KR101597762B1 - Composition for treating wound containing stellera chamaejasme extract or fraction thereof and method for treating wound in a subject - Google Patents

Abstract

Provided are a composition for treating a wound, comprising a Stellera chamaejasme L. extract or a fraction thereof, and a method for treating a wound of an object. The Stellera chamaejasme L. extract is a composition extracted from an aerial part of Stellera chamaejasme L., and the Stellera chamaejasme L. extract is obtained by an ethanol extract. The wound is on skin caused by incisions, abrasions, heat or lacerations, fractions, contusions, burns, or amputations.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a composition for treating a wound comprising a roots extract, or a fraction thereof, and a method for treating a wound of the individual,

A composition for treating a wound comprising a roots extract, or a fraction thereof, and a method for treating a wound of an individual comprising administering to said individual an amount of said composition effective to treat a wound.

The skin acts as a shield to protect the body from the outside. When a wound occurs in the skin, the wound is filled with blood by the natural healing action of the body, and the granule of the platelet is reduced, and the activation of the hageman factor starts and the wound healing process proceeds. Coagulation of blood is a temporary defensive action that protects exposed wound tissues and provides a basis for cells to move during the healing process.

Wound healing and / or healing occurs in three major stages. The first step is an inflammatory phase characterized by inflammation at the traumatic site. Inflammation can be generally short-lived in the absence of significant infection. The second stage is the proliferative phase, which is characterized by epithelialization, angiogenesis, granulation tissue formation and collagen deposition. Angiogenesis with new capillary formation is used to deliver nutrients and maintain granulation tissue formation. Without the formation of new capillaries into the wound, essential nutrients do not reach the wound, creating a wound that is not chronically healed. The surface of the injured wound is covered with a layer of keratinocytes, resulting in a new epidermis and a re-epithelialization in which the epithelium is reconstructed. When the re-epithelization is completed, a series of processes are performed in which the wound area is reduced through an increase in connective tissue and a reorganization. The final stage of wound healing is the maturational phase in which fibroblasts differentiate into collagen. During maturation, the junk cells and capillaries of the convalescent tissue gradually disappear, and scarring can occur if the cells and capillaries of these tissues are hyperplasia or not normally degraded.

Since it is important not only to treat wounds quickly during wound healing, but also to treat them without side effects or scars, efforts are being made to find substances with this effect.

An aspect of the present invention provides a composition for treating a wound comprising an extract of P. roots, or a fraction thereof as an active ingredient.

Another aspect provides a method of treating a wound in a subject comprising administering to the subject an amount of the composition as described above effective to treat the wound.

One aspect is a composition for treating a scar including an extract of P. roots, or a fraction thereof as an active ingredient, wherein the P. roots extract is extracted from the ground part of the roots.

The P. primrose extract may be an extract extracted with a solvent from the whole, part of, or a material derived from the above-ground part of the root. Said part may be stem, leaf, flower, or petal of the root crop. The plant may be grown in mountainous areas of Mongolia. The area may be near Ulaanbaatar. The root can grow to a height of about 30 to 40 cm. The length of the leaf of the root is about 15 to 27 mm, and the leaf may be lanceolate. The surface of the leaf may be green and its backside may be bluish gray. Both sides of the leaves have no hairs and petiole is short. The whole, part of, or a material derived therefrom of the above ground part of the root used in the extraction may be ground or chopped or suitably dried.

The solvent may be water, an alcohol such as a C1-C6 alcohol, or a mixture thereof. The C1-C6 alcohol may be methanol, ethanol, propanol, isopropanol, 1,3-propanediol, butanol, pentanol, hexanol and the like. The solvent may be, for example, a mixture of water and an alcohol, that is, an aqueous solution of an alcohol. The alcohol concentration of the alcohol aqueous solution may be 1 to 99.5 (v / v)%, for example, 10 to 99.5 (v / v)%, 1 to 70 (v / 5 to 25 (v / v)%, 7 to 20 (v / v)%, 5 to 25 (v / v)%, or 10 to 20 (v / v)%. The alcohol aqueous solution may be an aqueous ethanol solution.

