KR102610933B1 - Composition for hair or scalp comprising lysate of Lactobacillus plantarum - Google Patents

Abstract

This specification describes a composition for hair or scalp containing a lysate of Lactobacillus plantarum as an active ingredient. The composition can exhibit significantly excellent hair growth promotion and hair loss prevention effects.

Description

Composition for hair or scalp comprising lysate of Lactobacillus plantarum}

The present specification relates to a composition for hair or scalp containing an eluate of Lactobacillus plantarum.

With the recent trend of avoiding chemical raw materials, insects, and animal raw materials, there is a demand for materials of natural origin. Microbial resources are sustainable resources in that they are reproducible and are highly useful materials that utilize the unique characteristics of microorganisms adapted to various environments. Among these, lactic acid bacteria are a material that can be used without risk to the human body and are used as probiotics in the food field.

Republic of Korea Patent Publication No. 10-2014-0055397

From one perspective, the problem to be solved by the present invention is to provide a composition with excellent hair growth promotion and hair loss prevention effects.

From another perspective, the problem to be solved by the present invention is to provide a composition with excellent hair growth promotion and hair loss prevention effects without toxicity or irritation to the human body.

From another perspective, the problem to be solved by the present invention is to provide a composition with excellent hair growth promotion and hair loss prevention effects using ingredients of natural origin.

From another perspective, the problem to be solved by the present invention is to provide a composition that is excellent in promoting the proliferation of hair follicle dermal papilla cells and inhibiting their death.

From another perspective, the problem to be solved by the present invention is to provide a composition that is excellent in promoting the proliferation of outer root sheath cells and inhibiting their death.

In one aspect, the present invention relates to a composition for hair or scalp containing a lysate of Lactobacillus plantarum as an active ingredient.

From another perspective, the present invention (a) cultivates Lactobacillus plantarum,

(b) centrifuging the cultured Lactobacillus plantarum to remove insoluble components, and

(c) Hair containing as an active ingredient an eluate of Lactobacillus plantarum, which includes eluting the Lactobacillus plantarum obtained in (b) by heating it at 80°C to 120°C for 10 to 30 minutes; or It relates to a method of manufacturing a composition for the scalp.

From one perspective, the present invention can provide a composition with excellent hair growth promotion and hair loss prevention effects.

From another perspective, the present invention can provide a composition with excellent hair growth promotion and hair loss prevention effects without toxicity or irritation to the human body.

From another perspective, the present invention can provide excellent hair growth promotion and hair loss prevention effects by using ingredients of natural origin.

From another perspective, the present invention can provide a composition that is excellent in promoting the proliferation of hair follicle dermal papilla cells and inhibiting their death.

From another perspective, the present invention can provide an excellent composition that promotes the proliferation of outer root sheath cells and inhibits their death.

Figure 1 is a graph showing the effect of Lactobacillus plantarum on promoting hair follicle dermal papilla cell proliferation.
Figure 2 is a graph showing the effect of Lactobacillus plantarum on promoting outer root sheath cell proliferation.
Figure 3 is a graph showing the inhibitory effect of Lactobacillus plantarum on outer root sheath cell death.
Figure 4 is a fluorescence micrograph showing the inhibitory effect of Lactobacillus plantarum on outer root sheath cell death.

Hereinafter, embodiments of the present application will be described in more detail with reference to the attached drawings. However, the technology disclosed in this application is not limited to the embodiments described herein and may be embodied in other forms. However, the embodiments introduced here are provided to ensure that the disclosed content is thorough and complete and that the spirit of the present application can be sufficiently conveyed to those skilled in the art. In order to clearly express each component in the drawing, the size of the component, such as width or thickness, is shown somewhat enlarged. Additionally, for convenience of explanation, only some of the components are shown, but those skilled in the art will be able to easily understand the remaining components. Additionally, a person skilled in the art will be able to implement the ideas of this application in various other forms without departing from the technical spirit of this application.

In one embodiment of the present invention, a composition for hair or scalp containing a lysate of Lactobacillus plantarum as an active ingredient can be provided.

In one example, the Lactobacillus plantarum may be derived from the tea tree. In one example, the Lactobacillus plantarum is Camellisa sinensis ) may have been isolated and identified from the leaves.

In one example, the Lactobacillus plantarum may be the strain APsulloc 331266 (accession number KCCM11181P). The strain may have been isolated and identified from tea tree leaves, and may exhibit significantly superior hair growth promotion and hair loss prevention effects among several Lactobacillus plantarum strains isolated and identified from tea tree leaves.

