KR100908745B1 - Hair growth promoter using low temperature nano extraction method and manufacturing method thereof - Google Patents

Abstract

A hair growth promoting agent using low nano extraction is provided to absorb the agent into hair follicle, be useful in hair loss prevention and hair growth and overcome shedding phenomenon. A hair growth promoting agent using low temperature nano extraction comprises 0.03-5.0 weight part of nicotinic acid amide, 0.01-5 weight part of salicylic acid, 0.05-10 weight part of Poria cocas Wolf., 0.03-8 weight part of Morus alba L., 0.05-10 weight part of Artemisia iwayomogi Kitamura, 0.5-30 weight part of sea mustard, and 0.5-30 weight part of Laminaria. A method for the hair growth promoting agent using the low temperature nano extraction comprises: a step of adding nicotinic acid amide in alcohol and sequentially adding salicylic acid, benzoic acid and lavender oil to obtain main solution; a step of putting together ethanol, purified water, pine needle, Poria cocas Wolf., Morus alba L., etc.; a step of extracting with 20-50% of alcohol liquid at 15-60°C for 24-48 hours; a step of filtering and centrifuging; a step of concentrating in a vacuum compression concentrator at 30-50°C for one to five hours to remove ethanol; and adding main solution.

Description

Hair Growth Promoter Using Low Temperature Nano Extraction Method And Manufacturing Method Thereof}

The present invention relates to a hair growth promoter using a low temperature nano-extraction method and a manufacturing method thereof, and more specifically, the hair growth promoting agent using the low-temperature nano extraction method extracted with a nano-size of 1000 ~ 3000Å at 15 ~ 60 ℃ using an organic solvent. The present invention relates to a hair growth promoting agent using a low-temperature nano-extraction method, including amide, salicylic acid, Bongyeong extract, lettuce extract, jinjin mugwort extract, seaweed extract, and kelp extract.

With the development of modern science, human beings are getting better every day and the dream of prolonging their lives is becoming a reality. Nevertheless, the solution of alopecia is still the task of humankind and is being researched and developed in many countries, research institutes and companies. The hair loss prevention and regrowth is a product that has a global marketability.

Human hair has a cosmetic function in addition to functional aspects such as protecting the head from sunlight, absorbing shock from external shock, and maintaining body temperature. In recent years, the number of female hair loss patients has increased due to changes in lifestyles such as westernized eating habits and frequent hair pollution such as perm and dyeing, and adolescent hair loss due to the fact that smoking, academic stress, and puberty itself are accelerated by an average of three years. Patients are also increasing.

Alopecia is a disease that is lost in the area where hair is normally present. It occurs mainly in the head and is classified into non-alopecia areata and alopecia areata according to the damage of hair follicles. No specific drug has been found. Minoxidil has been developed and used as an antihypertensive agent. After Dargie et al. Reported in 1977 the occurrence of hirsutism after oral administration of minoxidil, the topical coating of minoxidil began to be used as a treatment for alopecia.

This has resulted in the release of numerous unproven hair regrowth, hair growth and tonic products worldwide, and the damage caused by unproven products has long been left to consumers suffering from hair loss. Therefore, the development of new hair loss preventing agent and hair growth promoting agent is urgently needed.

In order to solve such a problem, there is known a technique related to a method for manufacturing a hair regrowth using a natural extract and a topical coating agent and a pill (Korean Patent Registration No. 476,731). However, the method of preparing the hair regrowth has a problem that the effect of hair growth is reduced because the natural extract particles acting on the hair root.

Accordingly, an object of the present invention is a hair growth accelerator using a low-temperature nano extraction method that solves the problems of side effects caused by conventional unchecked hair loss preventing agent and hair growth promoter by manufacturing a hair growth promoter extracted from natural herbs and seaweeds at low temperature nanoscale And the purpose is to provide a method for producing the same.

In order to solve the above object, the present invention, as a hair growth promoter, nicotinic acid amide, salicylic acid, bongyeong extract, lettuce extract, jinjin mugwort extract, seaweed extract, and kelp extracted at a nano size at 15 ~ 60 ℃ using an organic solvent It provides a hair growth promoting agent using a low-temperature nano extraction method comprising an extract.

In addition, the present invention is a method for producing a hair growth promoter, the main solution (A) in which nicotinic acid amide is added to ethanol, and benzoic acid and lavender oil are sequentially added to salicylic acid to dissolve the ethanol, purified water, and seaweed in seaweed, kelp, pine needles, Boiled with baekbaek leaf, black soybean, woobangeun, black jima, sewage, Cheongung, Bongyeong, Sangbaekpi, Injin mugwort, and Chihwang, and extracted with 20 ~ 50% alcoholic water as an extraction solvent at 15 ~ 60 ℃ for 24 ~ 48 hours. After the extract was prepared, the resultant was filtered with an ultrafilter, again filtered with a nanofilter, separated by centrifugation, placed in a vacuum depressurizer, and concentrated at a temperature of 30-50 ° C. for 1-5 hours to evaporate ethanol. After the addition, and then added to the extraction solution (B) to lower the alcohol concentration to 0.5 to 5%, to provide a method for producing a hair growth promoter using a low-temperature nano extraction method comprising obtaining a uniform solution of greenish brown.