The extraction is carried out in an amount of 3 to 10 (volume / weight) times, for example, 3 to 7 (volume / weight) times the extraction solvent for the whole or part of the ground portion of the root, , 3 to 5 (volume / weight) times, 5 to 10 (volume / weight) times, or 4 to 10 times. For example, 3 to 10 L of the extraction solvent may be added to 1 kg of the whole, part of, or the material of the above-ground part of the plant.

The extraction may be performed by warmed liquid extraction, pressurized liquid extraction (PLE), microwave assisted extraction (MAE), subcritical extraction (SE), or a combination thereof . The subcritical extraction may be subcritical water extraction (SWE). Sub-critical water extraction is also referred to as superheated water extraction or pressurized hot water extraction (PHWE). The warmed liquid extraction may be a reflux extraction.

The extraction may be carried out at a temperature of 4 ° C to 70 ° C, for example 4 ° C to 50 ° C, 4 ° C to 40 ° C, 4 ° C to 30 ° C, 10 ° C to 70 ° C, 15 ° C to 70 ° C, 10 ° C to 50 ° C, 10 ° C to 50 ° C, 4 ° C to 40 ° C, 4 ° C to 30 ° C, 10 ° C to 40 ° C, 10 ° C to 35 ° C, or 10 ° C to 30 ° C. The extraction time may vary depending on the temperature selected and may range from 1 hour to 2 months, such as 1 hour to 1 month, 1 hour to 15 days, 1 hour to 10 days, 1 hour to 5 days, 1 hour to 3 days 5 hours to 5 days, 5 hours to 3 days, 5 hours to 2 days, 5 hours to 2 days, 1 hour to 1 day, 5 hours to 1 month, 5 hours to 15 days, 5 hours to 10 days, Hour to 1 day, 10 hours to 1 month, 10 hours to 15 days, 10 hours to 10 days, 10 hours to 5 days, 10 hours to 3 days, or 10 hours to 2 days. The extraction may include mixing the whole of the ground part of the root, a part thereof, or a material derived therefrom into the solvent, and leaving the mixture for a predetermined time. The setting may include moderate agitation. The extraction may be repeated one or more times, for example, 1 to 5 times.

The above extraction can be performed by separating the plant residue and the extract by a known method such as filtration. The extraction can also include removing the solvent from the resulting extract by known methods such as concentration under reduced pressure. The extraction may also comprise preparing the dried extract by drying, such as lyophilization, of the resulting extract.

In the above composition, the term "fraction" refers to a substance, i.e., a fractioned substance, in which the P. primrose extract is divided into components. The fraction may be obtained by solvent fractionation. The solvent fractionation may be to mix the root rot extract with the solvent and to separate the material present in the solvent. The fraction may be a hexane fraction, an ethyl acetate fraction, a butanol fraction, or a combination thereof obtained by successively fractionating the above-described P. pernolus extract in water, followed by fractionation with hexane, ethyl acetate, and butanol.

Specifically, the hexane fraction is prepared by mixing the P. pernobacterium extract with water, mixing the mixture again with hexane, allowing to stand for a certain period of time, separating the hexane layer, separating the fraction from the separated hexane layer May be obtained. Separation of the fractions can include removal of hexane from the hexane layer. The ethyl acetate fraction is obtained by mixing the hexane fraction with water, mixing the mixture with ethyl acetate, allowing to stand for a certain period of time, separating the ethyl acetate layer, and separating the fraction from the separated ethyl acetate layer . Separation of the fractions can include removal of the ethyl acetate from the ethyl acetate layer. The butanol fraction may be prepared by mixing the ethylacetate fraction with water, mixing the mixture again with butanol, allowing to stand for a certain period of time, separating the butanol layer, and separating the fraction from the separated butanol layer . Separation of the fractions may include removing butanol from the butanol layer. Conditions for such fractionation, such as temperature conditions, pressure conditions, time, amount or concentration of solvent used, stirring, and the like, may be as described for the extracts used to make the Rootgrass extracts described above. The fractionation may be repeated one or more times, for example, 1 to 5 times.

