KR20180082351A - A composition for promotion of hair growth comprising extracts of Eclipta prostrata - Google Patents

Abstract

The present invention relates to a composition for promoting hair growth comprising infused oil obtained by extracting an oil component of Eclipta prostrata using vegetable oil. The present invention relates to a method for manufacturing Eclipta prostrata infused oil using vegetable oil. The infused oil contains both vegetable oil component and extracted component from Eclipta prostrata so as to have an excellent effect of promoting hair growth, thereby being used in cosmetics, pharmaceuticals and foods. The method comprises the steps of: mixing leaves of Eclipta prostrata and vegetable oil; extracting the mixture; and filtering the extract.

Description

[0001] The present invention relates to a composition for promoting hair growth,

The present invention relates to a composition for promoting hair growth, which comprises a herb extract using a vegetable oil, and a method for producing the composition.

The hair of the human body is about 110, 150 thousand, and it is formed in "hair follicle". There is a nipple in the hair follicle, where small blood vessels are distributed to supply the nutrients needed to grow hair, and beside the papilla there is a bore hole that supplies the hair with shine oil. Hair follicles are composed of several different epithelial cells and dermal papilla cells (DPCs). DPCs are mesenchymally-derived fibroblasts located at the base of hair follicles and play an important role in hair growth. In particular, minoxidil has been reported to have proliferative and anti-apoptotic effects on DPCs. Similarly, several reports have shown an increase in hair growth through the proliferation of DPCs. Each hair has a different cycle and grows through anagen, catagen, and telogen, and then falls off. This cycle repeats over 36 years, with 50100 hairs averted out on a daily basis. In general, alopecia refers to an abnormal increase in the number of hair that has fallen due to a shortening of the percentage of growing hair and an increase in the number of retrogressive or resting hair among these cycles.

The side effects of chemotherapy include leukocytopenia, vomiting, diarrhea and alopecia, and chemotherapy induced alopecia is reported to occur in about 65% (Trueb RM, 2010). 5-fluorouracil is a type of antimetabolite that inhibits thymidylate synthase and induces apoptosis and cell death (Longley DB et al., 2003).

Although many studies on hair loss have been conducted in this way, the mechanism of essential hair loss is not well known. Efforts have also been made to treat hair loss. Nonetheless, so far only two drugs (finasteride and minoxidil) have been approved in the Food and Drug Administration (U.S.A) for the treatment of hair loss. The 5α-reductase inhibitor, finasteride, has been used to promote hair growth in male androgenetic alopecia. Minoxidil, an anti-hypertensive agent, can promote hair growth by opening the ATP-sensitive K + -channel. However, the effects of drugs are limited and transient due to unpredictable effects and side effects. There is a need for better new treatments to prevent hair loss and promote hair growth. In the case of preparations such as minoxidil and trichosaccharide, which have been known to prevent hair loss and promote hair growth and have the effect on hair growth, there is a lack of clear efficacy, There is a problem of side effects, and it is urgent to develop a composition having safety and efficacy.

In addition, extraction and purification of oil components are considered to be important techniques for use as a functional food, a pharmaceutical composition, and a cosmetic composition as main components of plant resources such as Hansuksu, camellia, and oilseed rape. When water is used in the extraction of the oil components, the oil components are not extracted sufficiently. Therefore, the extraction is performed using an organic solvent, but the harmfulness of the organic solvent has been questioned.

The infused oil refers to an oil obtained by extracting an oil component of a plant using an oil as an extraction solvent in order to solve the problem of oil component extraction as described above. The infused oil has a synergistic effect with the functional ingredient of the oil used as a solvent and the effective ingredient of the oil extracted from the plant to extract the oil component of the plant, so that the content of the active ingredient is increased and the functionality is improved. It can be applied to various products.

Accordingly, the present inventors have confirmed the hair-improving effect in the active ingredient of the infused oil extracted with camellia oil or rapeseed oil and developed a cosmetic composition and a pharmaceutical composition using the same.

Korean Patent No. 10-1238706

It is an object of the present invention to provide a method for producing fused oil, which is a herbicide using vegetable oil.

Another object of the present invention is to provide a cosmetic composition for facilitating hair growth comprising fused oil as an active ingredient.

Another object of the present invention is to provide a health functional food for promoting hair growth, comprising fusid oil as an active ingredient.

The present invention provides a method for producing fused oil,

Specifically,

Mixing the herb leaves with plant oil at a ratio of 1: 10;

Extracting the mixture by hot-pressing for 1 hour in a water bath at 50 to 70 ° C; And

And filtering the extract to form a fusible oil.