In one example, the eluate may be a heated eluate obtained by heating and eluting Lactobacillus plantarum cells. The heating can be performed at 80°C to 120°C for 10 to 30 minutes. The heated extract is a natural ingredient that has little irritation or toxicity when applied to the human body, and can be significantly effective in promoting hair growth and preventing hair loss.

In one example, the eluate is (a) cultured Lactobacillus plantarum,

(b) centrifuging the cultured Lactobacillus plantarum to remove insoluble components, and

(c) It can be prepared by heating the Lactobacillus plantarum obtained in (b) above for 10 to 30 minutes at 80°C to 120°C to elute it.

The composition according to one embodiment of the present invention is a natural ingredient and has excellent safety as it has little irritation or toxicity when applied to the human body. In addition, it can promote the proliferation of hair follicle dermal papilla cells and outer root sheath cells and inhibit their death, showing a significantly excellent hair growth promotion effect and hair loss prevention effect.

In one embodiment of the present invention, the composition may be a composition for external skin application.

The skin external composition may be, for example, a cosmetic composition.

The composition according to one embodiment of the present invention may be formulated containing a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, for example, solutions, gels, solids, pasty anhydrous products, emulsions obtained by dispersing the oil phase in the water phase, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) and non-ionic forms. It may be provided in the form of an ionic vesicular dispersion, or in the form of a cream, skin, lotion, powder, ointment, spray or conceal stick. It can also be used in the form of foam or in the form of an aerosol composition further containing compressed propellant. These compositions can be prepared according to conventional methods in the art.

Additionally, the composition according to one embodiment of the present invention may contain a substance that promotes skin absorption to increase the skin improvement effect.

In one example, the cosmetic composition may include the Lactobacillus plantarum extract in an amount of 0.1% to 20% by weight based on the total weight of the composition.

In one example, the composition can be used as a scalp or hair composition. Therefore, the composition may be used in formulations for use on the scalp and hair together, such as shampoo, rinse, scalp treatment, hair treatment, treatment for both scalp and hair, treatment for anti-hair loss hair, treatment for damaged hair, leave-in ) It can be formulated as a scalp or hair, scalp and hair combination product, such as a conditioner, wash-off conditioner, or hair essence.

The composition of each of these formulations may contain various ingredients that are mixed into a typical scalp or hair composition according to the various formulations described above or suitable for the final purpose, and the types and amounts of these ingredients can be easily selected by a person skilled in the art. .

In one example, the composition may include the Lactobacillus plantarum extract in an amount of 0.1% to 20% by weight based on the total weight of the composition.

In addition, the composition according to one embodiment of the present invention contains fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, and ionic types. or non-ionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other commonly used in cosmetics. It may contain auxiliaries commonly used in the fields of cosmetology or dermatology, such as ingredients. The auxiliary agent may be introduced in an amount commonly used in the fields of cosmetics or dermatology, for example, 0.001 to 5% by weight, for example, 0.001 to 3% by weight, based on the total weight of the composition.

In one embodiment of the present invention, the composition may be a pharmaceutical composition.

The pharmaceutical composition according to the present specification may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, soft or hard capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, or sucrose. Alternatively, it is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The pharmaceutical dosage form of the composition of the present specification may be used in the form of a pharmaceutically acceptable salt thereof, and may be used alone or in combination with other pharmaceutically active compounds, as well as in an appropriate combination. The salt is not particularly limited as long as it is pharmaceutically acceptable, and examples include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, and methanesulfonic acid. , benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, etc. can be used.

The composition of the present specification can be administered parenterally or orally depending on the purpose, and can be administered in one to several divided doses to be administered in an amount of 0.1 to 500 mg, preferably 1 to 100 mg per kg of body weight per day. there is. The dosage for a specific patient may vary depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, severity of disease, etc.

The pharmaceutical composition according to the present specification can be formulated into oral dosage forms such as powders, granules, tablets, soft or hard capsules, suspensions, emulsions, syrups, aerosols, external skin preparations such as ointments and creams, suppositories, and injections according to conventional methods. It can be formulated and used in any form suitable for pharmaceutical preparations, including sterilized injection solutions.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

[Preparation Example 1] Preparation of Lactobacillus Plantarum APsulloc 331266 eluate

1. Isolation of Lactobacillus plantarum strains

200 g of tea leaves were washed twice in distilled water to remove foreign substances. The washed tea leaves were shaken off, mixed with table salt equivalent to 8% of the weight of the tea leaves, and left at room temperature for 3 hours. Tea tree leaves pickled in table salt were mixed with 1000 ml of 1% fructooligosaccharide solution and cultured in an incubator at 32°C for 3 days. After 3 days, it was checked whether the pH of the culture medium fell below 5, and if the pH was below 5, it was taken and cultured in Difco Lactobacilli MRS Agar® medium. At this time, the culture was cultured for 2 days in a chamber under anaerobic conditions at 32°C, and then colonies showing white colonies were taken, and Lactobacillus plantarum APsulloc 331266 (accession number: KCCM11181P) was isolated from tea tree leaves in the same manner as above.