Separation of the fractions may be accomplished by known methods such as filtration. The fractionation may also include removing the solvent from the fraction obtained by known methods such as concentration under reduced pressure. The fractionation may also include concentration and / or drying of the obtained fractions. The concentration may be reduced pressure concentrated. The drying may include vacuum drying, boiling drying, spray drying, room temperature drying or freeze drying.

The term "wound" as used herein refers to a tissue that is cut, torn, broken, burned, traumatized, Means injury to the human body resulting from a disorder or disease that causes injury. The term "tissue" refers to a mass of cells in the body that together form a particular function. Tissues can include eyes, mucus, lungs, kidneys, heart, viscera, tendons, vascular tissues, bones, skin, connective tissue, and nerves, such as spinal cord. The wound may be an open wound whose surface is open, or a closed wound whose surface is not open. An example of such a wound may be an open wound in the skin. The wound may include lesions, sores, necrosis, and ulcers. The necrosis may be associated with dead tissue resulting from infection, injury or infarction. The ulcer may be a local defect or exfoliation of the surface of the organ or tissue produced by the dissection of the necrotic tissue.

An example of such a wound is the epidermis of the skin; Dermis; Epidermis and dermis; Or the damaged skin of the epidermis, dermis and subcutaneous fat layer.

In addition, examples of such wounds include cuts, incisions (e.g., surgical incisions), abrasions, lacerations, fractures, contusions, burns, , ≪ / RTI > or amputations.

The "treat " provided by the present invention may be to provide wound healing at a reduced time compared to natural healing. The treatment may include improvement and / or alleviation of the wound. In addition, the treatment may include treatment of a wound and / or a wound-related disease. Such treatment may mean healing and / or regeneration of damaged tissue caused by a wound. The wound treatment may include the meaning of skin regeneration. In addition, the treatment may be to maintain the original composition of the damaged tissue. In addition, the treatment may be to promote healing and / or regeneration of the damaged tissue while minimizing complications and / or scarring of wound related diseases.

The composition may be for promoting the pre-stage of the wound healing process. In addition, the composition may be one that promotes the proliferative and / or mature phase during the wound healing step. Thus, the composition may be for treating wounds in the proliferative and / or maturation phase.

The composition may be present in an amount of from 0.001% to 80%, such as from 0.01% to 60%, from 0.01% to 40%, from 0.01% to 30%, from 0.01% to 20% %, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20% From 0.05% to 10%, from 0.05% to 5%, from 0.1% to 60%, from 0.1% to 40%, from 0.1% to 30%, from 0.1% to 20% % To 10% by weight, or 0.1% to 5% by weight of the root extract or fraction thereof.

The composition may be a pharmaceutical composition. The composition may comprise a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, the lubricant may be magnesium stearate, talc, or combinations thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low substituted hydroxy cellulose, or a combination thereof. The disintegrant may be carboxymethylcellulose calcium, starch glycolate sodium, calcium monohydrogen phosphate anhydrate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or combinations thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The composition may be formulated into a parenteral dosage form. The parenteral dosage form may be an injection or an external preparation for skin.

The external skin preparation may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof.

The external preparation for skin is usually used as a component used in external skin preparations such as cosmetics or medicines such as an aqueous component, an oily component, a powder component, an alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, an antiseptic, , Coloring agents, various skin nutrients, or a combination thereof, and may be suitably blended as necessary.

The external preparation for skin may be a metal blocker such as sodium edetate, sodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate or gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridine, Vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, fructose, fructose and other herbal medicines, various herbal medicines, tocopherol acetate, glycyrrhizic acid, Sugars such as trehalose and the like can also be appropriately compounded.

The composition may be one for promoting expression of a collagen gene in a cell. The composition may increase the expression of collagen, or may increase the activity of the collagen produced by the composition. The composition may also increase the activity or expression of an up-stream enzyme or protein that produces collagen. In one embodiment, the composition may be one that increases the expression and / or activity of collagen and has wound healing efficacy. The collagen may be collagen (type I) or collagen (type III). The gene encoding the collagen may be selected from the group consisting of COL1A1 and COL3A1.

The composition may increase the production of keratinocyte differentiation factors. The composition may, for example, facilitate expression of the differentiation factor gene of keratinocytes. The differentiation factor may be filaggrin (FLG), loricrin (LOR), or involucrin (IVL). The composition may promote migration of keratinocytes to the wound.