In addition,

Mixing the herb leaves with plant oil at a ratio of 1: 10;

Extracting the mixture by vacuum low temperature method; And

And filtering the extract to form a fusible oil.

As used in the present invention, the term "Hansikcho, " is a perennial plant native to the rice fields of the central and southern regions of Korea, where moisture is present in the brook, and is also referred to as Jangjang, Mokwoon, It contains saponin and tannin, gumminess, echulidine (vitamins A), vitamin A substances, nicotine and coumarin compounds wedelolactone and demethylwedelolactone-7-glucoside.

When water is used in the extraction of the oil components from the above-mentioned extract, the oil components are not extracted sufficiently. Therefore, the extraction is carried out using an organic solvent, but the harmfulness of the organic solvent has been a problem.

The term " infused oil " as defined in the present invention refers to oil obtained by extracting an oil component of a plant by using oil as an extraction solvent in order to solve the problem of oil component extraction as described above. In the present invention, the " infusidine oil " The infused oil has a synergistic effect with the functional ingredient of the oil used as a solvent and the effective ingredient of the oil extracted from the plant to extract the oil component of the plant, so that the content of the active ingredient is increased and the functionality is improved. And can be applied to various products.

Specifically, the present invention uses vegetable oil for extracting the oil component from the persimmon, and more specifically, camellia oil or rapeseed oil can be used.

In the above-described method for producing fused oil, it is preferable to use an on-precipitation method, and preferably, the extraction is carried out in a water bath at 60 ° C for 1 hour.

The present invention provides a cosmetic composition for accelerating hair growth, comprising fusidic oil, which is Hansikcho, prepared by the above method, as an active ingredient.

In the present specification, hair loss refers to a phenomenon in which the hair falls off from the scalp or hair is tapered or tapered. The term "hair loss prevention" means prevention and suppression of the hair loss phenomenon as described above, Not only promotes the production of hair but also allows the existing hair to grow healthily.

Generally, hair growth occurs in the growing phase, and is induced by the delay from the dormancy to the growth phase and from the growth phase to the regressor. The hair cycle can be divided into three main stages known as growth phase, regressor phase, and quiescent phase. During the growing season, hair growth takes place as the hair follicles grow deep into the skin with rapid cell proliferation. The next phenomenon is the retrogressive period, which is a transitional period in which disruption of cell division is prominent, during which the hair follicles gradually regress and hair growth stops. In the next pause, the regressive hair follicle contains germs with densely packed dermal papilla cells. The onset of a new growth phase in dormancy is induced by rapid cell proliferation in the abdomen, swelling of the papilla and synthesis of basement membrane elements.

Therefore, it is necessary to prevent hair loss by promoting or prolonging the growing period, that is, to prevent hair loss or to induce regrowth of hair, that is, to promote hair growth, and the composition according to the present invention is effective in preventing hair An effect of improving the existing hair such as thickening, and an effect of generating new hair.

The concentration of the fusing oil is preferably 1 to 100 μg / ml, but is not limited thereto. The fusing oil of the present invention is extracted from a natural product and thus has almost no toxicity.

The composition may be used in hair cosmetics such as hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair conditioner, hair treatment, hair cream, hair nutrition cream, hair moisturizing cream, Hair dyes, hair dyes, hair dyes, hair dyes, hair dyes, hair dyes, hair dyes, hair dyes, hair dyes, hair dyes, hair moisturizers, Mousse, and hair spray. However, the present invention is not limited thereto.

The cosmetic composition may be prepared by dissolving or dispersing at least one compound selected from the group consisting of a fatty substance, an organic solvent, a solubilizer, a thickening agent, a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, Such as cosmetics or skin, such as fillers, sequestering agents, chelating agents, preservatives, vitamins, barrier agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient commonly used in cosmetics And may contain adjuvants conventionally used in the scientific field. Such adjuvants are introduced in amounts commonly used in the cosmetics or dermatological fields.

The cosmetic composition according to the present invention can be, for example, a cosmetic composition, and the external form of the cosmetic composition contains a cosmetically or dermatologically acceptable medium or base. It may be in any form suitable for topical application, for example, as a solution, a gel, a solid, a paste anhydrous product, an emulsion obtained by dispersing the oil phase in water, a suspension, a microemulsion, a microcapsule, In the form of a non-ionic follicle dispersing agent, or in the form of creams, skins, lotions, powders, ointments, sprays or conical sticks. These compositions may be prepared according to conventional methods in the art. The composition according to the invention may also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic composition of the present invention is not particularly limited in the form of the cosmetic composition of the present invention. For example, the cosmetic composition of the present invention can be used in a variety of forms such as a softening agent, a convergent lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, Water, a pack, a powder, a body lotion, a body cream, a body oil and a body essence.

