KR20070111765A - Extract of scutellariae and hair growing promotor as a main ingredient thereof, and detecting method of hair growing - Google Patents

Abstract

An extract of Scutellaria baicalensis GEORGI is provided to promote hair growth and prevent hair loss by promoting growth of a dermal papilla cell without toxicity, thereby screening the hair growth-promoting agents by using the dermal papilla cell. An extract of Scutellaria baicalensis GEORGI is prepared by drying Scutellaria baicalensis GEORGI, heating the dried Scutellaria baicalensis GEORGI in water, filtering the hot water extract, concentrating the filtered solution under reduced pressure, freeze-drying the concentrate for 72 hours, solubilizing the freeze-dried extract with PBS(phosphate buffered saline) to 10 mg/ml, filtering the solubilized solution with a filter and sterilizing the filtered solution. The hair growth-promoting effects of the Scutellaria baicalensis GEORGI extract are analyzed by separating the dermal papilla cell from a 2 day-rat, and measuring cell growth and growth factor expression.

Description

Extract of Scutellariae and hair growing promotor as a main ingredient describes, and detecting method of hair growing}

1 is a view showing the results of alkaline phosphatase activity measurement of isolated dermal papilla cells,

Figure 2 is a diagram showing the results of gene expression investigation of isolated dermal papilla cells,

Figure 3 shows the results of gene expression measurement in dermal papilla cells.

The present invention relates to a golden extract and a method for inspecting the hair growth promoting agent and hair growth promoting effect as a main component, in particular the extract obtained from gold, a natural native plant of Korea and hair growth promoting agent and plant extract as a main component It is about a test method.

The incidence of hair loss in modern people who are exposed to environmental factors and excessive stress is increasing, and many of the problems are pointed out due to the climate of modern society that emphasizes interpersonal relationships and appearance. Due to this trend, the demand and marketability of hair regrowth are growing very much, but the basic research in related fields is very insufficient. Minoxidil, the most widely used worldwide, has been used for more than 30 years, but its range and mechanism are still unknown. The development of new hair growth agents can be achieved through the understanding of the mechanism of action related to hair loss and hair growth at the molecular level and the search for substances that can regulate activity at each point of action. In particular, in order to develop an effective hair repellent that can be applied in clinical practice, it is essential to establish a suitable system for predicting the results of preclinical studies. Conventionally, by applying a substance that is expected to be effective to the experimental animal to observe the degree of hair growth, the first method was to select a functional substance effective for hair growth.

This method is very difficult to observe the efficacy of a large amount of samples at the same time, has the disadvantage of having to pay the cost of long-term observation and laboratory animal breeding. Phenomenologically hairy conditions can be observed, but individual differences must be taken, and the mechanism of action must be followed by the process of taking tissue or conducting a separate study.

Therefore, in order to observe the mechanism of action more effectively and search for agonists in a short time, the use of cells directly related to hair growth may be very useful.

In addition, an example of such a hair promoter is disclosed in Korean Patent Publication No. 2001- 0010655 (February 15, 2001).

That is, in the above document, a hair growth composition containing sesame flower extract and tretinoin as an active ingredient is proposed.

In addition, although Korean native extracts, including golden water extracts, are frequently used to promote hair growth, most of the specific principles of action have not been elucidated.

Therefore, the inventors of the present invention under these backgrounds have confirmed that the golden extract has an effect on hair loss prevention and hair growth promotion, the present invention using natural plant extracts rather than artificial compounds will have high utility as a hair growth promoter without side effects.

Thus, the present inventors isolated and cultured the dermal papilla cells from the two-day-old rats as suitable cells so that hair loss and hair growth mechanisms can be easily observed, and used for screening.

An object of the present invention has been made to solve the problems described above, causing the growth of dermal papilla cells through the increase in the expression of factors that promote the growth of dermal papilla cells, conventionally inducing hair retardation and hair growth promotion gold It is to provide an extract, a hair promoter having a main ingredient thereof, and a test method for the presence or absence of hair growth promoting effect.