The composition may increase the expression of the collagen gene itself or may increase the activity of the collagen produced by the composition. The composition may also increase the activity or expression of enzymes or proteins upstream of the collagen production.

Another aspect provides a method of treating a wound in a subject comprising administering to the subject an amount of the composition as described above effective to treat the wound. The composition is the same as described above.

Administration can be by any method known in the art. Administration can be by direct administration to an individual by any means, for example, by routes such as intravenous, intramuscular, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration . The administration can be systemically or locally administered. The administration may be topical administration to the site where the wound is present.

The subject may be a mammal, such as a person, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat.

The administration may be carried out in an amount of 0.1 mg to 1,000 mg, for example 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg.

Another aspect provides a cosmetic composition for improving wounds comprising an extract of P. roots or a fraction thereof as an active ingredient. The above-described root rot extract is the same as described above.

The term "for the improvement of a wound" may include a use for lowering or alleviating the degree of a wound. For example, the wound improvement may be to lower or alleviate the extent of the wound that has already occurred.

The cosmetic composition may be provided in all formulations suitable for topical application. For example, it may be provided as a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an oil phase in water, a suspension, a solid, a gel, a powder, a paste, a foam or an aerosol composition. Compositions of such formulations may be prepared according to conventional methods in the art.

The cosmetic composition may further comprise a moisturizing agent, an emollient agent, an ultraviolet absorber, an antiseptic, a bactericide, an antioxidant, a pH adjuster, an organic and inorganic pigment, a perfume, a cold agent or a limiting agent. The amount of the additional component such as the above-mentioned moisturizing agent can be easily selected by those skilled in the art within a range not to impair the purpose and effect of the present invention, and the amount thereof is 0.01 to 5% by weight, specifically 0.01 to 3% % ≪ / RTI >

The composition may be present in an amount of from 0.001% to 80%, such as from 0.01% to 60%, from 0.01% to 40%, from 0.01% to 30%, from 0.01% to 20% %, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20% From 0.05% to 10%, from 0.05% to 5%, from 0.1% to 60%, from 0.1% to 40%, from 0.1% to 30%, from 0.1% to 20% % To 10% by weight, or 0.1% to 5% by weight of the root extract or fraction thereof.

According to a composition for treating a wound according to one aspect, it can be used to treat an individual's wounds efficiently.

According to a method of treating a wound of an individual according to one aspect, it is possible to effectively treat a wound of the individual.

The cosmetic composition for improving the wound according to one aspect can be used for effectively improving the wound.

FIG. 1 shows the results of confirming that the root rot extract promotes the migration of keratinocytes.
FIG. 2 shows the results of confirming that the fraction of P. primrose extract promotes the migration of keratinocytes.
FIG. 3 shows the results of confirming that the extract of P. roots promotes the expression of COL1A1 and COL3A1 genes in fibroblasts.
FIG. 4 shows the results of confirming that the fraction of P. primrose extract promotes the expression of COL1A1 and COL3A1 genes in fibroblasts.
FIG. 5 is a result of confirming that the extract of P. roots promotes the expression of keratinocyte differentiation factor in keratinocytes.
FIG. 6 shows the result of clinically confirming that the pine root extract promotes the recovery of wounds in an animal experiment.
FIG. 7 is a result of quantitatively confirming that the extract of P. roots promotes recovery of wounds in an animal experiment.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Pine root extract and his Fraction  Produce

In this embodiment, the sprinkling of blood pool (Stellera chamaejasme ) and fractions thereof were prepared, and the effects of P. primrose extract and fractions on wound healing were examined.

1. Peel root extract and its Fraction  Produce

(1) Preparation of Pea Root Extract

The root rot extract was grown in Ulaanbaatar, Mongolia. The collected stems were washed with water and dried. The dried bloomed stem was cut into 2 ~ 3cm by using a cutting blade. 100 g of the finely pulverized stem and 1 L of 95% ethanol were added to a glass Erlenmeyer flask and mixed. The mixture was extracted by stirring at room temperature (about 20 ° C) for 48 hours at 150 rpm.