The hair growth promoter using the cold nano extraction method of the present invention and a method for producing the same are used for the low temperature nano extraction method, so that the nano-sized hair growth promoter is well absorbed into the hair follicles, thereby inhibiting the expression of TGF-β2, a hair growth cell degenerative protein, hair loss. Prevents and is useful for wool, hair growth, and hair growth. In addition, since herbal medicines and seaweeds are used as raw materials, various side effects of hormone therapy are prevented and shedding phenomenon is overcome. In addition, it contributes to the creation of employment and the local economy, as well as the vitalization of R & D in the bio industry, which can replace the local industry, and the effect of replacing hair loss, wool, hair growth, and hair growth-related products.

The present invention is a hair growth promoting agent using a low-temperature nano-extraction method, nicotinic acid amide, salicylic acid, bongyeong extract, lettuce extract, jinjin mugwort extract, seaweed extract, and kelp extract extracted at a nano size of 1000 ~ 3000Å at 15 ~ 60 ℃ using an organic solvent It includes.

The hair growth promoting agent using the low temperature nano-extraction method further includes pine needle extract, cypress leaf extract, black soybean extract, and right root extract.

The hair growth promoting agent using the low-temperature nano extraction method further includes black jima extract, sewage extract, cheongung extract, and goji extract.

Here, the organic solvent includes ethanol or ether.

Specifically, the hair growth promoting agent using the low-temperature nano-extraction method is added to the nicotinic acid amide to ethanol, benzoic acid as a preservative and to the salicylic acid in order to dissolve the main solution (A) dissolved in the ethanol, the purified water, And seaweed, seaweed, pine needles, pine needles, cypress leaves, black soybeans, woobangeun, black jima, sewage, cheongung, bongyeong, baekbaekpi, injin mugwort and jihwang after boiling with 30% alcoholic water as an extraction solvent, and less than 3% After the addition of the alcohol concentration to the extraction solution (B) was added to make a uniform solution and then separated by centrifugation, filtered by nanometer, sterilized and put in a container. Here, the size of each functional extract is a nano size of 1000 ~ 3000Å. The hair growth promoter is well absorbed by hair follicles and inhibits the expression of TGF-β2, a hair growth cell degenerative protein, to prevent hair loss, and is useful for wool, hair growth, and hair growth. In other words, since the low-temperature nano extraction method is used, the nano-sized hair growth promoter is well absorbed by hair follicles and is useful for hair loss prevention, wool, hair growth, and hair growth. In addition, the hair growth promoter using the low temperature nano-extraction method overcomes the shedding phenomenon. Shading is a phenomenon in which newly growing healthy hair pushes out resting hair which should be removed anyway, which is a normal process that occurs between two to six weeks after hair loss treatment, and hair falls out more than usual at this time.

The nicotinic acid amide is one of the water-soluble vitamin B complexes, like nicotinic acid, and exists in vivo as nicotinamide nucleotides, NAD + and NADP +, which are coenzymes of this vitamin, and is involved in many oxidation and reduction reactions as coenzymes of dehydrogenase . Another function of nicotinamide nucleotides is the reduction of flavincoenzyme, in which case proton addition or desorption is involved in the 4-membered pyridine ring carbon atom. The nicotinic acid amide is hydrolyzed by nicotinamidase and the resulting nicotinic acid is reused for NAD synthesis. In addition, the nicotinic acid amide is methylated in the liver to form n-methylnicotinic acid amide, which is mixed with urine and discharged.

Since salicylic acid has the action of antipyretic and analgesic as o-oxybenzoic acid, it is internalized with sodium salt or calcium salt, and is used to improve solubility, stability, pharmacology, etc. by combining salicylic acid with various drugs.

The order is to protect the intestines and dried the fungal nucleus of Bokryeong, diuretic and lowering blood sugar, sedation and the like.

The baekbaekpi is a mulberry bark anti-inflammatory action, antioxidant action, skin soothing action, to prevent aging.

The jinjin mugwort is called cheonjae wormwood or Injincho and jaundice, soy sauce, cold cold, anti-inflammatory diuretics, etc. may be taken. The phosphorus mugwort contains dimethylescholetin, scopoline, essential oils, aromatic oxycarboxylic acids, flavonoids, oxycoumarins and the like in the outpost.

The seaweed is rich in dietary fiber and potassium, calcium, iodine, etc., to promote metabolism, postpartum cooking, constipation, obesity prevention, iron and calcium supplements are excellent.