The cosmetic composition as described above may be applied to the skin, or may be applied to the skin using a micro needle or the like.

In addition, the compositions of the present invention may be made into a hair composition comprising a carrier or carrier mixture suitable for application to the hair. The carrier comprises a vehicle component commonly used in a carrier or other carrier or hair protective composition, and may preferably be in the form of a liposome. Is present at about 0.5% to 99.5%, preferably about 5.0% to 99.5%, most preferably about 10.0% to 90.0%, of the composition of the total composition. As a carrier, the solvent is selected by the copolymer to be used, regardless of whether the formulated hair composition remains on the hair after use such as hair spray, mousse, tonic, etc., or whether it is cleaned, such as shampoos, conditioners and the like. Suitable solvents for use in the present invention include water, lower alcohols (ethanol, isopropanol, etc.), hydroalcoholic mixtures, hydrocarbons such as isobutane, hexane and decene, acetone, halogenated hydrocarbons such as Freon, Dibutyl phthalate and the like), volatile silicone derivatives, siloxane (phenylpentamethyldisiloxane, methoxypropylheptamethylcyclotetrasiloxane, chloropropylpentamethyldisiloxane, hydroxypropylpentamethyldisiloxane, octamethylcyclotetrasiloxane, decamethyl Cyclopentasiloxane, and the like), and mixtures thereof.

In addition, the present invention provides a health functional food for preventing hair loss or promoting hair growth comprising fused oil as an active ingredient.

The concentration of the fusing oil is preferably 1 to 100 μg / ml, but is not limited thereto.

The health functional food is preferably powder, granule, tablet, capsule or beverage, but is not limited thereto.

The food of the present invention can be used as it is or in combination with other food or food ingredients, and can be suitably used according to conventional methods.

There is no particular limitation on the kind of the food. Examples of foods to which the fusible oil can be added include milk, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Tea, a drink, an alcoholic beverage, and a vitamin complex, and includes foods in a conventional sense.

The beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are 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. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.001 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention relates to a composition for promoting hair growth comprising an infused oil obtained by extracting an oil component of Hansuk Ecology using a vegetable oil. The infused oil contains both the vegetable oil component and the extracted component from the haniksikcho so that the effect of promoting hair growth is excellent and can be utilized in cosmetics, pharmaceuticals and foods.

Fig. 1 is a process drawing showing the step of producing a myrrh.
FIG. 2 is a visual observation of the hair growth effect after treating the mice with a Hanchonchos extract extracted with a control, minoxidil, camellia oil and camellia oil.
FIG. 3 shows the result of image analysis of hair growth effect using Image J after treatment of mice with a extract of Han, Suk Cho, which was extracted with a control, minoxidil, camellia oil and camellia oil.
FIG. 4 is a graph showing the results of analyzing the hair growth effect using Image J after treating the mice with the extract of Han, Suk Cho, which was extracted with the control, minoxidil, camellia oil and camellia oil.
FIG. 5 is a graph showing the results of analyzing the hair growth effect using Image J after treating the mice with the extract of Han, Suk Cho, which was extracted with the control, minoxidil, rapeseed oil and rapeseed oil.
FIG. 6 is a result of observation of a skin follicular breakfast in a mouse treated with a control group, minoxidil, camellia oil, extracts of a mushroom extract extracted with camellia oil, extracts of rapeseed oil and rapeseed oil.
FIG. 7 is a diagram illustrating an antibody array process for examining expression of proteins involved in hair growth and degeneration expressed in a mouse or the like.
FIG. 9 is a scan image of protein expression rates involved in the growth and degeneration of hair follicles in the skin, such as a control, minoxidil, camellia oil, and mice coated with a extract of Hansikoso extract of camellia oil.
FIG. 9 is a flow chart of a process for manufacturing a hair product (essence) containing an extract of Han Silicin (infused oil) extracted with camellia oil.
10 is a view showing a manufacturing process of a hair product (shampoo) containing an extract of Hansikoso extract (infused oil) extracted with camellia oil.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more fully explain the present invention to those skilled in the art.

<Example 1> Preparation of Hansikucho extract using camellia oil or rapeseed oil

<1-1> Manufacture of Hansik's herb extract using onion method

Two groups of 40g each were collected on the electronic scales. The quantified Hanwiche leaf was mixed with rape oil or camellia oil (400ml) to a concentration of 10%. The mixture was extracted with a hot water bath at 60 ° C for 1 hour. After extraction, large leaves were filtered by 0.45 μm filter paper and secondary filtered through a 0.2 μm membrane filter. The extract was dispensed and labeled and then refrigerated (Fig. 1).