Another object of the present invention is to provide a hair growth promoting agent which is derived from natural plants and is non-toxic to humans.

Another object of the present invention is to examine a method for examining the effect of hair loss prevention and hair growth by screening plant extracts having the effect of causing the growth of dermal papilla cells by increasing the expression of factors that promote the growth of dermal papilla cells. To provide.

In order to achieve the above object, the extracting method of the golden composition according to the present invention comprises the steps of preparing dried gold, heat extraction with 5 times of water with respect to 1 kg of dried gold, and filtering the filtrate in the filtering step. Freeze-drying for 72 hours after concentration under reduced pressure, dissolving the golden hot water extract in the drying step to a final concentration of 10 mg / ml using PBS (Phosphate buffered saline) and the solution dissolved in the dissolution step To filter and sterilize the filter characterized in that it comprises the step of extracting the golden composition.

In the present invention is characterized in that the golden extract extracted by the extraction method of the golden composition.

In addition, the present invention is characterized in that it is a hair growth promoter containing the golden extract as a main component.

In the present invention, it is characterized in that the wool extract containing the golden extract as a main component.

In addition, the present invention is characterized in that it is a hair restorer containing the golden extract as a main component.

In addition, the test method of the hair growth promoting effect according to the present invention in order to achieve the above object is isolated from the dermal papilla cells from the 2 days old rats by measuring the growth and expression of growth factors to prevent hair loss and promote hair growth It is characterized by screening.

The above and other objects and novel features of the present invention will become more apparent from the description of the specification and the accompanying drawings.

First, the concept of the present invention will be described.

The inventors of the present invention completed the present invention by demonstrating the effect of preventing hair loss and promoting hair growth in vitro by measuring growth promotion of hair papilla cells and increased expression of related factors in golden hydrothermal extract. .

Golden (Scutellaria baicalensis) is a perennial herb belonging to the Lamiaceae family. It grows at the foot of the mountain and is grown as a herb. Its height is about 60cm and several square stems come out from the root, lanceolate leaves face each other, the edges are flat, and the lip-shaped purple flowers bloom in July-August at the end of the stem. . The fruit is in the calyx and is round. In Korea, there are about 10 species, including the thimble flower (S. indica) and the thimble flower (S. fauriei) of the thimble flower (―Scutellaria). do.

This is a medicinal herb that is often used for the purpose of lowering heat and detoxification. When taking the root and peeling the root, it is yellowish like golden (黃 金) and treats frequent heat poisoning or chills in the body. It is used for the symptoms of heat in the lungs, and it treats boils and tumors in the body, such as thirst and jaundice, dysentery, diarrhea, indigestion, and nausea caused by fever. Unusually commonly used and cold medicine is classified as a medicine that should be prohibited to pregnant women, if the golden fry is known to have a good therapeutic effect even when suffering from pregnancy anxiety.

That is, in the present invention, gold as described above is used.

The inventors of the present invention measured the growth of the dermal papilla cells and the related factors, namely VEGF (Vascular Endothelial Growth Factor), KGF (Keratinocyte Growth Factor), IGF (Insulin Growth Factor) by measuring the expression of in vitro To determine the effect of hair loss prevention and hair growth in (in vitro), from the present invention was devised a gold extract as a material for hair loss prevention and hair growth promoting agent.

Meanwhile, the hair loss prevention and hair growth promoting screening method utilized in the present invention is made by measuring growth promotion and growth factor expression increase of dermal papilla cells, and these methods are test methods that can simultaneously prove screening and mechanism of action.

The hair is buried about 3-5mm in the skin and accumulated on the epidermis and dermis. This part is called a blanket. There is dermal papilla, the tissue that controls the hairs on top of the follicles, and there are hair cells that make the hairs just above the papillas and continue to divide, producing new hairs and pushing them up. The dermal papilla cells have a period of active growth, anagen, catagen, and telogen cycles, and when signals are received from adjacent cells after the resting phase, they enter the growth phase and regenerate the cells. This results in the formation of new hair.

