used in various fields such as pharmaceuticals, cosmetics, food, quasi-drugs, and beauty.

Description

A composition for preventing hair loss or promoting hair growth comprising an extract of a caterpillar pupae {Composition for preventing hair loss or promoting hair growth comprising extract pupae of male bees}

The present invention relates to a composition for preventing hair loss or promoting hair growth, comprising an extract of a honey bee pupa.

Alopecia, in which hair falls out from the scalp, is caused by various factors, and internal factors such as genetic constitution or the action of male hormones, or external factors such as mental stress in daily life and accumulation of lipid peroxide in the scalp It is known that these factors are involved in a complex manner and cause hair loss symptoms. Recently, as well as male pattern hair loss, hair loss in young people, including obese hair loss in women, is gradually spreading. In order to improve this hair loss phenomenon, many types of hair growth or hair restorers are on the market.

Hair growth can be divided into an anagen phase, a catagen phase, and a telogen phase. It atrophys rapidly and the growth activity of the hair follicle stops, and during the resting period, the growth activity of the hair follicle stops completely. At this time, new growth phase hair is formed at the base of the follicle in the resting phase, and the existing hair in the resting phase is pushed and detached by the newly emerged hair in the growth phase or is detached by a mechanical action such as combing.

Therefore, in order to obtain an effective hair loss prevention and wool effect, it should be possible to simultaneously exert an anagen-inducing effect that promotes the transition from the resting phase to the anagen phase and a degenerative-phase inhibitory effect that delays the transition from the anagen phase to the catagen phase.

The main ingredients used in commercially available hair growth or hair restorers include vasodilators such as capronium chloride and minoxidil to circulate sufficient blood to the scalp, or to inhibit the action of male hormones that attach to the dermal papilla and cause hair loss. There are female hormones such as estrogen, estraziol, and progesterone for this purpose, or male hormone activation inhibitors such as pentadecanoic acid and finasteride. For example, there are two types of drugs approved by the U.S. Food and Drug Administration (FDA) as a treatment for hair loss. Minoxidil, a topical treatment, is due to its vasodilatory action, and Propecia, which is for oral use, has finasteride as the subject matter of inhibiting the activation of male hormones. Therefore, it is widely used as a formulation with excellent anti-hair loss effect.

However, in the case of minoxidil, not only does it depend on imports of raw materials from overseas, but it also takes a long time for skin absorption, hair loss recurs when use is stopped, and there is a disadvantage that there is little treatment effect after hair loss has completely progressed, Serious problems with existing products are being highlighted, such as reports that Propecia is prescribed only to adult males and can cause a decrease in sperm count and sexual dysfunction. Moreover, since the mechanism of action of most of the existing hair loss treatment agents is deeply related to male hormones, the efficacy does not appear for women, or it contains serious problems that may cause birth defects. Therefore, in recent years, due to these problems, attention has been focused on natural ingredients for promoting hair growth and preventing hair loss.

Republic of Korea Patent No. 10-1431965

It is an object of the present invention to provide a pharmaceutical composition for preventing hair loss or promoting hair growth, comprising an extract of the pupa of the present invention.

Another object of the present invention is to provide a composition for external application for skin for preventing hair loss or promoting hair growth, which includes an extract of the pupa of the present invention.

Another object of the present invention is to provide a cosmetic composition for preventing hair loss or promoting hair growth, comprising an extract of a wasp pupa.

Another object of the present invention is to provide a quasi-drug composition for preventing hair loss or promoting hair growth, comprising an extract of the pupa of the present invention.

Another object of the present invention is to provide a food composition for preventing hair loss or promoting hair growth, comprising an extract of a honey bee pupa.

As one aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing hair loss or promoting hair growth, comprising an extract of the honey bee pupa.

As used herein, the term "bee pupa" is obtained by producing 16 to 20-day-old bee pupa from April to early September 2016 using Western species of bees whose scientific name is Apis mellifera.

As used herein, the term "extract" refers to an extract obtained by extraction treatment, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a prepared or purified product of the extract, or a mixture thereof, such as the extract itself and the extract Includes extracts of all formulations that can be formed using

Drying of the extract in the present invention may be carried out by a known method as long as useful components are not destroyed from the honey bee pupa, for example, it may be carried out by a method of natural drying in the shade, or it may be carried out in a freeze-drying or dry oven. In addition, crushing or pulverization can be pulverized by crushing or pulverizing enough to sufficiently extract useful components of the honey bee pupa in the subsequent extraction process. The drying, crushing, or pulverizing processes may be performed in reverse order or repeated if necessary.

In the extraction of the present invention, the extraction method is not particularly limited, and extraction may be performed according to a method commonly used in the art.

In the present invention, the honey bee pupa extract is water, C ₁ to It can be obtained by extraction with a C _{4 lower alcohol or a mixed solvent thereof.} In addition, the lower alcohol may be ethanol or methanol. More specifically, it may be a 50% ethanol extract of honey bee pupa.

In the present invention, the term "hair loss" refers to a phenomenon in which hair falls off from the scalp or a condition in which hair is sparse or thin, and prevention of hair loss means to prevent and suppress the phenomenon of hair loss as described above, and promote hair growth This means not only promoting the generation of new hair, but also making the existing hair grow healthy.