Next, the mixture was passed through a filter paper (Whatman, NO.2, 8 mu m) to obtain a filtrate. The resulting filtrate was concentrated under reduced pressure using a rotary vacuum concentrator (EYELA, N-1100) and freeze-dried (Operon, FDCF-12003) was performed for 48 hours to remove remaining solvent. 3 g of root grass extract was obtained.

 (2) of the root rot extract Fraction  Produce

Add 5 g of dried Peppermint Extract and 100 mL of water into an Erlenmeyer flask, suspend, add to a separatory funnel (250 mL), add 100 mL of n-hexane (Extra pure grade, 95% or more) And allowed to stand at room temperature for 3 hours. The above mixture was subjected to the above-mentioned procedure three times by repeating the above procedure only by taking the hexane layer using a separating funnel. The obtained hexane fraction was concentrated under reduced pressure (EYELA, N-1100) to obtain hexane fraction.

In addition, ethyl acetate was added to the water fraction from which the hexane fraction was removed in the same manner, and the mixture was subjected to the same procedure to obtain an ethyl acetate fraction.

In addition, water-saturated butanol was added to the water fraction from which the ethylacetate fraction had been removed in the same manner and mixed, followed by the same procedure to obtain a butanol fraction.

The P. primrose extract and its fractions were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mg / ml and used in the following experiment.

2. Increased migration of human keratinocytes

Whether the P. primrose extract or its fractions increased the migration of human keratinocytes was confirmed as follows.

In a well of a 24-well microtiter plate containing 1 ml of DMEM (Dulbecco's Modified Eagle's Media: Hyclone) medium containing 2.5 (v / v)% fetal bovine serum, HaCaT human keratinocytes 2 x 10 < ⁵ > cells / well. Next, the keratinocytes were cultured in an incubator at 37 ° C and 5% CO ₂ until the confluency reached 80%, and then a single cell layer in the culture medium was scratched with a p10 pipette tip to induce "scratch-damage" Respectively.

Next, on the scraped cell monolayer, 200 μl of serum-free DMEM and the above-mentioned P. grubu extract or its fraction were added at a concentration of 5 μg / ml medium and 10 μg / ml medium. Thereafter, human keratinocytes were cultured for 24 hours under the same conditions as the culture conditions described above. As a negative control, DMSO used to dissolve the extract was treated in the same amount. GW501516 (Sigma Aldrich, USA) or Centella asiatica extract, which is a PPAR? Activity, was used as a positive control. After that, the cell layer was stained with a crystal violet reagent, and the extent to which the scratch scar was recovered was confirmed. The centilla mushroom extract was purchased from BioSpectrum (Korea).

FIG. 1 shows the results of confirming that the root rot extract promotes the migration of keratinocytes.

FIG. 2 shows the results of confirming that the fraction of P. primrose extract promotes the migration of keratinocytes.

As shown in FIGS. 1 and 2, scratch damage of the cells treated with negative control remained relatively unhealed after 24 hours, whereas in cells treated with the root rot extract or fraction, cell proliferation of wound edges and Movement Scratches - The damage was healed and the area of scratches decreased after 24 hours. In Fig. 1, GW represents GW501516.

3. In fibroblasts COL1A1  And COL3A1  Increased gene expression

Whether the P. primrose extract or its fraction promotes the expression of collagen gene in human fibroblasts was confirmed as follows.

Wells were inoculated into wells of a 6-well microtiter plate containing 2 ml of DMEM (Dulbecco's Modified Eagle's Media: Hyclone) medium containing 2.5% (v / v) dermal fibroblasts: HDF, American Type Culture Collection, USA) was added at 2 × 10 ⁶ cells / well and cultured under the same conditions as above until confluency reached 80%.