In addition to various pigments such as carotene, xanthophylls, and chlorophyll, the kelp contains a lot of carbohydrates, such as manit and laminarin, which are made by carbon assimilation, and alginic acid, which are components of cell walls, and iodine, vitamin B2, glutamic acid, etc. Contains amino acids The kelp contains laminin, which lowers blood pressure.

The pine needles are an ingredient of oxyl palmitic acid is effective for skin, beauty and heart strengthening.

The white leaf has a hemostatic action.

In the black soybean, aggregation of platelets is suppressed.

The right root is the root of burdock soothes the sore face and treats sore throat, boil, mange and ringworm. It has been reported that pharmacological action also acts against the various filamentous fungi that cause cancer, activate liver function, purulent bacteria and skin disease.

The black jima is a black sesame to promote the recovery of the skin mucous membranes, greatly reduce the cholesterol level in the blood, and activate the bowel movements.

The sewage is tonic action, hematopoietic function strengthening, fatigue recovery action.

The uterus has a sedation, blood pressure lowering action, antibacterial action.

The sulfur is lowering blood sugar, diuretic, antibacterial action.

The content of the nicotinic acid amide of the hair growth promoting agent is 0.03 to 5.0 parts by weight, the content of the salicylic acid is 0.01 to 5 parts by weight, the content of the extract of the Bulgyeong extract is 0.05 to 10 parts by weight, and the content of the lettuce extract is 0.03 to 8 parts by weight. , The content of the jinjinjin mugwort extract is 0.05 to 10 parts by weight, the content of the seaweed extract is 0.5 to 30 parts by weight, and the content of the kelp extract is 0.5 to 30 parts by weight.

When the content of each of the components is less than the above range, it is difficult to exhibit hair growth, and when the content of each of the ingredients exceeds the above range, side effects may be exhibited.

In addition, the content of the pine needle extract of the hair growth promoter is 0.5 to 10 parts by weight, the content of the cypress leaf extract is 0.5 to 10 parts by weight, the content of the black soybean extract is 0.05 to 8 parts by weight, and the content of woobangeun extract 0.03 to 5 parts by weight.

When the content of each of the components is less than the above range, it is difficult to exhibit hair growth and skin soothing action, and when the content of each of the above components exceeds the range, side effects may be exhibited.

In addition, the content of the black jima extract of the hair growth promoter is 0.05 to 8 parts by weight, the content of the sewage extract is 0.03 to 5 parts by weight, the content of the cheongung extract is 0.03 to 5 parts by weight, and the content of the sulfuric acid extract is 0.03-7 weight part.

When the content of each component is less than the above range, it is difficult to exhibit hair growth and skin pharmacological action, and when the content of each of the above components exceeds the range, side effects may be exhibited.

In addition, according to the present invention, a method for preparing a hair growth promoting agent using the low-temperature nano extraction method includes adding nicotinic acid amide to ethanol, and sequentially adding benzoic acid and lavender oil to salicylic acid to dissolve the main solution (A) into the ethanol, purified water, and seawater. Boil it with kelp, pine needles, cypress leaves, black soybeans, right roots, black jima, sewage, Cheongung, Bongyeong, Sangbaekpi, Phosphorus mugwort and zucchini, and then extract with 20 ~ 50% alcohol water for 15 ~ 60 ℃ for 24 ~ 48 hours. Each extract was prepared by extracting from the filtrate, followed by filtration with an ultrafilter, followed by filtration with a nanofilter, followed by separation through centrifugation, and then into a vacuum vacuum concentrator, followed by concentration at a temperature of 30 to 50 ° C. for 1 to 5 hours. After evaporation of ethanol, the resultant is added to the extraction solution (B) having the alcohol concentration lowered to 0.5-5%, to obtain a greenish brown uniform solution.

Here, the content of the nicotinic acid amide is 0.03 to 5.0 parts by weight, the content of the salicylic acid is 0.01 to 5 parts by weight, the content of the seaweed extract is 0.5 to 30 parts by weight, the content of the kelp extract is 0.5 to 30 parts by weight, The content of pine needle extract is 0.5 to 10 parts by weight, the content of the cypress leaf extract is 0.5 to 10 parts by weight, the content of the black soybean extract is 0.05 to 8 parts by weight, the content of the right root extract is 0.03 to 5 parts by weight, The content of the black jima extract is 0.05 to 8 parts by weight, the content of the sewage extract is 0.03 to 5 parts by weight, the content of the cheongung extract is 0.03 to 5 parts by weight, and the content of the extract is 0.05 to 10 parts by weight, and The content of the turmeric extract is 0.03 to 7 parts by weight.

The above description of each extract and the content of each extract uses the preceding description.

In addition, the method for preparing a hair growth promoter using the low temperature nano extraction method is to perform the sorting, washing, drying, or shredding of the raw materials as preparation before extraction.