2. Culture and elution of Lactobacillus plantarum

The isolated and identified Lactobacillus plantarum APsulloc 331266 was stored in a cryotube at 10% by weight in skim milk, inoculated into a medium (MRS Broth), and cultured for 24 hours at 37°C. When the bacterial concentration in the liquid medium ^{reached 5} The washed ^bacterial cells were resuspended in PBS at a concentration of ⁵

[Test Example 1] Proliferation effect of hair follicle and papilla cells (MTT assay)

The proliferation effect in human dermal papilla cells (hDPCs) was confirmed using the eluate prepared in Preparation Example 1.

1. hDPCs isolation and culture

Dermal papilla cells were isolated from the occipital scalp tissue using a microscope and cultured in a culture dish coated with type 1 collagen (collagen type I) for 14 days. Culture medium containing DMEM (Dulbecco's modified eagles medium; HyClone GE Healthcare) supplemented with antibiotics (1%), fungizone (0.1%), and 20% FBS, 5% CO _{2 ,} at 37°C for 72 hours. After culturing, the medium was replaced with DMEM medium supplemented with 10% FBS, and when the cells had grown to more than 90% of the dish, the cultured cells were collected using 0.25% trypsin/10mM EDTA (Welgene) and added with 10% FBS. Added DMEM medium, 5% CO _{2 ,} and cultured at 37°C for 72 hours (until cells grew to more than 90% of the dish) and used for subsequent experiments.

2. Culture to confirm hDPCs cell proliferation effect

2000 hDPCs/well were inoculated into a 96 well plate and cultured at 37°C with 5% CO ₂ for 24 hours. Here, 10% by volume of the eluate prepared in Preparation Example 1 was treated and cultured at 37°C in 5% CO ₂ for 72 hours. An untreated group was used as a control, and the same amount of 5% FBS as the above eluate was treated as a positive control.

3. Measurement of cell proliferation: Viability over 120% compared to control

MTT solution (Sigma, 70㎍/well) was added to each well of the 96 well plate and cultured at 5% CO ₂ at 37°C for 3 hours. Afterwards, formazan was dissolved in DMSO and the absorbance was measured at 570 nm using a microplate reader. In the measured results, the relative cell proliferation rate is shown in Figure 1, with the value of the control group being 100.

From the results in Figure 1, it can be seen that the APsulloc 331266 strain of Preparation Example 1 exhibits an excellent hair follicle dermal papilla cell proliferation effect.

[Test Example 2] Effect of outer root sheath cell proliferation

The proliferation effect of outer root sheath keratinocytes (ORS keratinocytes) was confirmed using the eluate prepared in Preparation Example 1.

1. Isolation and culture of outer root sheath cells (ORS keratinocytes)

Outer root sheath cells were isolated from the occipital scalp tissue using a microscope and cultured on a culture dish coated with type 1 collagen (collagen type I) for 14 days. Culture medium containing DMEM (Dulbecco's modified eagles medium; HyClone GE Healthcare) supplemented with antibiotics (1%), fungizone (0.1%), and 20% FBS, 5% CO _{2 ,} for 3 days at 37°C. The cells were cultured, replaced with a medium dedicated for outer root sheath cell culture (EPILIFE MEPI500CA, Invitrogen), and after confirmation of confluent, they were treated with trypsin/EDTA (T/E) and subcultured.

2. Culture to confirm the cell proliferation effect of outer root sheath cells

After inoculating 20,000 outer root sheath cells/well in a 96 well plate, the cells were cultured for 24 hours at 5% CO _{2 and} 37°C. Here, 10% by volume of the eluate prepared in Preparation Example 1 was treated and cultured at 37°C in 5% CO ₂ for 72 hours. As a control, an untreated group was used, and as a positive control, growth factors (EpiLife S-012-5, invitrogen) added to outer root sheath cell culture medium (EPILIFE MEPI500CA, invitrogen) were used in the same amount as the eluate. did.