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

Thus, the present inventors separated the dermal papilla cells from the rats 2 days old, and used the versican markers in the isolated cells to measure the hair growth promoting effect.

Growth of dermal papilla cells is caused by various growth factors. Representative growth factors include VEGF (Vascular endothelial growth factor), KGF (keratinocyte growth factor), and IGF (Insulin Growth Factor). These growth factors are important for maintaining the growth of dermal papilla cells. Induction of growth of the dermal papilla cells through increased expression of these growth factors can induce hair loss prevention and hair growth by conventionally having the effects of maintaining the growth phase and delaying the degenerative phase of the hair cell cycle.

Therefore, the present inventors confirmed the increased expression of VEGF, KGF, IGF after treatment of the isolated golden papilla cells.

The inventors of the present invention, in order to confirm the hair loss prevention and hair growth promoting effect of the golden extract, using the following method in vitro experiments using the dermal papilla cells directly isolated from rats 2 days old. It was.

1) Isolation of dermal papilla cells

2) Measurement of increased expression of VEGF, KGF and IGF via RT-PCR.

Such assays are very useful as in vitro screening methods that can be utilized prior to in vivo assays.

Next, an embodiment of the present invention will be described.

The inventors of the present invention conducted the experiment in the following order.

1) Preparation of Golden Extract

2) Isolation and identification of dermal papilla cells

3) VEGF, KGF, IGF expression measurement by RT-PCR

Hereinafter, the experimental content of the present invention will be described in detail.

1. Preparation of Extract

Filtration was carried out by heat extraction with 5 times of water to 1kg of dried golden gold, and the filtrate was concentrated under reduced pressure and freeze-dried for 72 hours. The golden hot water extract was dissolved in PBS (Phosphate buffered saline) to a final concentration of 10 mg / ml and filtered and sterilized with a filter (0.22 μm pore size, Millipore).

2. Isolation and identification of dermal papilla cells

Separation of dermal papilla cells was carried out using a sample of rat skin tissue 2 days old. The sample was pulverized with a dissecting knife and then reacted while rotating in a 37 ° C. incubator for 4 hours with the addition of 1 mg / ml collagenase I solution. The cells were blown for 1 minute to separate the cells from the finished tissue solution and allowed to stand at room temperature for 30 minutes. Then, the supernatant was carefully placed on 5 ml of Histopaque 1083 solution and centrifuged for 20 minutes at 2500 rpm.

After centrifugation, the intermediate cell layer was taken and placed in DMEM (Dulbecco's modified Eagle's medium) containing 15% fetal bovine serum and incubated under conditions of 5% CO ₂ and 37 ^° C.

The first 5 days after separation were left unobserved, and the medium was changed every 2 days thereafter. When primary cultures were filled with culture plates, dermal papilla cells were harvested with 0.25% trypsin and subcultured with single cell suspension.

Some cells were subjected to alkaline phosphatase assay for biochemical identification (FIG. 1), and the expression of the dermal papilla cell-specific genes was confirmed by extracting total RNA using the RT-PCR method (FIG. 2).

1 is a view showing the results of alkaline phosphatase activity measurement of isolated dermal papilla cells, Figure 2 is a view showing the results of the gene expression of the isolated dermal papilla cells.

Alkaline phosphatase activity is already known to be very high in dermal papilla cells. In order to confirm whether the isolated cells are dermal papilla cells, the isolated dermal papilla cells (denoted DP) and fibroblasts having a similar shape as a control group (chineses) hamster ovary cell) and melanocyte B16BL6, a kind of skin cell, were set up and enzyme activity was measured in three cells. As can be seen in Figure 1, the enzyme activity was 30 times stronger than CHO cells, 10 times stronger than in melanocytes.

It is also known that Versican and Nexin genes are characteristically expressed in dermal papilla cells.

Therefore, the present inventors set up CHO (chineses hamster ovary cells), which are fibroblasts with similar shapes as control cells, to identify whether the cells are separated from the dermal papilla cells, and investigate gene expression in the two cells. It was.