Specifically, the method for producing hair growth promoter using the low temperature nano extraction method is to extract the raw material by applying a step low temperature nano extraction method to the target object using ultrasonic waves after the screening and washing process using natural herbal ingredients and seaweeds to prevent hair loss prevention products It is intended to be manufactured in a way that maximizes the effective and extraction of specific components.

The hair growth promoter manufacturing method using the low temperature nano extraction method will be described with drawings.

1 is a process chart showing a method for preparing hair growth promoter using a low temperature nano-extraction method of the present invention.

As shown in Figure 1, after preparing algae, natural herbal medicines, herbal medicines, and other materials, the preparation work before extraction, such as sorting, washing, drying, crushing.

Then, each raw material is extracted with an organic solvent containing ethanol and distilled water.

After extracting the raw materials, the ultrafiltration process is performed, and the extract which is difficult to be separated by normal filtration is filtered by using dialysis paper using a sulfate sheet, a bladder membrane, and colloidion.

Thereafter, a nanofiltration process is performed using a nanofilter on the extract obtained by the ultrafiltration process.

Then, the resultant was separated by centrifugation, placed in a vacuum vacuum concentrator, and concentrated, and the ethanol was evaporated to lower the alcohol concentration to produce an extract (B).

Thereafter, the extraction solution (B) is added with the nicotinic acid amide to the ethanol, and the benzoic acid and the lavender oil are sequentially added to the salicylic acid and added to the dissolved main solution (A). Produces a solution, a product.

In addition, the hair loss management system of the present invention consultation and shooting; Primary sampu; Scalp steamer; Scalp peeling and oxygen supply; Infrared irradiation; Ampoule application; Scalp massage; And secondary sampu; sequentially.

In the counseling and photographing step, when a baldness visits, a person consults about eating habits and hair hygiene, and photographs a bald area with a digital camera. At this time, shoot with the hair enlarger.

In the first sampu step to remove the first contaminants such as foreign substances by scalp cleaning before management.

The scalp steamer step facilitates the removal of fine contaminants remaining in the scalp (mogu) by steam and absorption of the hair growth promoter ampoule.

In the scalp peeling and oxygen supplying step, oxygen is supplied to the scalp and the contaminant filling and scalp such as foreign matter in the scalp (mogu) relaxed by steam.

In the infrared irradiation step helps to circulate blood using infrared rays.

In the ampoule application step, the hair growth promoter ampoule for nourishing the scalp is finely sprayed with an oxygen jet spray.

In the scalp massage step to massage the scalp to improve the blood circulation of the scalp to increase the absorption of the hair growth promoter ampoule.

Finally, in the second shampoo phase, the final shampoo after absorbing the hair growth promoter ampoule creates a personal style.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

EXAMPLES Material Extraction and Methods

After sorting each material (natural herbal medicine and algae), it is prepared by washing, drying, crushing, etc. for detailed selective extraction according to the functional material of each raw material. It was made to include the extract. The extracted active ingredients are again subjected to primary extraction and concentration using ultrafiltration (ultrafiltration, MWCO, 20,000), followed by extraction and concentration according to molecular weight in the second order through nanofiltration (nanofiltration, MWCO, 10,000). As an example of to prepare a hair growth promoter (product name: JW12) using a low temperature nano-extraction method (see Fig. 2).
The composition and content of the extract in 100 ml of hair growth promoter (product name: JW12) using the low temperature nano extraction method of the present invention are as follows.
The content of nicotinic acid amide is 0.1g, the content of salicylic acid is 0.1g, the content of seaweed extract is 3g, the content of seaweed extract is 4g, the content of pine needle extract is 2.5g, the content of cypress leaf extract is 2.5g, the content of black soybean extract 1g of silver, 0.5g of zigzag extract, 1g of black jimma extract, 0.5g of sewage extract, 0.5g of cheonsung extract, 0.3g of cheongung extract, 0.3g of Bongyeong extract, 0.8g of lettuce extract g, the content of Insam mugwort extract is 0.5g, and the content of the sulfuric acid extract contains 0.3g, 0.2g of benzoic acid as a preservative, 0.1g of lavender oil as a flavoring agent, 30g of ethanol, and the rest is purified water and seawater Put it.

Experimental Example 1

Content of experiment

end. Establish standardization of extraction, separation, purification and concentration of functional ingredients from seaweeds and natural herbs through low temperature nano extraction

(1) In order to prevent hair loss and maximize the effects of wool and hair growth, functional bioactive substances that require specific heat treatment of raw materials were extracted and obtained at 50 ° C or below.

(2) Functional bioactive materials requiring organic solvent extraction were extracted from 50% organic solvent.

(3) Natural herbal medicines were separated / filtered secondly after the first hot water extraction.