3. Measurement of cell proliferation: Viability over 120% compared to control

MTT solution (Sigma, 70㎍/well) was added to each well of the 96 well plate and cultured at 5% CO ₂ at 37°C for 3 hours. Afterwards, formazan was dissolved in DMSO and the absorbance was measured at 570 nm using a microplate reader. In the measured results, the relative cell proliferation rate is shown in Figure 2, with the control value set to 100.

From the results in Figure 2, it can be seen that the APsulloc 331266 strain of Preparation Example 1 exhibits an excellent outer root sheath cell proliferation effect.

[Test Example 3] Effect of suppressing death of outer root sheath cells

Using the eluate prepared in Preparation Example 1, the effect of suppressing the death of outer root sheath keratinocytes (ORS keratinocytes) induced by DKK-1, a known hair loss inducing factor, was confirmed.

1. Cell culture

The outer root sheath cells isolated and cultured as in Test Example 2 were inoculated at 20,000 cells/well on a 96 well plate and an 8 chamber slide, and cultured for 24 hours at 5% CO _{2 and} 37°C. After starvation for 24 hours, the cells were treated with 50 ng/ml of DKK, 50 ng/ml of DKK + 10% by volume of the eluate prepared in Preparation Example 1, respectively. An untreated group was used as a control, and a positive control group was used. Growth factors (EpiLife S-012-5, invitrogen) added to outer root sheath cell culture medium (EPILIFE MEPI500CA, invitrogen) were used in the same amount as the eluate.

2. MTT assay

MTT solution (Sigma, 70㎍/well) was added to each well of the 96 well plate treated with the test substance and incubated for 3 hours at 37°C in 5% CO ₂ . Afterwards, formazan was dissolved in DMSO and the absorbance was measured at 570 nm using a microplate reader. In the measured results, the relative cell proliferation rate (***) is set to 100 for the negative control group, and the relative cell proliferation rate () is set to 100 for the group treated with only DKK-1. The results are shown in Figure 3.

3. TUNEL assay (8 chamber slide)

The 8 chamber slides treated with the above test substances were cultured for 24 hours, fixed using 4% paraformaldehyde, washed, and then treated with triton-X100 0.05% (in PBS). After reacting using TUNEL reaction mixture (Roche kit), the reaction mixture was washed, stained with DAPI (4',6-diamidino-2-phenylindole), and observed under a fluorescence microscope. A fluorescence micrograph of the cells is shown in Figure 4.

From the results in Figures 3 and 4, it can be seen that the APsulloc 331266 strain of Preparation Example 1 exhibits an excellent effect of inhibiting outer root sheath cell death.

A formulation example of the composition according to one aspect of the present specification is described below, but various other formulations can also be applied, and this is not intended to limit the present specification, but is only intended to provide a detailed description.

[Formulation Example 1] Tablet

100 mg of the eluate of Preparation Example 1 of the present invention, 400 mg of lactose, 400 mg of corn starch, and 2 mg of magnesium stearate were mixed, and then tableted according to a conventional tablet manufacturing method.

[Formulation Example 2] Capsule

100 mg of the extract of Preparation Example 1 of the present invention, 400 mg of lactose, 400 mg of corn starch, and 2 mg of magnesium stearate were mixed, and then filled into a gelatin capsule according to a conventional capsule manufacturing method to prepare a capsule.

[Formulation Example 3] Granules

50 mg of the eluate from Preparation Example 1 of the present invention, 250 mg of anhydrous crystalline glucose, and 550 mg of starch were mixed, formed into granules using a fluidized bed granulator, and then filled into bags.

[Formulation example 4] Drink formulation

After mixing 50 mg of the eluate of Preparation Example 1 of the present invention, 10 g of glucose, 0.6 g of citric acid, and 25 g of liquid oligosaccharide, 300 ml of purified water was added, and 200 ml of each bottle was filled. After filling the bottle, it was sterilized at 130°C for 4 to 5 seconds to prepare a drink.

[Formulation Example 5] Injectable

Injections were prepared by a conventional method according to the composition shown in the table below.

Formulation Ingredients content Eluate of Preparation Example 1 10-50mg Sterile distilled water for injection Appropriate amount pH regulator Appropriate amount

[Formulation Example 6] Soft lotion (skin lotion)

Soft lotion was prepared in a conventional manner according to the composition shown in the table below.