To the thus cultured cells were added 1, 5, and 10 μg / ml of the above-described P. nipponu sp. Extract, respectively, at a concentration of 5 μg / ml and 10 μg / ml, respectively Lt; / RTI > for 24 hours under the same conditions as described above. As a negative control, DMSO used for dissolving the extract was treated in the same amount, and KCPC extract (BioSpectrum, Korea) was used as a positive control. After 24 hours, the expression of collagen gene was confirmed by RT-PCR assay. Specifically, after 24 hours, total RNA was harvested from the cells using TRIzol reagent (Invitrogen, Carlsbad, Calif., USA) and reverse transcribed, and then RT-PCR was performed as follows. First, the RNA was reverse transcribed using reverse transcriptase for cDNA synthesis. RT-PCR was performed using the following specific primers as primers. The relative mRNA expression level of each gene was normalized to the beta -actin value.

FIG. 3 shows the results of confirming that the extract of P. roots promotes the expression of COL1A1 and COL3A1 genes in fibroblasts.

FIG. 4 shows the results of confirming that the fraction of P. primrose extract promotes the expression of COL1A1 and COL3A1 genes in fibroblasts.

As shown in FIG. 3 and FIG. 4, it can be seen that the extracts of P. pernifolia or fractions thereof are remarkably effective in activating the expression of at least one gene selected from the group consisting of COL1A1 and COL3A1 which are collagen synthesis-related factors.

beta -actin COL1A1 COL3A1 Forward primer SEQ ID NO: 1 SEQ ID NO: 3 SEQ ID NO: 5 Reverse primer SEQ ID NO: 2 SEQ ID NO: 4 SEQ ID NO: 6

4. Increased keratinocyte differentiation factor in human keratinocytes

It was confirmed whether or not the pine root extract increased the production of keratinocyte differentiation factors filaggrin, lorikreen and inbolucrine, as follows.

Fetal bovine serum is 2.5 (v / v)% a DMEM containing a (Dulbecco's Modified Eagle's Media: Hyclone) of the medium 1 ml This example HaCaT human keratinocyte cells in a well of a 6-well plate culture medium with 2 × 10 ⁶ cells / well . Next, the cells were cultured in a 5% CO ₂ incubator at 37 ° C until confluency reached 80%.

The pine root extract was added to the DMEM medium at a concentration of 1 μg / ml, 5 μg / ml, and 10 μg / ml, respectively, and cultured for 24 hours. Then, total RNA was obtained from the cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and RT-PCR analysis was performed. Specifically, the obtained RNA was reverse-transcribed with reverse transcriptase using the following specific primers. Each relative mRNA expression level was normalized to a beta -actin level.

FIG. 5 is a result of confirming that the extract of P. roots promotes the expression of keratinocyte differentiation factor in keratinocytes. As shown in FIG. 5, it can be seen that the extract of P. perioli is remarkably effective in increasing the expression of at least one gene selected from the group consisting of keratinocyte differentiation factors FLG, LOR, and IVL.

Forward primer Reverse primer beta -actin SEQ ID NO: 1 SEQ ID NO: 2 Pillar Green (FLG) SEQ ID NO: 15 SEQ ID NO: 16 Rorikreen (LOR) SEQ ID NO: 17 SEQ ID NO: 18 In the Bolivar (IVL) SEQ ID NO: 19 SEQ ID NO: 20

6. Improve wound healing in animal experiments

It was confirmed whether or not the above-mentioned P. primrose extract promotes recovery of a wound site in an animal experiment as follows .

6.1 Experimental animals

Sprague-Dawley (SD) rats (male, 7 weeks old) were obtained from Koatech Co., Ltd. and maintained at 25 ± 1 ° C and 50 ± 10% humidity. In addition, diets and water of the normal diet were allowed to be ingested freely, and all experimental animals were adapted to the animal room environment for one week and then used in the experiment. The dietary dose, dose and method of administration to the experimental group are shown in Table 4 below.

Test group composition and dosage of test substance, administration method Experimental group Test substance Dosage and Methods G1 none - G2 Vehicle - G3 Root Extract 3% (v / v), once a day G4 Centipede extract 3% (v / v), once a day

6.2 Wound inducement and treatment

SD rats were fasted for 24 hours before anesthesia and 100 ml of water were fed. One hour before inducing scars in all experimental groups, 100% of Zoletil and 100% of rumun were mixed in a ratio of 1: 2, and all the experimental groups were anesthetized with intramuscular injection using 0.1 cc / kg of the obtained mixture. Thereafter, the rats in the back of the rats were removed with an electric epilator in all experimental groups. And 10% of povidone-iodine and 70% of ethanol were applied to the back, respectively, and then disinfected using a surgical scissors disinfected at the back of the back, and a 2 cm x 2 cm square sized full- full-thickness excisional wound).