In general, hair growth occurs in the growth phase, and is promoted by induction from the resting phase to the growth phase and delay from the growth phase to the regression phase. The hair cycle can be divided into three main stages known as growth phase, catagen phase, and telogen phase. During the growth phase, hair follicles grow deep into the skin along with rapid cell proliferation and hair formation occurs. The next phenomenon is the catagen stage, which is a transition period in which cell division is markedly interrupted. In this process, hair follicles gradually degenerate and hair growth is stopped. In the next phase, the telogen, the degenerative hair follicles contain germs with densely packed dermal papilla cells. The initiation of new anagen phenomena in the resting phase is induced in the embryo by rapid cell proliferation, expansion of the dermal papilla and the synthesis of basement membrane elements.

Therefore, it is necessary to prevent hair loss, that is, prevent hair loss, or induce hair regrowth, that is, promote hair growth by promoting or extending the growth phase. The composition of the present invention includes at least one of an effect of preventing existing hair from falling out, an effect of improving existing hair such as thickening, and an effect of generating new hair. Dermal papilla cells are mesodermal-derived fibroblasts located at the base of hair follicles and are thought to play an important role in hair growth through interaction with the matrix cells of the hair follicles (Jahoda CA, et al., 1984; Oliver RF, et al., 1986). In addition, it has been found that finasteride, a type 2 5α-reductase inhibitor, improves hair loss by reducing DHT in hair loss patients (Van Neste et al., 2000), and this fact indicates that 5α-reductase is an important target for hair loss treatment. tell me

In one embodiment of the present invention, it was confirmed that the pupa extract restores the growth of dermal papilla cells reduced by DHT (5α-dihydrotestosterone) treatment, which induces male pattern hair loss in HFDPC (Human Follicle Dermal Papilla Cells), which are human papilla cells. . In addition, it was confirmed that the male pupa extract inhibited male pattern hair loss by restoring cAMP activity and IGF-1 production involved in hair growth and inhibiting TGF-β1 production. In addition, it was confirmed that the apoptosis inhibition rate was improved by 23.6% by the treatment of the honeysuckle pupa extract in the hair cells (HaCaT) treated with LPS to induce inflammatory hair loss, and the expression of inflammatory cytokines TNF-α and IL-6 It was confirmed that inflammatory hair loss was inhibited by inhibiting the activity of caspase-3 that induces cell death of hair follicles. In addition, in order to evaluate the efficacy of wool, mitosis inhibitor (TBM, tolbutamide) was treated to fibroblasts (NIH3T3), and then the apoptosis inhibition rate of fibroblasts (NIH3T3) was improved by 25.9% by the treatment of the honey bee pupa extract. It was confirmed, and it was confirmed that the pupa extract had an effect on promoting hair growth by increasing the production of collagen type I related to the effect of wool.

Therefore, the composition containing the male pupa of the present invention can prevent hair loss and induce hair growth by activating dermal papilla cells, hair cells, and fibroblast cells, and suppress male pattern hair loss and inflammatory hair loss and have a hair growth (hair growth) promoting effect. have.

In addition, in an embodiment of the present invention, it was confirmed that the pupa extract has an activity of scavenging DPPH and ABS radicals, and the composition including the extract of pupa of the present invention also has antioxidant activity.

The honeysuckle pupa extract may be included in an amount of 0.00001 wt% to 30 wt% based on the total weight of the pharmaceutical composition, specifically, 0.0001 wt% to 10 wt%. More specifically, it is 0.002% by weight to 3% by weight.

For administration, the composition of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredients described above.

The carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention may be administered orally or parenterally during clinical administration, but may be administered topically directly to the skin as an external preparation in general.

In addition, when the pharmaceutical composition of the present invention is formulated, it may be formulated using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant.

Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations include at least one excipient in the active ingredient of the present invention, for example, starch, calcium carbonate, sucrose, lactose and It is prepared by mixing gelatin, etc. In addition to simple excipients, lubricants may also be used. Liquid formulations for oral administration include suspensions, solutions, emulsions and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances and preservatives are used. may be included. Formulations for parenteral administration include topical formulations such as creams, lotions, ointments (semi-solid external preparations), microemulsions, gels, pastes, and transdermal preparations (TTS).

The composition of the present invention can be used by methods such as applying or dispersing directly to the hair or scalp.

The hair to which the composition of the present invention is applied includes hair roots and follicles of the head, hair and eyelashes and eyebrows, beard, armpits, pubic hair, and all parts of the body having hair roots and follicles.

A suitable dosage of the composition of the present invention may vary depending on the patient's condition and weight, the degree of disease, drug form, and time, but may be appropriately selected by those skilled in the art. In addition, if necessary, it can be administered by applying 1 to 5 times a day.

As another aspect, the present invention provides a composition for external application to the skin for preventing hair loss or promoting hair growth, comprising an extract of the pupa of water.

In the present invention, the descriptions of the terms "pupae", "extract", "hair loss", and "hair growth" are the same as described above.