Formulation Ingredients Content (% by weight) Eluate of Preparation Example 1 0.2 glycerin 3.0 Butylene glycol 2.0 propylene glycol 2.0 Carboxy vinyl polymer 0.1 PEG-12 nonylphenyl ether 0.2 Polysorbate 80 0.4 ethanol 10.0 Triethanolamine 0.1 Preservatives, coloring, flavoring Appropriate amount Purified water remaining amount

[Formulation example 7] Nutritional lotion (milk lotion)

Nutritional lotion was prepared in a conventional manner according to the composition shown in the table below.

Formulation Ingredients Content (% by weight) Eluate of Preparation Example 1 1.0 glycerin 3.0 Butylene glycol 3.0 propylene glycol 3.0 Carboxy vinyl polymer 0.1 beeswax 4.0 Polysorbate 60 1.5 Caprylic/Capric Triglyceride 5.0 squalane 5.0 Sorvitasequiolate 1.5 liquid paraffin 0.5 cetearyl alcohol 1.0 Triethanolamine 0.2 Preservatives, coloring, flavoring Appropriate amount Purified water remaining amount

[Formulation Example 8] Massage Cream

Massage cream was prepared by a conventional method according to the composition shown in the table below.

Formulation Ingredients Content (% by weight) Eluate of Preparation Example 1 2.0 glycerin 8.0 Butylene glycol 4.0 liquid paraffin 45.0 beta glucan 7.0 carbomer 0.1 Caprylic/Capric Triglyceride 3.0 beeswax 4.0 Cetearyl Glucoside 1.5 Sorbitan sesquioleate 0.9 Vaseline 3.0 paraffin 1.5 Preservatives, coloring, flavoring Appropriate amount Purified water remaining amount

[Formulation Example 9] Preparation of hair lotion

Hair lotion is prepared according to a conventional method with the composition listed in the table below.

ingredient Content (% by weight) Eluate of Preparation Example 1 2.0 Purified water remaining amount glycerin 3.0 Butylene glycol 3.0 liquid paraffin 7.0 beta glucan 7.0 carbomer 0.1 edelweiss extract 2.0 Caprylic Capric Triglyceride 3.0 squalane 5.0 Cetearyl glucoside 1.5 Sorbitan Stearate 0.4 polysorbate 1.2 antiseptic Appropriate amount incense Appropriate amount pigment Appropriate amount triethanol amine 0.1

[Formulation Example 10] Preparation of shampoo composition

A shampoo composition was prepared according to a conventional method with the composition listed in the table below.

Ingredients (% by weight) Content (% by weight) Polyquaternium-10 0.5 Sodium Laureth Sulfate 20 Eluate of Preparation Example 1 0.3 incense 1.0 Methylparaben 0.1 cetyl alcohol 0.5 sodium chloride 0.8 Purified water To 100

[Formulation Example 11] Preparation of hair treatment

Hair treatments were prepared according to conventional methods with the compositions listed in the table below.

Classification (weight%) Content (% by weight) Eluate of Preparation Example 1 0.5 Stearamidopropyldimethylamine 2 glycerin 0.5 Spices 0.5 antiseptic 0.03 Cetostearyl alcohol 5 lactic acid One Distilled water to 100

Korea Microbial Conservation Center (overseas) KCCM11181P 20110328

Claims (11)

A composition for promoting hair growth or preventing hair loss comprising a lysate of Lactobacillus plantarum as an active ingredient. A composition for promoting proliferation and inhibiting death of hair follicle dermal papilla cells, comprising a lysate of Lactobacillus plantarum as an active ingredient. A composition for promoting proliferation and inhibiting death of outer root sheath cells comprising a lysate of Lactobacillus plantarum as an active ingredient. According to any one of claims 1 to 3,
The composition wherein the Lactobacillus Plantarum is a strain with accession number KCCM11181P. According to any one of claims 1 to 3,
A composition in which the Lactobacillus plantarum is isolated and identified from the tea tree. According to any one of claims 1 to 3,
A composition in which the eluate of Lactobacillus plantarum is obtained by heating and eluting cells of Lactobacillus plantarum. According to clause 6,
The composition, wherein the heating is performed at 80°C to 120°C for 10 to 30 minutes. According to any one of claims 1 to 3,
The composition is a cosmetic composition. According to any one of claims 1 to 3,
The composition is a pharmaceutical composition. A method for producing the composition according to any one of claims 1 to 3, comprising:
The method is:
(a) Culture Lactobacillus plantarum ,
(b) centrifuging the cultured Lactobacillus plantarum to remove insoluble components, and
(c) A composition containing as an active ingredient an eluate of Lactobacillus plantarum, which includes eluting the Lactobacillus plantarum obtained in (b) by heating it at 80°C to 120°C for 10 to 30 minutes. Manufacturing method. delete