6.3 Observation of gross wound changes

The extracts of each of the above P. periplus and Centella asiatica (Kinship) extracts known to be effective in wound healing were applied to the scarred skin once daily for 3 days at 3% (v / v). The centilla extract is identical to that described in Example 1, 2. Scars were taken at 1, 4, 8, 11, and 14 days after each test material was applied to the wound, and the area of the wound was measured and analyzed using ImageJ (NIH, USA).

6.4. Histological examination

On the 14th day after the administration of each test substance, all test groups were inhaled with ether and an administration site of the test substance was fixed with a 10% neutral buffered formalin solution. For histopathological examination, the fixed skin tissue was cut longitudinally in a square of 2 cm x 2 cm horizontally and vertically so that the wound surface could be examined evenly. The cut tissue was embedded in paraffin according to the general tissue treatment method, and a piece of tissue was formed to a thickness of about 3 to 4 쨉 m and attached to a slide glass. Hematoxylin-eosin staining was performed on the adhered sections, and the stained sections were examined with an optical microscope (Olympus BX53, Japan). The interpretation of the injured tissue was performed by scoring with the evaluation method known in the art (Nisbet et al., Implantation of functionalized thermally gelling xyloglucan hydrogel within the brain: associated neurite infiltration and inflammatory response: Tissue Eng Part A. ) and Barati et al., Histopathological and clinical evaluation of Kombucha tea and Nitrofurazone on cutaneous full-thickness wounds in healing in rats: an experimental study: Diagn Pathol (2013). The criteria for each finding at the time of reading are shown in Table 5 below, and the results of the reading are shown in Table 6.

As shown in Table 6, it can be seen that the Rootgrass extract is prominent in fibrous tissue formation, angiogenesis and epithelialization.

FIG. 6 shows the result of clinically confirming that the pine root extract promotes the recovery of wounds in an animal experiment. FIG. 7 is a result of quantitatively confirming that the extract of P. roots promotes recovery of wounds in an animal experiment. Also, as shown in Figs. 6 and 7, it can be seen that the root rot extract promotes re-epithelization of the injured area and has a remarkable effect of restoring wounds wound.

Histopathological parameters
(histopathological parameter) score 0 One 2 3 Inflammation none slightly
(mild) middle
(moderate) Remarkable
(marked) Fibrous tissue formation No or minimal fibroblast
(fibroblast) A few
Fibroblast Many fibroblasts Mostly fibroblasts Angiogenesis
(angiogenesis) none Less than 5 vessels per high power field (HPF) 6 to 10 per HPF
blood vessel Over 10 vessels per HPF Epithelialization none Partial focal defective complete

Experimental group Histopathological parameters Inflammation Fibrous tissue formation Angiogenesis Epithelialization G1 2.6 2.4 2.3 1.6 G2 2.5 2.4 2.2 1.7 G3 2.4 2.6 2.5 1.8 ^* G4 2.3 ^* 2.7 ^* 2.5 1.6

^* Is P <0.05 means statistical significance with G1.

Formulation example  1: Preparation of pharmaceutical preparations

1. Preparation of Soft Capsules

A soft capsule was prepared by mixing 150 mg of the root extract or fraction, 2 mg of palm oil, 8 mg of palm oil, 8 mg of palm oil, 4 mg of yellow pearls and 6 mg of lecithin and filling with 400 mg per capsule according to a conventional method.

2. Preparation of tablets

The granules were formed by mixing 150 mg of the root rot extract or fractions, 100 mg of glucose, 50 mg of red ginseng extract, 96 mg of starch and 4 mg of magnesium stearate, and then 30% ethanol was added thereto, followed by drying at 60 ° C Tablets were prepared by tabletting using a tablet machine.

3. Preparation of Granules

The granules were formed by mixing 150 mg of the root rot extract or fractions, 100 mg of glucose, 50 mg of red ginseng extract and 600 mg of starch and 100 mg of 30% ethanol to form granules, followed by drying at 60 ° C to form granules, Lt; / RTI &gt; The final weight of the contents was 1 g.