As used herein, the term "external preparation" is a preparation provided for external use, and there are external acid preparations, external tablets, external preparations, ointments, warning preparations, suppositories, etc. include

The external preparation for skin according to the present invention may be a parenteral administration preparation formulated in solid, semi-solid or liquid form by adding commercially available inorganic or organic carriers, excipients and diluents. The preparation for parenteral administration may be a transdermal dosage form selected from the group consisting of drops, ointments, lotions, gels, creams, patches, sprays, suspensions and emulsions, but is not limited thereto.

Carriers, excipients and diluents that may be included in the external preparation include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition for external application for skin according to each formulation can be appropriately selected and formulated by those skilled in the art without difficulty depending on the formulation or purpose of use of other external preparations for skin other than the extract of the present invention. can happen

The composition for external application for skin of the present invention may contain 0.0001 to 30% (w/v) of the active ingredient relative to the total weight of the composition. If it is included in less than 0.0001% (w/v), the effect of preventing hair loss cannot be substantially expected. On the other hand, when it is included in excess of 30% (w/v), the overall processability of the composition, such as solubility in solvents, may decrease, and thus its use for various purposes such as formulations may be limited.

As yet another aspect, the present invention provides a cosmetic composition for preventing hair loss or promoting hair growth, comprising an extract of the pupa of water.

In the present invention, the descriptions of the terms "pupae", "extract", "hair loss", and "hair growth" are the same as described above.

The cosmetic composition is a hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair conditioner, 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 dryer, hair preservative, hair dye, hair wave agent, hair bleach, hair gel, hair glaze, hair dresser, hair lacquer, hair moisturizer, It is preferred that the formulation is selected from the group consisting of hair mousse and hair spray, but is not limited thereto.

In addition, the cosmetic composition of the present invention is not particularly limited in its formulation, for example, softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam , cleansing water, packs, powders, body lotions, body creams, body oils, and cosmetics such as body essences.

The cosmetic composition may be a scalp hair care composition for preventing hair loss or promoting hair growth.

As used herein, the term "scalp and hair care composition" refers to a composition that prevents or alleviates damage to the scalp and hair caused by physical or chemical factors. The composition of the present invention is preferably shampoo, conditioner, essence, nutrition, serum, massage cream, lotion or pack, but is not limited thereto.

The cosmetic composition as described above may be applied in the form of being applied to the skin, or may be applied in a form that is absorbed into the skin by using a microneedle or the like.

The cosmetic composition comprises a fatty substance, an organic solvent, a solubilizer, a thickener, a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, an ionic or non-ionic emulsifier, cosmetology or skin, such as fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any other ingredient commonly used in cosmetics. It may contain adjuvants commonly used in the scientific field. The adjuvant is introduced in an amount generally used in the field of cosmetology or dermatology.

The appearance of the cosmetic composition contains a cosmetically or dermatologically acceptable medium or base. It is in any formulation suitable for topical application, for example solutions, gels, solids, kneaded dry products, emulsions obtained by dispersing the oily phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or, ionic (liposomes) and It may be provided in the form of a non-ionic vesicular dispersant, or in the form of a cream, skin, lotion, powder, ointment, spray or concealer stick. These compositions can be prepared according to conventional methods in the art. The composition according to the invention can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

In addition, the composition of the present invention may be prepared into a hair composition comprising a carrier or carrier mixture suitable for application to the hair. The carrier may include a solvent and other carrier or vehicle components commonly used in hair care compositions.

In the cosmetic composition of the present invention, the active ingredient may be included in any amount (effective amount) according to the use, formulation, purpose of blending, etc. as long as it can exhibit hair loss activity, and the usual effective amount is to be based on the total weight of the composition. When it may be included in the range of 0.001 weight % to 99.99 weight %. Here, "effective amount" refers to an amount of an active ingredient capable of inducing a hair loss effect. Such an effective amount can be determined empirically within the ordinary ability of one of ordinary skill in the art.

As yet another aspect, the present invention provides a quasi-drug composition for preventing hair loss or promoting hair growth, comprising an extract of the pupa of water.

In the present invention, the descriptions of the terms "pupae", "extract", "hair loss", and "hair growth" are the same as described above.

The term "quasi-drug" as used in the present invention refers to articles with a milder action than pharmaceuticals among articles used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases of humans or animals, for example, in the Pharmaceutical Affairs Act. According to the report, quasi-drugs exclude products used for medicinal purposes, and include products used for the treatment or prevention of diseases in humans and animals, and products with minor or no direct action on the human body.

The quasi-drug composition of the present invention is a quasi-drug formulation for preventing hair loss or promoting hair growth. Hair tonic, hair cream, hair lotion, hair shampoo, hair conditioner, hair conditioner, hair spray, hair aerosol, pomade, powder, gel, hair pack, hair Treatment, eyebrow hair growth agent, eyelash hair growth agent, eyelash nutrition agent, or pet shampoo and pet rinse can be prepared in various forms, such as solution, brush, emulsion, oil, wax, aerosol, etc., and the scalp such as a wig or hat Or it may be used for manufacturing or processing a device for hair, but is not limited thereto.