<1-2> Preparation of Hansik Sekwajo extract using vacuum low temperature method

In order to prepare the extract of Hansikucho, Hansikucho was infused into camellia oil or rapeseed oil by vacuum low temperature (low temperature high pressure) method. The ratio of inflorescence to rapeseed oil was 1:10. After removing the residue of the infused Hanchuksikcho extract through 0.45 μm filter paper, it was filtered again through a 0.2 μm membrane filter.

<Example 2> Liposome formulation development

end. Liposome preparation

1) Mare is melted in a 40 ° C water bath.

2) Add lecithin, auxiliary emulsifier, etc. to purified water and dissolve at 75 ℃.

3) After cooling to about 50 ° C, mix with the sample and stir into the microfluidizer.

※ Microfluidizer pressure setting: Open the pressure gauge valve, turn the pressure control switch to 25,000 psi and lock the pressure gauge valve.

4) When pressure setting is completed, put the mixed material into Product Inlet Reservoir and start Liposome.

I. Liposome operation

1) Pull the Intensifier On / Off switch and the Microfluidizer's pump will start working.

2) Stop pump 7 times and discard the product. (Removing foreign matter from the line)

3) After turning on the Intensifier switch, first collect the product through the discharge hose.

4) Place the primary product back into the Inlet reservoir, turn on the Intensifier switch, and collect the product secondarily.

5) Put the secondary product back into the Inlet reservoir, turn on the Intensifier switch and collect the product third.

Using the above procedure, a liposome formulation having a particle size of 180 nm was obtained.

&Lt; Example 3 > Evaluation of Hair Growth Efficacy of Hansik's Herb Extract (Infused Oil)

end. Evaluation of Hair Growing Effect of Hansukscho using C56BL / 6N

 1) C57BL / 6N mouse

In addition to the proliferation of subcutaneous dermal papillary cells in the growth phase, the activation of melanocytes in the upper part of the dermal papilla coincides with the activation of the dermal papilloma. However, since it has a pink character when it enters the dormant period, And C57BL / 6N mice, which have the merit that melanin is produced only in the growth phase of the hair follicle cycle, and which has a tendency to grossly estimate the hair cycle.

- Hairy traits of C57BL / 6N mice

In C57BL / 6 female mice, the hair follicles develop after birth, after 2 weeks, after 3 weeks, after 4 weeks, after 6 weeks, after 6 weeks, after 7 weeks, after 12 weeks of growth, and in female, There are characteristics. In addition to genetic factors, environmental factors such as sex, temperature, sunlight, and nutritional factors also affect the hair cycle.

In the case of C57BL / 6, which is a standardized model, C57BL / 6 was started after 1 day after epilating, 3 days after growth, II after 4 days, IIIa-c after 5 days, IV after 6 days, V after 8-12 days After 17 days, and after 25 days. Thus, 6-week-old female C57BL / 6N mice were selected as experimental animals for resting period, in which the hair growth inhibitory effect was observed and the change of pigmentation of hair and hair growth was best observed.

2) Hair removal of mouse

 - Tools and materials: hair removal cream, clipper, laboratory instrument, ether

 - Mice were anesthetized by induction of anesthesia for 5-10 minutes with 0.08 ml (80ul) / L ether of 1.9% concentration. After the anesthesia, the mouse was firstly depilated with a clipper, and the depilatory cream was applied, and after 5 minutes, the second epilating was performed again.

3) Sample application

(N = 5), minoxidil (n = 5), camellia oil (n = 5), hanchikcho (camellia oil extract, n = 5) and rapeseed oil (n = 5) and Hankyucho (extracted with rapeseed oil, n = 5) were used. The sample was quantitated with a pipette to 100 ul and applied to the dorsal skin of the depilated mouse. After application, it was absorbed well by hand to be absorbed.

4) Isolate skin tissue

 The mice divided into groups were sequentially anesthetized by ether anesthesia and euthanized to collect skin. The skin was divided into two pieces, one was frozen sample for antibody array analysis and the other piece was stored in 10% formalin fixative for histological examination.

I. Analysis of Hair Growth Efficacy of Hansukscho using Image J

The area of the epilating area of the extracted rectangle was analyzed using Image J (FIG. 2 and FIG. 3). Data by group and date were obtained and averaged. The standard deviation was added and plotted (Figs. 4 and 5).

As a result, minoxidil, camellia oil, camellia oil extract, and Hanwikcho were applied on epilated C57BL / 6 mice. Image intensities of hair growth for 16 days were analyzed by Image J. As a result, &Lt; 0.05, and showed similar hair growth effect to that of the positive control group, minoxidil. No significant increase was observed in the camellia oil alone.