(4) The concentration of the extract was tested for efficacy at 25% or 50% concentration (water and alcohol removal) using a general vacuum evaporation at 40 ° C.

I. Analysis of General Components of Extracts

Component analysis was conducted in 1st (2006/08/10), 2nd (2006/10/01), 3rd (2006/12/01), and 4th (2007/02/02).

(1) Analysis

 1) Elemental Analysis

ICP (Inductively Coupled Plasma Emission Spectrometer, Thermo Jarrel Ash Co., IRIS / AP) was used to investigate the elemental content and content of the sample. Sample (3 g) was added H ₂ O ₂ 2 to HN ₃ 10 and digested with Microwave (Malstone Co.,) and filled up with standard amount 20 with 2-3% HNO ₃ .

 2) Free Sugar Analysis

   HPLC (High Performance Liquid Chromatography, Waters Co., Sugarpak Icolumn) was used to investigate the free sugar component and content of the samples.

   Sample 1 was filtered through a microfilter and used as the sample.

 3) amino acid analysis

   An automatic amino acid analyzer (Automatic Amio Acid Analyzer, Pharmacia Biotech Co., Biochrom 20, Na form column) was used to investigate the amino acid content and content of the sample.

   The sample was hydrolyzed with 6N hydrochloric acid (HCl), filtered through a microfilter, and used as a sample.

 4) Microbial Analysis

   In order to analyze the microorganisms in the sample, the presence of Escherichia coli, bacteria, actinomycetes, yeast, and fungi was analyzed by the general Bergy's mannul method and 3M Petrifilm.

(2) Analysis result

 1) Elemental analysis (ppm) of the product (JW12; hair growth promoter)

Element JW0810 JW1001 JW1201 JW0202 K 1513.8 1766.5 1765.2 1577.5 Ca 466.4 485.5 505.4 407.9 Mg 708.1 780.7 740.6 1224.4 P 99.6 107.9 110.5 62.2 B 17.3 29.4 14.7 0 Mo 0.3 0.2 0.3 1.1 Si 9.6 11.9 9.8 8.2 Fe 7.3 6.3 5.5 3.2 Mn 9.9 10.5 10.9 9.4 Cu 0.6 0.4 0.4 0.7 CD 0 0 0 0 Cr 2 2.3 2.3 2.2 Pb 0 0 0 0 Zn 0.3 0.2 0.3 0 Ni 1.7 1.8 1.9 1.7 Ti 0 0 0 0 Al 0.4 0.4 0.3 0

2) Analysis of free sugar (%) of the product (JW12; hair growth promoter)

Name JW0810 JW1001 JW1201 JW0202 Glucose 9.97 8.56 9.40 8.92 Sucrose 13.47 13.58 10.27 6.52 Fructose 10.73 11.42 15.26 15.05 Maltose 0.00 0.00 0.00 0.00 Lactose 0.00 0.00 0.00 0.00

3) Microbiological analysis (cfu / ml) of the product (JW12; hair growth promoter)

Microorganism name JW0810 JW1001 JW1201 JW0202 Escherichia coli 0.00 0.00 0.00 0.00 General bacterial count 0.00 0.00 0.00 0.00 Actinomycetes 0.00 0.00 0.00 0.00 Yeast 0.00 0.00 0.00 0.00 Mold water 0.00 0.00 0.00 0.00

4) Amino acid analysis of the product (JW12; hair growth promoter)

No. Name JW0810 JW1001 JW1201 JW0202 One Asp 157.09 104.53 136.79 130.27 2 Thr 30.03 18.54 21.98 20.32 3 Ser 28.47 17.65 22.66 20.61 4 Glu 279.48 189.90 252.21 240.09 5 Pro 42.69 29.41 31.63 31.42 6 Gly 35.20 23.06 36.24 26.51 7 Ala 57.13 36.15 0.00 0.00 8 Cys 41.00 19.22 20.91 19.02 9 Val 0.00 28.99 4.29 4.57 10 Met 0.00 0.00 0.00 0.00 11 Ile 18.87 12.53 20.47 20.27 12 Leu 26.95 16.89 26.15 24.49 13 Tyr 0.00 0.00 16.29 14.79 14 Phe 19.15 10.99 0.00 0.00 15 His 0.63 6.42 0.00 0.00 16 Lys 42.68 24.93 33.93 31.52 17 Amm 171.52 147.99 169.98 165.06 18 Arg 0.00 0.00 0.00 0.00

5) Catechin analysis of product (JW12; hair growth promoter) (unit: mg%)

(-) EGC (+) C (-) EC (-) EGCG (-) ECG 67.314 3.094 3.872 9.118 0.956

6) Vitamin component of the product (JW12; hair growth promoter) (unit: mg / 100g)

Vitamin b1 Vitamin b2 Vitamin b6 Vitamin c 0.109 16.678 3.459 5.902

All. Long term preservation test of extract

JW12 was divided into 1st (2007/02), 2nd (2007/04), 3rd (2007/06), and 4th (2007/08), and the change of organic acid and phenolic acid were tested at two-month intervals. Table 7, Figure 3; Table 8, Figure 4).