As yet another aspect, the present invention provides a food composition for preventing hair loss or promoting hair growth, comprising an extract of the pupa of water.

In the present invention, the descriptions of the terms "pupae", "extract", "hair loss", and "hair growth" are the same as described above.

The food composition may have a function of preventing hair loss or promoting hair growth.

The food composition of the present invention may include the form of pills, powders, granules, needles, tablets, capsules or liquids, and there is no particular limitation on the type of food to which the pupa extract of the present invention can be added, for example, For example, various beverages, chewing gum, tea, vitamin complexes, and health supplements are available.

Other ingredients may be added to the food composition in addition to the honey bee pupa extract, and the type is not particularly limited. For example, it may contain, as an additional ingredient, various herbal extracts, food-logically acceptable food supplements or natural carbohydrates, such as conventional food, but is not limited thereto.

As used herein, the term "food supplement additive" refers to a component that can be supplementally added to food, and is added to prepare food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplement additive of the present invention.

Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Hygin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used.

The food composition of the present invention may include a health functional food. The term "health functional food" used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients useful for the human body. Here, the term "functionality" refers to obtaining a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and during the manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, unlike general drugs, there are no side effects that may occur when taking the drug for a long time by using food as a raw material, and it can be excellent in portability.

The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food, the active ingredient of the present invention may be added in an amount of 1 to 50% by weight, preferably 5 to 10% by weight of the raw material composition, but is not limited thereto. However, in the case of long-term intake for health and hygiene purposes or for health control, the above amount may be used below the above range.

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health functional foods in the ordinary sense.

The composition containing the pupa extract is recognized for its efficacy in inhibiting and preventing hair loss and promoting hair growth, so it can be used in various fields such as pharmaceuticals, cosmetics, food, quasi-drugs, and beauty.

1 shows the process of extracting the honey bee pupa.
2 is a result of HFDPC cytotoxicity test.
Figure 3 shows the HFDPC cell proliferation rate during co-treatment with the pupa extract and DHT.
4 is a result of HaCaT cytotoxicity test.
Figure 5 shows the HaCaT cell proliferation rate when the extract and LPS were co-treated with a wasp pupa.
6 is a result of NIH3T3 cytotoxicity test.
Figure 7 shows the NIH3T3 cell proliferation rate when the extract and TBM were co-treated with a wasp pupa.
Figure 8 shows the recovery of cAMP activity upon simultaneous treatment of HFDPC cells with DHT and honeysuckle pupa extract.
Figure 9 shows the inhibition of TGF-β1 production when HFDPC cells are treated with DHT and a honey bee pupa extract.
10 shows the results of recovery of IGF-1 production when HFDPC cells are simultaneously treated with DHT and a wasp pupa extract.
11 shows the inhibitory effect of TNF-α production upon simultaneous treatment with LPS and larvae pupa extract on HaCaT cells.
12 shows the IL-6 inhibitory effect upon simultaneous treatment with LPS and larvae pupa extract on HaCaT cells.
13 shows the caspase-3 inhibitory effect of co-treatment with LPS and a wasp pupa extract on HaCaT cells.
Figure 14 shows the collagen type 1 production recovery effect upon simultaneous treatment with the honey bee pupa extract and TBM.
15 shows a measurement result of a subject's scalp condition.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.

<Examples 1 to 4> Preparation of extract of pupa of honeybee

Example 1

Bee pupae between 16 and 20 days of age were produced and freeze-dried from April to early September 2016 using Western species of bees bred in Jeollanam-do Insect Promise Research Institute. Then, 2L of a 50% ethanol solvent was added to 100 g of the pupa powder, and the mixture was stirred and extracted at room temperature for 24 hours. Then, the obtained extract was filtered using filter paper. Again, 2L of a 50% ethanol solvent was added to the filtered extract, followed by repeated extraction with stirring at 4°C for 24 hours. Thereafter, the obtained extract was filtered using filter paper, concentrated using a rotary vacuum concentrator, and then freeze-dried to obtain a 50% ethanol extract of chrysanthemum pupae.

Example 2

A 70% ethanol extract of honey bee pupa was prepared in the same manner as in Example 1 except that 70% ethanol was used.

Example 3

A 100% ethanol extract of honey bee pupa was prepared in the same manner as in Example 1 except that 100% ethanol was used.

Example 4

A water bee pupa water (distilled water) extract was prepared in the same manner as in Example 1, except that water (distilled water) was used.

<Experiment method>

1. Measurement of antioxidant activity of honey bee pupa extract

1-1. Measurement of DPPH scavenging activity (%)

The test solution was prepared at a concentration of 40 mg/mL by adding each extraction solvent to the dry sample, and then filtered with a 0.45 μm syringe filter. The filtered sample was diluted by concentration and used as a test solution, and ascorbic acid was used as a control. A 0.1 mM DPPH (1,1-diphenyl-2-picrylhydrazyl) solution was prepared, and 190 μL of 0.1 mM DPPH solution was mixed with 10 μL of test solution for each concentration of the honey bee pupa extract, and reacted for 30 minutes at room temperature with light blocked. After 30 minutes, absorbance was measured at 517 nm using a spectrophotometer, and the DPPH radical scavenging ability of each sample was calculated using the following formula and expressed as a percentage. After measuring the same test solution three times, the average value was expressed.