Minoxidil, rapeseed oil and rapeseed oil extracts were applied to epilated C57BL / 6 mice for 16 days. The results showed that hair growth potential of Hanwikcho extracted from rapeseed and rapeseed oils was lower than control Day4 and day8, but not on day 14 and day 16, respectively.

<Example 4> Histological analysis of hair follicles for evaluating hair growth potential of the extract of Han licorice (infused oil)

H & E staining was used to compare histologic changes of hair follicles between groups. A concrete method is as follows.

end. Tissue specimen production

1) Fixed

 When the supply of oxygen to the cell is stopped, the cell membrane component which is holding the hydrolitic enzymes is not maintained, and the hydrolytic enzyme comes out and the cell organelles digestion, so that the cell structure is completely destroyed . To prevent this, treatment with formalin at a certain concentration causes intracellular components to remain in that state without further degradation.

 The goal of tissue fixation is to denaturation and coagulation of proteins in tissues to give the tissues some degree of hardness while at the same time stopping the action of enzymes and preserving the state of cells or tissues in a fixed state .

2) Tissue triming and tissue processing (Tissue processing)

 It is a process of finishing some of the entire organization to make it an organizational slide. The tissue is trimmed to a size suitable for tissue processing, embedding, and dissection, and classified into cassettes in groups.

 Tissue processing is the process of dehydration and transparency, and it is necessary to wash the tissue after it is fixed. Dewatering gradually removes moisture in tissues with a high concentration of ethanol solution. In addition to ethanol, other organic solvents such as acetone are used. However, there is a disadvantage that the fat tissue in the tissue is dissolved due to the dehydration process. Once the dehydration is complete, the transparency process begins, followed by the use of xylene, which increases the penetration of paraffin through the tissue. Since the tissue processing process takes about 16 hours in total, it is performed using an automatic tissue processor.

* Organizational Processor Protocol

70% EtOH 3-6 hours

80% EtOH 1 hours

90% EtOH 1 hours

95% EtOH 1 hours

100% EtOH 1 hours

100% EtOH (2 steps) 1 hours

100% EtOH (3 steps) 1 hours

Xylene 1 hours

Xylene (Step 2) 1 hours

Xylene (Step 3) 1 hours

Paraffin 1 hours

3) Tissue embedding

 In order to observe the tissue under an optical microscope, its thickness must be thin enough to allow light to pass through. For this purpose, a microtome is used to cut the tissue to a uniform thickness. Since the tissue is not rigid enough to be thinly cut and irregular in shape, first paraffin is infiltrated into the tissue, And cut using a periodic machine. The process of infiltrating paraffin into tissue and making it hard is called embedding.

 After selecting the tissue mold for the tissue, pour the paraffin over the tissue mold and arrange the tissue appropriately. Let the cut side go down.

 Place the cassette on the mold and follow the paraffin. Place the mold on the chill plate and cure so that the cassette and paraffin are fixed slowly.

4) Tissue cutting

로 박절한다. 박절된 조직을 충분히 펴지도록 항온 수조에 옮긴다 이때 조직이 찢어지거나 다른 artifact가 발생하지 안도록 조심스럽게 작업을 하여야한다. 만약 조직이 찢어지거나 주름이 심하게 발생하면 조직 박절 날을 교체하여야 한다. It is a thin cutting process to see the tissue under a microscope. A special machine, a microtome, is used to cut the embedded paraffin tissue thinly. The paraffin tissue should be stored in a post-mortem freezer and should be cold enough to allow good dissection. Also, paraffin tissues should be kept frozen to dissect as thin as desired. The frozen tissue is fixed in the paralysis, the handle is turned, and the paraffin is cut off until the tissue comes out. After that, the tissue is frozen and stored at the desired size.

. Transfer the discarded tissue to a thermostatic bath so that it is fully stretched. Care should be taken to avoid tissue tearing or other artifacts. If tissue is torn or wrinkled excessively, tissue cutting edges should be replaced.

 If the tissue is placed in a constant-temperature bath, the tissue is spread out for easy observation. If it is stretched sufficiently, it should be transferred to the tissue using a slide glass. At this time, if the tissue is moved to the slide glass later, the tissue is excessively stretched and torn.

5) Tissue Dyeing

 When the tissue is cut thin enough to transmit light, the contrast of the tissue components is very low, and when the optical microscope is observed, the structures in the tissue are not distinguished. Staining the tissue components to enhance the color contrast effect of the tissue components. A typical tissue sample is stained using two types of dyes, hematoxylin and eosin, to easily distinguish the cell nucleus and cytoplasm.

① Remove the tissue paraffin with xylene.

(2) The water-soluble dye is hydrated while being treated with a low-concentration alcohol solution in a high-concentration alcohol so as to stain the tissue well.