As shown in FIG. 2, Actin genes were identically expressed in CHO cells and DP cells, and Nexin and Versican genes, which are gene markers of dermal papilla cells, were expressed only in the dermal papilla cells isolated by the present inventors.

3. RT-PCR in dermal papilla cells

As mentioned in 2 above, isolate and identify the dermal papilla cells identified and seeded on a 6-well culture plate at a concentration of 5x105 cells / well, and incubate until the cell's conflucency is about 80-90%. The cultured cells were treated with a gold extract at a concentration of 50 μg / ml.

When culturing cells, DMEM medium containing both 15% serum (FBS) and antibiotics (penicillin) was used, and RPMI1640 medium without 5% serum (FBS) was used for material treatment. 24 hours after the material treatment, the cells were collected and the total RNA of the cells was extracted. RT-PCR was performed on the extracted RNA and the product was confirmed by electrophoresis, thereby measuring gene expression levels of VEGF, KGF, and IGF.

By measuring the expression of growth factors and marker genes affecting the growth of dermal papilla cells, Korean native plants that have the effect of directly promoting hair growth could be screened.

As a result of the screening, it was confirmed that the expression of VEGF, KGF, and IGF was markedly increased in RNA of the cells treated with the golden extract.

In addition, it was confirmed that the gold extract promoted the expression of growth factors and extracellular matrix protein genes of dermal papilla cells and epithelial cells more significantly than that of minoxidil-treated cells. As a result, as shown in Figure 3, as a result, the golden extract was expected to directly act on the dermal papilla cells of the hair growth tissue to promote cell growth .

In addition, as can be seen in Figure 3, during the golden extract treatment, growth factors related to vascular expansion, VEGF, keratinocyte growth factor KGF, insulin-like growth factor IGF expression was increased compared to the control group, positive control It was confirmed that the efficacy was superior to that in the minoxidil treatment group. 3 is a diagram showing the results of gene expression measurement in dermal papilla cells. In Figure 3 Control is not treated with the extract control, Sample is a gold extract treated group.

In the case of VEGF, the gene expression was increased by 1.83-fold increase and 2-fold increase of Minoxidil when treated with gold extract, and KGF was increased by 2.77-fold, which proved more effective in the growth factor expression than Minoxidil, which was increased about 2 times. It became. IGF increased 1.36-fold in the Minoxidil-treated group, but increased 1.73-fold in the golden extract group.

As mentioned above, although the invention made by this inventor was demonstrated concretely according to the said Example, this invention is not limited to the said Example and can be variously changed in the range which does not deviate from the summary.

As described above, according to the golden extract according to the present invention and the hair growth promoting agent having a main component thereof, and the test method of the hair growth promoting effect, it is possible to prevent hair loss and promote hair growth by promoting the growth of dermal papilla cells, which are hair growth tissues. Is obtained.

In addition, according to the golden extract according to the present invention, the hair promoter having a main component thereof, and the inspection method of the hair growth promoting effect, it is possible to obtain a hair regrowth material derived from natural plants and harmless to the human body, and the effect of hair growth promoting The effect of knowing the presence or absence is obtained

Claims (6)

Preparing dried gold, Filtration by heat extraction with 5 times the water for 1kg of the dried golden, Freeze-drying the filtrate in the filtration step under reduced pressure for 72 hours, Dissolving the golden hot water extract in the drying step to a final concentration of 10 mg / ml using PBS (Phosphate buffered saline); and Extracting the golden composition comprising the step of extracting the golden composition by filtration and sterilization of the solution dissolved in the dissolving step. Golden extract extracted by the extraction method of claim 1. Hair growth promoting agent comprising the golden extract of claim 2 as a main component. A woolen agent comprising the golden extract of claim 2 as a main component. Hair growth agent containing the golden extract of claim 2 as a main component. A method for testing the presence or absence of hair growth effects, characterized by screening the golden extract to prevent hair loss and promote hair growth by separating dermal papilla cells from rats 2 days old and measuring cell growth and expression of growth factors.

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