1-2. ABTS radical scavenging activity measurement

ABTS working solution was prepared by reacting 7 mM ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) with 2.45 mM potassium persulfate in the dark for 16 hours. The prepared ABTS working solution was mixed with distilled water. After standing for 10 minutes, the absorbance was adjusted so that the absorbance at 734 nm was 0.80 ± 0.02. Mix 190 μL of ABTS working solution with 10 μL of test solution for each concentration of honey bee pupa extract and react at room temperature for 30 minutes, then use a spectrophotometer. Absorbance was measured at 734 nm The ABTS radical scavenging ability of each sample was calculated by the following formula and expressed as a percentage, and the same test solution was measured three times and then expressed as an average value.

2. Efficacy evaluation of male pattern hair loss prevention

2-1. cell culture

Human Follicle Dermal Papilla Cells (HFDPC) were purchased from Promo cells, and a dedicated medium, Follicle Dermal Papillar Cell Growth Medium (Ready-to-use) (Cat. No. C-26501), was subcultured once every 3 days. Cells were used by culturing in an incubator at 37° C. in which 5% CO _{2 was maintained.}

2-2. Cytotoxicity test (MTS assay)

^{HFDPC cells were seeded at a concentration of 1x10 4} cells/ml in a 96-well plate and cultured for 24 hours. Samples were treated by concentration (0, 50, 100, 200ug/ml), and after 24 hours of incubation, ^{10 μl of CellTiter 96 ⓡ} AQueous One Solution Reagent (MTS) (Promega, USA) was added and incubated for 2 hours in an incubator at 37°C. did The absorbance was measured at 490 nm using a microplate reader (EPOCH2, BioTek, USA) to determine the cell viability for each concentration compared to the cell viability of the untreated group.

2-3. Cell proliferation efficacy test (MTS assay)

Cells were cultured in the same manner as in the cytotoxicity test, and samples were treated at different concentrations (0, 50, 100, 200 μg/ml) and at the same time treated with DHT (5α-dihydrotestosterone) at 10 ^-5 M (=10 μM) concentration. After 24 hours of incubation, 10uL of CellTiter 96ⓡ AQueous One Solution Reagent (MTS) (Promega, USA) was added and incubated for 2 hours in an incubator at 37°C. The absorbance was measured at 490 nm using a microplate reader (EPOCH2, BioTek, USA) to determine the cell viability for each concentration compared to the cell viability of the untreated group.

2-4. Hair growth factor transcript expression test (TGF-β1, IGF-1, cAMP)

HFDPC was seeded in a 6-well plate at a concentration of 1×10 ⁶ cells/ml and cultured until the cells in the well reached 80-90%. Samples were treated in a medium containing 10 ^-5 M (=10 μM) of DHT (5α-dihydrotestosterone) by concentration (0, 50, 100, 200 μg/ml), and cells and medium were collected after 24 hours of incubation. Total RNA was extracted from the cells using Trizol reagent (Invitrogen, USA). The extracted total RNA was quantified by measuring absorbance at 260 nm and 280 nm using a Microplate reader (EPOCH2, BioTek, USA). RT-qPCR is amplification (denaturation: 15 seconds at 95°C, annealing: 15 seconds at 54°C, extension using Real-time PCR machine (Applied Biosystems, Thermo) using ^{AccuPower ⓡ 2X GreenStarTM qPCR Master Mix (BioNeer))} : It was carried out at 72°C for 15 seconds, 40 cycles). The C(t) value obtained as a result was corrected with the C(t) value of GADPH to quantify the gene expression level relative to the negative control group. The primers used for PCR are shown in Table 1.

3. Efficacy evaluation of inflammatory hair loss prevention

3-1. cell culture

HaCaT (Human Keratinocyte) was purchased from ATCC (ATCC, USA) and DMEM medium containing 10% FBS (fetal bovine serum; Hyclone) and 1% penicillin-streptomycin (Sigma Aldrich, St. Louis, MO, USA) ( Dulbecco's Modified Eagle Medium (Hyclone, Dallas, TX, USA) was subcultured once every 3 days. Cells were used by culturing in an incubator at 37° C. in which 5% CO _{2 was maintained.}

3-2. Cytotoxicity test (MTS assay)

^{HaCaT cells were seeded at a concentration of 1x10 4} cells/ml in a 96-well plate and cultured for 24 hours. Samples were treated according to concentration (0, 500, 1000, 2000㎍/ml), and after 24 hours of incubation, ^{10uL of CellTiter 96 ⓡ} AQueous One Solution Reagent (MTS) (Promega, USA) was added and incubated for 2 hours in an incubator at 37°C. did The absorbance was measured at 490 nm using a microplate reader (EPOCH2, BioTek, USA) to determine the cell viability for each concentration compared to the cell viability of the untreated group.