③ First dye with hematoxtlin aqueous solution. At first, the staining is done by differentiating the staining intensity of the excessively stained specimen with an alcohol solution after dark staining.

④ Secondary dyeing with eosin. Hematoxylin stains the nucleus of the cell and eosin stains the cytoplasm.

⑤ If water is present after the specimen is dyed, the propagation of fungi or bacteria is easy, and the tissue may be destroyed. In order to prevent this, dehydration is carried out by passing through high alcohol concentration again, and cover glass is covered with balsam.

6) Hematoxylin & Eosin staining

Dyeing reagents are prepared and stained to stain the tissue. Dyeing is a process to look at the tissue well and the staining protocol can be changed according to the situation, but the general protocol is as follows

① xyleneⅠ 5 minutes

② xyleneⅡ 5 minutes

③ 100% ethanol 3 minutes

④ 95% ethanol 3 minutes

⑤ 90% ethanol 3 minutes

⑥ 80% ethanol 3 minutes

⑦ 70% ethanol 3 minutes

  ⑧ Washing water for 5 minutes

⑨ Hematoxylin 5 minutes

⑩ Washing water for 3 minutes

⑪ Eosin 2 minutes

⑫ 80% ethanol 2-3 dipping

- Visual confirmation of dyeability

⑬ 95% ethanol 3 minutes

⑭ 100% ethanol 3 minutes

⑮ Xylene III 5 minutes

 Xylene IV 5 minutes

I. Microscopic observation and evaluation

 Observe the tissue using an optical microscope. There are a variety of methods to analyze according to the type of experiment depending on the type of tissue. The depth of hair follicles, the number of hair follicles seen in one field of view, and the stage of growth of the hair follicles should be evaluated in order to evaluate the hair growth potential of this experiment. The higher the number of hair follicles suggesting the entry into the growth phase, the greater the hair growth potential of the sample. The smaller the number of follicles entering the retrograde phase, the slower the induction of retrograde regeneration of the sample. Through the microscope, we look at the tissue as a whole and compare the number and depth of hair follicles with how the hair follicles have entered the stage.

In general, the hair follicles grow from the boundary between the epidermis and the dermis to the dermis during the growing season. When entering the growing stage, dermal papilla is formed from dermal papilla fibroblast, from which inner root sheat is formed and hair grows in it. Growing hair follicles grow vigorously with hair shaft and develop sebaceous gland, an accessory. During the growing stage 6, the hair follicles reach the subcutaneous layer from the dermal layer and the hair protrudes out of the pore at the stage 8.

As a result of observing the hair follicle of the dorsal skin of the mouse to which the sample was applied through a microscope at a magnification of 40 times, the hair follicle was least observed in the control group and the overall hair follicle was in the initial stage of growth. In the case of minoxidil, the number of hair follicles was higher than control, and the growth period was more advanced than control. In the camellia oil group, the growth phase was similar to that of minoxidil and the number of hair follicles was observed. In the case of Camellia sinensis group, hair follicles were most frequently observed and many hair follicles that entered into growth period were also observed. There was not much hair follicle entering the retrogression. In the case of rapeseed oil and oilseed rape, many hair follicles were observed at the growing stage compared with control, but not as much as camellia rapeseed (Fig. 6). In addition, most of the growing follicles were observed in the camellia group. The number of total hair follicles, including the growing follicles, was highest in the camellia group, which is thought to be due to the growth period of the hair follicle itself. Especially when the last stage of the growing season and the short period of long anagen were observed to be less observed. In the case of camellia oil, rapeseed oil, and oilseed rape, hair follicles were observed more frequently than those of controls (Table 1)

Comparative evaluation of hair follicles by sample Stage of hair follicle sample ID Long anagen Anagen # Telogen # Others control One + + + 2 + + + 3 - ++ + 4 + + - Minoxidil One + + - 2 + + + 3 + + + 4 + + + 5 + + - Camellia oil One + ++ + 2 + + - 3 - ++ + 4 - - + 5 + + - Dongbaek One - +++ ++ 2 + ++ + 3 - ++ + 4 - +++ ++ 5 - +++ ++ Rapeseed oil One + ++ + 2 + + ++ 3 + + + 4 + ++ + 5 + + + A rapeseed drill One + + + 2 + + - 3 - ++ + 4 - ++ + 5 + ++ +

< Example  5> Infused  Analysis of protein expression involved in growth and degeneration of hair using Antibody array for evaluation of hair growth potential

An antibody array was constructed as shown in Fig. 7 in order to observe the protein expression by incising the dorsal skin of the mouse to which the sample was applied.