 (1) Change of organic acid according to storage period

Storage Time (Month) Organic acid (Unit: mg%) Oxalic Citric Malic Malonic Succinic Fumaric JW-02 February 25.20 388.14 255.58 59.85 880.79 0.51 JW-04 month 25.16 304.57 248.95 58.94 1430.37 0.67 JW-06 25.07 305.67 253.04 58.08 1425.55 0.64 JW-08 25.01 316.95 243.61 79.75 1466.08 0.59

(2) Phenolic Acid Changes According to Storage Period

Storage Time (Month) Phenolics (Unit: mg%) Caffeic Chlorogenic p-coumaric Salicylic Ferulic Cinamic JW12-02February 5.39 1.00 0.86 0.84 1.69 0.13 JW12-04 9.44 1.26 1.01 1.00 1.67 0.11 JW12-June 9.21 1.41 1.01 1.03 1.60 0.11 JW12-08 9.21 1.41 1.01 1.03 1.60 0.11

la. Consignment institution (Department of Oriental Medical Materials Engineering, Kyung Hee University)

(1) Measurement of the proliferative effect of hair forming cells on JW12

  1) Experimental material

    Human hair follicle cells, human dermal papilla cells (HHDPC) were used as hair-forming cells, and cell culture medium was cultured using MSC and subcultured once every 3 days.

2) Sample Preparation

As a test substance, the final concentration of JW12, a natural product extract, was used at a final concentration of 0.05%, 0.1%, and 0.5%. H ₂ O was used as a negative control.

3) Cell growth rate measurement

    Proliferation evaluation as follows by quantifying the formation of formazan pigment by living mitochondria by 3- [4,5-dimethylthiazol-2-yl] -2 and 5-diphenyltetraolium bromide test (MTT) method Was carried out.

① The HHDPC cells were adjusted to an initial cell concentration of about 1 × 10 ⁴ cells / well per well using a 96-hole plate incubator.

② The treatment of sample (JW) was dissolved in H ₂ O as a solvent, adjusted to serum content at 5% concentration, and then diluted sequentially with MSC medium.

③ The medium was adjusted to an amount of about 200ml per well and incubated in 37 ° C. and 5% CO ₂ incubator for 3 days.

   ④ 50 ml of the MTT solution prepared at a concentration of 2 mg / ml was added per well, and left for 4 hours in the same incubator.

   ⑤ When the MTT reagent was determined to be absorbed into the intracellular mitochondria, the supernatant was removed, 150 ml of DMSO was added, and stirred for 10 minutes to uniformly dissolve formazan dye.

   ⑥ After that, the absorbance value was measured at 540 nm using an ELISA reader, and the cell number was measured according to a standard sample curve prepared in advance.

 (2) Regulation of TGF-β2 Expression, a Parental Growth Cycle Regulatory Protein by JW12

  1) Experimental material

   ① Human keratinocyte HaCaT was used as hair-forming cells, and cell culture medium was cultured using RPMI and subcultured once every 3 days.

   ② Human hair follicle cells, human dermal papilla cells (HHDPC) were used, and cell culture medium was cultured using MSC, and passaged once every 3 days.

2) Preparation of Sample

    JW12, a natural product extract, was used as a test substance and a final concentration of 0.1% or 1% was used.

3) Measurement of TGF-β2 Expression

① Incubate HaCaT and HHDPC in 60mm dish for 24 hours when each cell shows 70% confluency, and incubate in 0.5% FBS condition medium for 24 hours in 0.5% FBS condition medium to make final concentration 0.1% and 1%. After culturing for 24 hours by setting an untreated group (-) as a control group, each cell is harvested with TRIzol reagent.

  ② Isolate total RNA from each cell by chromoform extraction and isopropanol precipitation method and quantify RNA.

  ③ Synthesize TGF-β2 cDNA using reverse transcriptase from total RNA isolated using TGF-β2 specific primer using human TGF-β2 gene sequence and electrophores TGF-β2 DNA generated by PCR method.

(3) Research results

 1) Measurement of the proliferative effect of hair-forming cells on JW12 (see FIG. 5)

  ① The final concentration was treated to 0.05%, 0.1%, and 0.5%. As a result of measuring the cell proliferation rate of HHDPC at 48h, no cytotoxicity was observed at 0.05%, 0.1%, and 0.5% at 48 hours. Cell proliferation of about 16% was observed at the final concentration of 0.05%. JW12 showed proliferative effect in HHDPC.

   ② These results indicate that the hair growth promoting effect by JW12 has a significant activity in the prevention / treatment of hair loss, and thus can be considered to have the potential to develop a hair loss treatment using JW12.