3-3. Cell proliferation efficacy test (MTS assay)

Cells were cultured in the same manner as in the cytotoxicity test, and samples were treated at different concentrations (0, 500, 1000, 2000 μg/ml), and at the same time LPS (Lipopolysaccharide) was treated at 400 ng/ml concentration. After 24 hours of incubation, CellTiter 96 ^ⓡ 10uL of AQueous One Solution Reagent (MTS) (Promega, USA) was added and incubated for 2 hours in an incubator at 37°C. The absorbance was measured at 490 nm using a microplate reader (EPOCH2, BioTek, USA) to determine the cell viability for each concentration compared to the cell viability of the untreated group.

3-4. Inflammatory cytokine expression test related to hair follicle cell destruction (Caspase-3, IL-6, TNF-α)

HaCaT was seeded in a 6-well plate at ^{a concentration of 1×10 6} cells/ml and cultured until the cells in the well reached 80-90%. Samples were treated in a medium containing 400 ng/ml of LPS (Lipopolysaccharide) by concentration (0, 500, 1000, 2000 μg/ml), and cells and medium were collected after 24 hours of incubation. Total RNA was extracted from the cells using Trizol reagent (Invitrogen, USA). The extracted total RNA was quantified by measuring absorbance at 260 nm and 280 nm using a Microplate reader (EPOCH2, BioTek, USA). RT-qPCR was performed using AccuPower ^ⓡ 2X GreenStar ^TM qPCR Master Mix (BioNeer) using a Real-time PCR machine (Applied Biosystems, Thermo) for amplification (denaturation: 15 sec at 95 ℃, annealing: 15 sec at 54 ℃, extension: 15 seconds at 72° C., 40 cycles). The C(t) value obtained as a result was corrected with the C(t) value of GADPH to quantify the gene expression level relative to the negative control group. The primers used for PCR are shown in Table 1.

4. Wool efficacy evaluation

4-1. cell culture

NIH3T3 (mouse fibroblast cell) was purchased from ATCC (ATCC, USA) and DMEM medium containing 10% FBS (fetal bovine serum; Hyclone) and 1% penicillin-streptomycin (Sigma Aldrich, St. Louis, MO, USA) (Dulbecco's Modified Eagle Medium; Hyclone, Dallas, TX, USA) was subcultured once every 3 days. Cells were used by culturing in an incubator at 37° C. in which 5% CO _{2 was maintained.}

4-2. Cytotoxicity test (MTS assay)

In a 96-well plate, NIH3T3 cells were ^{seeded at a concentration of 1x10 4} cells/ml and cultured for 24 hours. Samples were treated by concentration (0, 100, 200, 400ug/ml), and after 24 hours of incubation, ^{10uL of CellTiter 96 ⓡ} AQueous One Solution Reagent (MTS) (Promega, USA) was added and incubated at 37°C for 2 hours in an incubator. . The absorbance was measured at 490 nm using a microplate reader (EPOCH2, BioTek, USA) to determine the cell viability for each concentration compared to the cell viability of the untreated group.

4-3. Cell proliferation efficacy test (MTS assay)

Cells were cultured in the same manner as in the cytotoxicity test, and samples were treated at different concentrations (0, 100, 200, 400㎍/ml), and at the same time, TBM (Tolbutamide) was treated at 410mM concentration and incubated for 24 hours, CellTiter 96 ^ⓡ AQueous One 10uL of Solution Reagent (MTS) (Promega, USA) was added and incubated for 2 hours at 37°C in an incubator. The absorbance was measured at 490 nm using a microplate reader (EPOCH2, BioTek, USA) to determine the cell viability for each concentration compared to the cell viability of the untreated group.

4-4. Wool-related factor expression test (collagen type I)

NIH3T3 was seeded in a 6-well plate at ^{a concentration of 1×10 6} cells/ml and cultured until the cells in the well reached 80-90%. Samples were treated in a medium containing 410 mM TBM (Tolbutamide) for each concentration (0, 100, 200, 400 μg/ml), and cells and medium were collected after 24 hours of incubation. Total RNA was extracted from the cells using Trizol reagent (Invitrogen, USA). The extracted total RNA was quantified by measuring absorbance at 260 nm and 280 nm using a Microplate reader (EPOCH2, BioTek, USA). RT-qPCR was performed using AccuPower ^ⓡ 2X GreenStar ^TM qPCR Master Mix (BioNeer) using a Real-time PCR machine (Applied Biosystems, Thermo) for amplification (denaturation: 15 sec at 95 ℃, annealing: 15 sec at 54 ℃, extension: 15 seconds at 72° C., 40 cycles). The C(t) value obtained as a result was corrected with the C(t) value of β-actin to quantify the gene expression level relative to the negative control group. The primers used for PCR are shown in Table 1.