Protein extraction and purification were performed according to the Raybiotech sample preparation protocol. Samples to be used for comparison of protein expression were prepared as shown in Table 2.

Samples and Concentrations sample  OD AVR. Average concentration.
(ug / ml) dilution factor Stock conc.
(ug / ul) vol (ul) total (ug) 1. Control group 0.1926
0.1960
0.1998 0.196 56.2 10 0.562 100 56.2 2. Minoxidil 0.1686
0.1729
0.1749 0.172 34.4 10 0.344 100 34.4 3. Camellia oil 0.2018
0.2012
0.2120 0.205 64.3 10 0.643 100 64.3 4. Camellia + Hanryun 0.2061
0.2081
0.2116 0.209 67.5 10 0.675 100 67.5 5. Rape oil 0.1544
0.1591
0.1576 0.157 20.7 10 0.207 100 20.7 6. Rapeseed + Hanryun 0.2120
0.2118
0.2160 0.213 71.8 10 0.718 100 71.8 7. Water + Soften 0.2342
0.2354
0.2379 0.236 92.3 10 0.923 100 92.3 8. Mayu 0.2934
0.2881
0.2940 0.292 143.2 10 1.432 100 143.2

Pierce BCA Protein Assay Kit was used to measure the concentration of protein in the skin such as mouse. The results were analyzed for antibody array data according to Table 3 below.

Analyze array data scanning detection Detection is made of one color (Cy-3 Streptavidin). Due to the nature of fluorescent dye sensitive to light, scanner GenePix 4000B (Agilent) Image analysis GenePix Pro 6.0 (Agilent) analysis Normalization Performs positive control normalization
How to perform the normalization recommended by Raybiotech
X (Ny) = X (y) * P1 / P (y)
Where:
P1 = mean signal intensity of POS spots on all arrays
P (y) = mean signal intensity of POS spots on Array "y"
X (y) = mean signal intensity for spot "X" on Array "y &
X (Ny) = normalized signal intensity for spot "X" on Array "y &

The results of the analysis are shown in Fig.

FGF R5 beta, VEGF-I, FGF-21, HGF, IGF-I, Shh-N and VEGF-D were analyzed for beta-catenin, -D showed an increase in expression compared with the control group in the Hankyucho group extracted from camellia oil and camellia oil, and the expression of FGF R3, IGF-I, and Shh-N was increased in the camellia oil (Table 4).

Expression of protein promoting growth phase Probe name Minoxidil / control Camellia oil / control Camellia + Hanryan / control beta-Catenin 1.021 0.928 0.947 FGF R3 1.237 1.044 0.977 FGF R4 1.087 0.978 0.945 FGF R5 beta 1.112 1.051 1.001 FGF-21 1.132 0.963 0.954 HGF 1.225 0.970 0.968 IGF-I 1.095 1.037 0.930 Shh-N 0.957 1.154 0.920 VEGF-D 1.056 1.182 1.348

IL-1Rb, IL-1R4 / ST2, IL-1R6 / IL-1, IL-1Rb, FGFR4, FGFR5 beta, FGF-21 , IFN-g, FGF R4, FGF-21, IL-1Ra, IL-1Rl, IL-1Rl, IL-1Rl, TACI / TNFRSF13B, TGF-b1 and TGFb2, The expression levels of IL-1R / IL-1R / IL-1R / IL-1R I and IL-1R I and IL- 1 R9, and TACI / TNFRSF13B (Table 5).

Expression of protein promoting retrogression Probe name Minoxidil / control Camellia oil / control Camellia + Hanryan / control FGF R3 1.237 1.044 0.977 FGF R4 1.087 0.978 0.945 FGF R5 beta 1.112 1.051 1.001 FGF-21 1.132 0.963 0.954 IFN-g 1.112 1.076 0.975 IL-1 Ra 1.341 0.939 0.838 IL-1 Rb 1.186 1.086 1,000 IL-1R4 / ST2 1.077 0.998 0.872 IL-1 R6 / IL-1 R rp2  / IL-1RL2 1.207 1.193 1.043 IL-1 R9 1.128 1.017 0.962 IL-1 RI 1.324 0.999 0.969 IL-1 RII 1.227 1.041 1.044 TACI / TNFRSF13B 1.196 1.140 0.973 TGF-b1 1.094 1.112 1.030 TGFb2 1.007 1.152 1.543

&Lt; Preparation Example 1 > Hair essence

Hair essence composition division Component name (Korean) INCI name










Raw material details Sunflower seed oil Sunflower Oil Camellia oil Camellia Japonica Seed Oil Argan oil Argan Oil Evening Primrose Oil Evening Primrose Oil Mayu Horse Fat Tocopherol Tocopheryl Acetate Butylene glycol 1,3-Butylene Glycol Spices ARTE-73H Moth extract Ecklonia Cava Extract Hanchuncho extract Eclipta Prostrata Leaf Extract