 2) Regulation of TGF-β2 Expression, a Parental Growth Cycle Regulatory Protein by JW

   ① Inhibition of the expression of TGF-β2 of the hair growth cell degenerative protein induced by JW12 in HaCaT cells (see FIG. 6).

6 (A) shows Total RNA from HaCaT treated with JW12; Figure 6 (B) is RT-PCR products by specific primer for TGF-β2.

   ② Inhibition of the expression of TGF-β2 of the hair growth cell degenerative protein TGF-β2 by JW12 in HHDPC cells (see FIG. 7).

Here, Figure 7 (A) is Total RNA from HHDPC treated with JW12; Figure 7 (B) is RT-PCR products by specific primer for TGF-β2.

As a result of electrophoresis of RNA isolated from HaCaT and HHDPC in FIGS. 6 and 7, 28S rRNA, 18S rRNA, and 5S rRNA were clearly separated and the same amount was separated through each rRNA band.

     RT-PCR was performed using each group of RNA isolated from each group in HaCaT and HHDPC cells as TGF-β2 primer. The results showed that the expression of TGF-β2 was higher in the HaCaT and HHDPC cells than in the group treated with JW12.

     In the HHDPC cell group treated with different concentrations of 0.1% and 1%, the group treated with 0.1% JW had higher expression of TGF-β2 than the group treated with 1% JW12.

     As a result, the cells treated with JW12 had an effect of inhibiting the expression of TGF-β2. Therefore, it is thought that JW12 has an effect of delaying the entry time of Anagen to Catagen in the hair cycle cycle (see FIG. 8).

(3) Hair growth promoting effect by JW12 in C57BL / 6 mouse model

   1) Experimental animals were used for 5 weeks of male C57BL / 6 mice for one week after adapting to the environment of the animal breeding room, and induced growth phase through hair removal, and observed the hair color of the hair follicles. Efficacy of the sample on hair growth was evaluated by histological observation such as number measurement.

   2) The hair growth change of C57BL / 6 mice was observed in a time-dependent manner in the allopesia model (see FIG. 9).

   ① HE promoted the hair growth cycle about 2 days compared to the trypsin treatment group which delayed the hair growth phase.

   ② At 9 days after depliation, the HE-treated group promoted the hair growth phase for about 1 day than the mice with normal hair growth cycle.

   ③ In other words, the HE-1 and HE-2 medicinal treatment groups showed about 11.4% more hair growth promoting effect than the hair growth delay group.

conclusion

  end. In order to evaluate the efficacy of hair loss prevention and treatment, hair follicle cells were cultured in a culture medium containing JW12 in the cytotoxic effect on dermal papilla cells, which are hair-forming cells. To evaluate the toxicity and prolifer-ation in hair follicle cells, JW12 showed no toxicity in the hair follicle cell group with 0.05% and 0.1% added, and the cell growth rate of about 16% was observed in 0.05%.

  I. To evaluate the efficacy of regulating hair growth cycle, to evaluate the efficacy of hair follicle cell growth by JW12 in investigating changes in gene expression rates for proteins known to induce degenerative phase (TGF-β2) during hair growth cycle, TGF-β2 gene expression, a protein that induces GI, was isolated from total hair follicle cells treated with JW12, and RT-PCR was performed using a TGF-β2 specific primer. In, it was observed that the expression level of TGF-β2 was significantly decreased compared to the control group not treated with JW12.

   All. These results show that cell growth rate and decreased expression of TGF-β2, a protein that induces hair follicle degeneration, in the hair follicle experimental group using the culture medium added with JW12. JW12 has the effect of promoting the growth of hair follicle cells and delaying cell degeneration. Appeared.

Experimental Example 2 Photographs of Experienced People with Hair Loss Management Program

10 to 16 are hair pictures of subjects whose hair growth is promoted through the hair loss prevention program of the present invention. 10 to 16 subjects were successfully hair growth through the hair loss prevention program.

The above description is merely illustrative of the technical idea of the present invention, and those skilled in the art to which the present invention pertains may make various modifications and changes without departing from the essential characteristics of the present invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention but to describe the present invention, and the scope of the technical idea of the present invention is not limited by these embodiments. The protection scope of the present invention should be interpreted by the following claims, and all technical ideas within the equivalent scope should be interpreted as being included in the scope of the present invention.

1 is a process chart showing a method for preparing hair growth promoter using a low temperature nano-extraction method of the present invention.

Figure 2 is a process chart showing the technical standards of cold nano extraction, separation and concentration products of the present invention.

Figure 3 is a graph showing the change in organic acid according to the storage period of the extract of the present invention.

Figure 4 is a graph showing the change in phenolic acid according to the storage period of the extract of the present invention.

Figure 5 is a graph measuring the proliferation effect of HHDPC hair-forming cells by the hair growth promoter JW12 of the present invention.