Primer sequence for hair loss prevention efficacy evaluation Gene Sequence for Primers (5'-3') TGF-β1 Forward GGC TAC TTT GCC AAC TAC TGC Reverse CTG CTC CAC CTT GTG TTG C IGF-1 Forward TCG CAT CTC TTC TAT CTG GCC CTG T Reverse GCA GTA CAT CTC CAG CCT CCT CAG A cAMP Forward GCA CAG GAG CAC AAC ATC AG Reverse CTC ATC TAC GTG CTC ATC GTG caspase-3 Forward CTG CTG GGG ATG GCC ACT GTG Reverse TCG CCT CGA GGA CAT CGC TCT C IL-6 Forward GGA GAC TTG CCT GGT GAA AA Reverse GTC AGG GGT GGT TAT TGC AT TNF-α Forward CGG GCA GGT CTA CTT TGG AG Reverse CAG GTC ACT GTC CCA GCA TC GADPH Forward CCA GGC GCC CAA TAC G Reverse CCA CAT CGC TCA GAC ACC AT collagen type I (mouse) Forward AGC TTT GTG GAT ACG CGG AC Reverse TAG GCA CGA AGT TAC TGC AAG β-Actin (mouse) Forward GAA GCT GTG CTA TGT TGC TCT A Reverse ATG CCA CAG GAT TCC ATA CCC

5. Statistical processing of data

All data obtained in the experiment were expressed as mean and standard error, and were tested using SPSS (SPSS Version 20.0 Inc. USA). As a result of one-way analysis of variance (ANOVA) (significance level: 0.05), if significance was observed, a multiple test of Dunnett's t-test was performed to confirm the significance between each test group for the control group (significance level). : one-sided 0.05). Duncan's multiple range test was performed to determine whether there was a difference between the test groups for the in vitro tyrosinase inhibition assay results (significance level: 0.05 on both sides).

<Experiment result>

<Experimental Example 1> Evaluation of antioxidant activity of honeybee pupa extract

As a result of testing the antioxidant effect (DPPH, ABTS radical scavenging ability) as shown in Tables 2 and 3 below, the water extract and ethanol extract prepared in Examples 1 to 4 were extracted using water and 50% ethanol as solvents. It was confirmed that the extract had a high antioxidant effect.

<Experimental Example 2> Cytotoxicity and cell proliferation effect of the honey bee pupa extract

As described above, the results of the dermal papilla cell (HFDPC), mother cell (HaCaT) and mouse fibroblast (NIH3T3) culture test results, as shown in FIGS. It was confirmed that there was no cytotoxicity up to 200 μg/ml, up to 2000 μg/ml in parental cells (HaCaT), and up to 100 μg/ml in mouse fibroblasts (NIH3T3) ( FIGS. 2 to 4 ).

In the dermal papilla cells (HFDPC) treated with DHT (5α-dihydrotestosterone) to induce male pattern hair loss, it was confirmed that the apoptosis inhibition rate was improved by about 3.3% by the treatment of the honey bee pupa extract ( FIG. 5 ). In addition, it was confirmed that the apoptosis inhibition rate was improved by 23.6% by the treatment of the pupa extract in hair cells (HaCaT) treated with LPS to induce inflammatory hair loss (FIG. 6). In addition, in order to evaluate the efficacy of wool, mitotic inhibitors (TBM, tolbutamide) were treated to fibroblasts (NIH3T3), and it was confirmed that the apoptosis inhibition rate of fibroblasts (NIH3T3) was improved by 25.9% by treatment of the honey bee pupa extract. was done (FIG. 7).

Therefore, through the above results, it was found that the pupa extract improves male pattern hair loss and inflammatory hair loss, and has a wool effect.

<Experimental Example 3> Expression analysis of male pattern hair loss-related transcriptome of the extract of the honey bee pupa in scalp cells

8 to 10 are results of confirming the expression of genes related to male pattern hair loss suppression targeting the extract of a wasp pupa. Human follicle dermal papilla cells (HFDPCs) are treated with DHT (5α-dihydrotestosterone), which induces male pattern hair loss, and at the same time, the pupa extract is added to the culture medium and cultured. The effect of inhibiting production and TGF-β1 production was confirmed. As a result, it was confirmed that the cAMP activity and IGF-1 production involved in hair growth were restored by the treatment of the wasp pupa extract ( FIGS. 8 and 10 ), and it was confirmed that the TGF-β1 production was inhibited ( FIG. 9 ).

Through the above results, it was found that the male pupa extract restored cAMP activity and IGF-1 production involved in hair growth, and suppressed TGF-β1 production, thereby suppressing male pattern hair loss.

<Experimental Example 4> Gene expression test related to the inhibition of inflammatory hair loss of the extract of the honey bee pupa in scalp cells

11 to 13 are results of confirming the expression of genes related to the inhibition of inflammatory hair loss in the extract of a wasp pupa. HaCaT (Human Keratinocyte), which is the parental cell, was treated with LPS (lipopolysaccharide) that induces inflammation, and at the same time, the extract of the pupa of honeybee was added to the culture medium and cultured, and then the expression of inflammatory cytokines was analyzed. As a result, it was confirmed that the expression of TNF-α and IL-6 corresponding to inflammatory cytokines was suppressed (FIG. 11, FIG. 12), and inhibited the activity of caspase-3 that induces cell death of hair follicles (FIG. 13) .

Through the above results, it was found that the pupa extract suppressed inflammatory hair loss by inhibiting the expression of inflammatory cytokines TNF-α and IL-6, and inhibiting the activity of caspase-3 that induces hair follicle cell death.