In the process of hair essence process, Hanwikcho was infused with camellia oil by vacuum low temperature method to make Hanchoncho extract. The ratio of Hwangsikcho: Camellia oil was increased to 1:10. After removing the residue of the infused Hanchuksikcho extract through 0.45 μm filter paper, it was filtered again through a 0.2 μm membrane filter. First, to make hair essence, sunflower oil and camellia oil, which are most abundant among raw material specifications, were mixed and stirred using a homomixer. After that, it was mixed with argan oil, evening primrose oil and camphor oil in order, and tocopherol and butyleneglycol was mixed with antioxidant due to high possibility of rancidity due to oil property. The raw materials were thoroughly stirred through a homomixer to ensure good mixing. As a result of the incense test after mixing the oil, it was considered that the oil-only fragrance might give some rejection to the person who uses the product. Therefore, the fragrance was added to hide the fragrance of oil alone. A flow chart of the hair essence is shown in Fig.

&Lt; Preparation Example 2 > Hair shampoo

Hair shampoo composition division Component name (Korean) INCI  persons
















Raw material details Purified water Water Sodium cocoyl glutamate Sodium Cocoyl Glutamate Decyl glucoside Decyl Glucoside Sodium cocoyl isethionate Sodium Cocoyl Isethionate Coco-glucoside Coco-glucoside PGE-150 Pentaerythritol tetrastearate PEG-150 Pentaerythrityl Tetrastearate &Lt; RTI ID = 0.0 &gt; phage-6 &lt; / RTI &gt; caprylic / capric glyceride PEG-6 Caprylic / Capric Glycerides &Lt; RTI ID = 0.0 &gt; phage-120 &lt; PEG-120 Methyl Glucose Dioleate Citric acid Citric Acid Polyquaternium-10 Polyquaternium-10 Caprylic glycol Caprylyl Glycol p-no formula p-Anisic Acid Moth extract Ecklonia Cava Extract Hose fat Horse Fat Hanchuncho extract Eclipta Prostrata Extract Camellia oil Camellia Japonica Seed Oil Spices Fragrance

The process of functional shampoo for hair improvement was first inffused with camellia oil by vacuum low temperature method to make extract of. The ratio of Hwangsikcho: Camellia oil was increased to 1:10. After removing the residue of the infused Hanchuksikcho extract through 0.45 μm filter paper, it was filtered again through a 0.2 μm membrane filter. First, in order to make a functional shampoo for hair improving, the phytotoxic powder was dispersed and dissolved using purified water. Since the powder can not enter the state, it must be dissolved. Then, polyquaternium-10 was dispersed with purified water of PHASE B. Mix together the PHASE C sodium cocoyl glutamate, decyl glucoside, sodium cocoyl isethionate, coco-glucoside, phage-120 methyl glucoside diolate, citric acid, p-anionic acid, hose fat ingredients Lt; / RTI &gt; Phage D purified water, phage-150 pentaerythrityl tetrastearate, and phage-6 caprylic / capric glyceride were added and mixed. Finally, infusion of Hansunikcho extract and fragrance were added to the camellia oil PHASE E. In the case of shampoos, the effect of surfactant is reduced if the oil component is added to the product using a surfactant. Therefore, in the case of functional shampoo for hair improvement, the addition of the extract of hanchoncho (oil phase) is added to ensure the stability and effectiveness of the product And it was added in a small amount. A process chart of the functional shampoo for hair improving is shown in Fig.

Claims (5)

Mixing the herb leaves with plant oil at a ratio of 1: 10;
Extracting the mixture by hot-pressing for 1 hour in a water bath at 50 to 70 ° C; And
And filtering the extract. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
Mixing the herb leaves with plant oil at a ratio of 1: 10;
Extracting the mixture by vacuum low temperature method; And
And filtering the extract. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
The method according to claim 1 or 2, wherein the plant oil is a rapeseed oil or a camellia oil.
A cosmetic composition for accelerating hair growth, comprising as an active ingredient, fusid oil, which is manufactured by the method of claim 1 or 2.
5. The method of claim 4,
The composition may be used in hair cosmetics such as hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair conditioner, hair treatment, hair cream, hair nutrition cream, hair moisturizing cream, Hair dyes, hair dyes, hair dyes, hair dyes, hair dyes, hair dyes, hair dyes, hair dyes, hair dyes, hair dyes, hair moisturizers, Wherein the composition is selected from the group consisting of mousse and hairspray.

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