Figure 6 is an electrophoresis picture showing the suppression of the expression of TGF-β2 hair growth cell induction phase in HaCaT hair-forming cells by the hair growth promoter JW12 of the present invention.

Figure 7 is an electrophoresis picture showing the suppression of the expression of hair growth cell degeneration phase protein TGF-β2 in HHDPC hair-forming cells by the hair growth promoter JW12 of the present invention.

8 is a photograph showing the regeneration of the hair growth cycle by the hair promoter JW12 of the present invention.

9 is an animal photograph showing the hair growth promoting effect of the C57BL / 6 mouse model by the hair growth promoter JW12 of the present invention.

10 to 16 are hair pictures of subjects whose hair growth is promoted through the hair loss prevention program of the present invention.

Claims (9)

Hair growth promoting agent using low temperature nano extraction method, including nicotinic acid amide, salicylic acid, Bongyeong extract, lettuce extract, jinjin mugwort extract, seaweed extract, and kelp extract extracted at 15 ~ 60 ℃ nano size of 1000 ~ 3000Å using an organic solvent. The method of claim 1, wherein the hair growth promoting agent using the low temperature nano extraction method hair growth promoter using a low temperature nano extraction method further comprises pine needle extract, cypress leaf extract, black soybean extract, and right root extract. The hair growth promoting agent of claim 1, wherein the hair growth promoting agent using the low temperature nano-extraction method further comprises black jima extract, sewage extract, cheon-ung extract, and sulfuric acid extract. According to any one of claims 1 to 3, The content of the nicotinic acid amide is 0.03 to 5.0 parts by weight, the content of the salicylic acid is 0.01 to 5 parts by weight, the content of the extract is 0.05 to 10 parts by weight, The content of the lettuce extract is 0.03 to 8 parts by weight, the content of the jinjin mugwort extract is 0.05 to 10 parts by weight, the content of the seaweed extract is 0.5 to 30 parts by weight, and the content of the kelp extract is characterized in that 0.5 to 30 parts by weight. Hair promoter using low temperature nano extraction method. According to claim 2, wherein the content of the pine needle extract is 0.5 to 10 parts by weight, the content of the cypress leaf extract is 0.5 to 10 parts by weight, the content of the black soybean extract is 0.05 to 8 parts by weight, and the content of the right root extract Hair growth accelerator using a low-temperature nano extraction method, characterized in that 0.03 ~ 5 parts by weight. According to claim 3, wherein the content of the black jima extract is 0.05 to 8 parts by weight, the content of the sewage extract is 0.03 to 5 parts by weight, the content of the cheongung extract is 0.03 to 5 parts by weight, and the content of the sulfuric acid extract is Hair growth accelerator using a low-temperature nano extraction method, characterized in that 0.03 ~ 7 parts by weight. Nicotinic acid amide was added to ethanol, benzoic acid and lavender oil were dissolved in order to dissolve salicylic acid. Sewage, Cheongung, Bongyeong, Sangbaekpi, Injin mugwort, and botanicals were boiled, extracted with 20 to 50% alcoholic water at 15 to 60 ℃ for 24 to 48 hours, and each extract was prepared and filtered through an ultrafilter. After filtration through the nano-filter again, separated by centrifugation, put into a vacuum vacuum concentrator and concentrated for 1 to 5 hours at a temperature of 30 ~ 50 ℃ evaporated ethanol, the alcohol concentration to 0.5 to 5% After adding to the lowered extraction solution (B), a method for producing a hair growth promoter using a low-temperature nano extraction method comprising obtaining a uniform solution of greenish brown. According to claim 7, wherein the content of the nicotinic acid amide is 0.03 to 5.0 parts by weight, the content of the salicylic acid is 0.01 to 5 parts by weight, the content of the seaweed extract is 0.5 to 30 parts by weight, the content of the kelp extract is 0.5 to 30 Parts by weight, the content of the pine needle extract is 0.5 to 10 parts by weight, the content of the cypress leaf extract is 0.5 to 10 parts by weight, the content of the black soybean extract is 0.05 to 8 parts by weight, the content of the right root extract is 0.03 to 5 Parts by weight, the content of the black jima extract is 0.05 to 8 parts by weight, the content of the sewage extract is 0.03 to 5 parts by weight, the content of the cheongung extract is 0.03 to 5 parts by weight, the content of the extract is 0.05 ~ 10 weight In part, the content of the extract of S. alba extract is from 0.03 to 8 parts by weight, the content of the extract of Injin mugwort is 0.05 to 10 parts by weight, and the content of the sulfuric acid extract is 0.03 to 7 parts by weight. room . The method of claim 7, wherein the method for preparing a hair promoter using the low temperature nano extraction method comprises selecting, washing, drying, or crushing raw materials as preparation before extraction.

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