<Experimental Example 5> Wool-related gene expression analysis of honey bee pupa extract in mouse fibroblasts

14 is a result of confirming the expression of a wool-related gene in the extract of the honey bee pupa. When NIH3T3 (mouse fibroblast cell), a mouse fibroblast cell, was treated with a mitosis inhibitor (TBM) and at the same time the extract was added to the culture medium and cultured. It was identified in the ethanol extract.

Through the above results, it was found that the pupa extract has a hair growth promoting effect by increasing the production of collagen type I related to the hair growth effect.

<Experimental Example 6> Sensory test evaluation of shampoo and scalp tonic formulations

A shampoo for alleviating hair loss having the composition shown in Table 4 was prepared.

Shampoo ingredients for alleviating hair loss Formulation 1 - Shampoo Raw material name Korean raw material name Raw material name Korean raw material name Water Purified water LDE laura hyde d.imay Jaguar C-13S Grilled Hydroxypropyl Trimonium Chloride EGMS clicoxviarate EDTA2Na Disodium EDIT EtOH ethanol Kathon CG Methylchloroisothiazolinone
Dichilisothiazolinone OriStar Propolis propolis extract Glycerin glycerin CS-770 Diechcon
Rio Res-3
Laureth-20 M-550 Polyquaternium-7 pf lavender oil lavender oil A-428 Ammonium Lauryl Sulfate CM10 ex Western Rose Flower Extract
chamomile
Jasmine
rosehip fruit
Hibiscus Flower Extract
tangerine peel
three hundred seconds
Eoseongcho
chestnut shell
stuttering 1ea-525 Ammonium Laureth Sulfate view group Ginseng Extract
Maekmun-dong
plantain
licorice
Astragalus
ginger Mitain CA cocaidopropyl betaine DL-Panthenol panthenol LES Na Disodium Laureth Sulfosuccinate Bee pupa 50% ethanol extract

A scalp tonic for alleviating hair loss having the composition shown in Table 5 below was prepared.

Scalp tonic ingredient for hair loss relief Formulation 2 - Scalp Tonic Raw material name Korean raw material name Raw material name Korean raw material name water Purified water Apcide-700 Yellow Tangerine Seed Extract
iris extract
perilla leaf extract Tyhol-5 Benzophenone-5 MIP-9 1,2-Hexanediol PG propylene glycol 1% Red 2 Red No. 2 CM10 Complex Western Rose Flower Extract
chamomile
Jasmine
rosehip fruit
Hibiscus Flower Extract
tangerine peel
three hundred seconds
Eoseongcho
chestnut shell
stuttering EtOH ethanol view group Ginseng Extract
Maekmun-dong
plantain
licorice
Astragalus
ginger OriStar Propolis propolis
extract Copper peptide-1 Coppertripeptide-1 Bee pupa 50% ethanol extract

In Table 6, the sensory test of the Shamponic formulation was evaluated. As a result, as shown in Table 6, it was confirmed that the sensory test results were the best when 1 wt % of the extract of the honey bee pupa was included in the shampoo.

In Table 7, the sensory test of the scalp tonic formulation was evaluated. As a result, as shown in Table 7, it was confirmed that the sensory test result was the best when 1 wt % of the extract of the honey bee pupa was included in the scalp tonic.

<Experimental Example 7> Measurement of scalp condition, hair thickness, and density of shampoo and scalp tonic formulations

Next, the subject's scalp condition, hair thickness, and density were measured.

As a result, as a result of using the shampoo and scalp tonic containing 50% ethanol extract of pupa of honeybee, it was confirmed that after 4 weeks, hair thickness and hair density increased compared to before use. It was found that there is (Table 8 and Figure 15).

Subject's hair thickness and density Jeon after 4 weeks 8 weeks 16 weeks Hair thickness (mm) 0.049±0.021 0.066±0.015 0.052±0.006 0.056±0.002 Hair density (Hairs/㎠) 70.00±12.75 83.00±11.51 85.00±28.94 85.00±29.15

Claims (7)

delete delete delete As a skin external composition for preventing hair loss or promoting hair growth, comprising 1% by weight of a honey bee pupa extract,
The bee pupa extract was extracted with 50% ethanol solvent,
The hair loss is male pattern hair loss or inflammatory hair loss,
The composition increases the production of collagen type I (collagen type I) to promote hair growth,
A composition for external application for skin for preventing hair loss or promoting hair growth. As a cosmetic composition for preventing hair loss or promoting hair growth, comprising 1% by weight of a honey bee pupa extract,
The bee pupa extract was extracted with 50% ethanol solvent,
The hair loss is male pattern hair loss or inflammatory hair loss,
The composition increases the production of collagen type I (collagen type I) to promote hair growth,
A cosmetic composition for preventing hair loss or promoting hair growth. As a quasi-drug composition for preventing hair loss or promoting hair growth, comprising 1% by weight of a honey bee pupa extract,
The bee pupa extract was extracted with 50% ethanol solvent,
The hair loss is male pattern hair loss or inflammatory hair loss,
The composition increases the production of collagen type I (collagen type I) to promote hair growth,
A quasi-drug composition for preventing hair loss or promoting hair growth. delete

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