Formulation example  2: Cosmetics  Preparation of composition

1. Manufacture of softening longevity (skin lotion)

Compounding ingredient Content (% by weight) Root extract or fraction 0.1 glycerin 3.0 Butylene glycol 2.0 Propylene glycol 2.0 Carboxyvinyl polymer 0.1 Phage-12 nonylphenyl ether 0.2 Polysorbate 80 0.4 ethanol 10.0 Triethanolamine 0.1 Preservative, coloring, fragrance Suitable amount Purified water Balance

2. Manufacture of nutrition lotion (milk lotion)

Compounding ingredient Content (% by weight) Root extract or fraction 0.1 glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxyvinyl polymer 0.1 Wax 4.0 Polysorbate 60 1.5 Caprylic / capric triglyceride 5.0 Squalane 5.0 Sorbitacecequioleate 1.5 Liquid paraffin 0.5 Cetearyl alcohol 1.0 Triethanolamine 0.2 Preservative, coloring, fragrance Suitable amount Purified water Balance

3. Manufacture of nutritional cream

Compounding ingredient Content (% by weight) Root extract or fraction 0.1 glycerin 3.0 Butylene glycol 3.0 Liquid paraffin 7.0 Beta Glucan 7.0 Carbomer 0.1 Caprylic / capric triglyceride 3.0 Squalane 5.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Polysorbate 60 1.2 Triethanolamine 0.1 Preservative, coloring, fragrance Suitable amount Purified water Balance

4. Manufacture of massage cream

Compounding ingredient Content (% by weight) Root extract or fraction 0.1 glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 45.0 Beta Glucan 7.0 Carbomer 0.1 Caprylic / capric triglyceride 3.0 Wax 4.0 Cetearyl glucoside 1.5 Sesquioleic acid sorbitan 0.9 Vaseline 3.0 paraffin 1.5 Preservative, coloring, fragrance Suitable amount Purified water Balance

5. Manufacture of pack

Compounding ingredient Content (% by weight) Root extract or fraction 0.1 glycerin 4.0 Polyvinyl alcohol 15.0 Hyaluronic acid extract 5.0 Beta Glucan 7.0 Allantoin 0.1 Nonyl phenyl ether 0.4 Polysorbate 60 1.2 Ethanol preservative 6.0 Good Preservative, coloring, fragrance Suitable amount Purified water Balance

6. Manufacture of ointment

Compounding ingredient Content (% by weight) Root extract or fraction 0.1 glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 15.0 Beta Glucan 7.0 Carbomer 0.1 Caprylic / capric triglyceride 3.0 Squalane 1.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Cetearyl alcohol 1.0 Wax 4.0 Preservative, coloring, fragrance Suitable amount Purified water Balance

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY &Lt; 120 > Composition for treating wound containing stellera chamaejasme          extract or fraction thereof and method for treating wound in a          subject <130> PN109096 <160> 20 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 agccatgtac gtagccatcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 ctctcagctg tggtggtgaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 tgagcggacg ctaaccccct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 cagacgggac agcactcgcc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 agccatgtac gtagccatcc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 tacggggcaa aaccgccagc 20 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 7 ttcgaggcac aaggcacaa 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 8 tccatggcca caacaactga 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 9 tcgagcccac cgggaacgaa a 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 10 aggcaactgg accgaaggcg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 11 tgactgtacc cggactggat 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 12 tggctccagt tcagacaagg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 13 agaagcctgt gccaactctc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 14 gggaatcgct gatgggaaca 20 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 15 agtgcactca gggggctcac a 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 16 ccggcttggc cgtaagtgtg t 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 17 gggtaccacg gaggcgaagg a 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 18 actgaggcac tggggttggg a 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 19 ggggcagctg aagcacctgg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 20 gagacgggcc acctagcgga 20

Claims (13)

A pharmaceutical composition for treating a wound containing an extract of P. roots as an active ingredient, wherein the P. roots extract is extracted from the ground part of the roots, wherein the roots extract is an ethanol extract, These wounds may be present in the skin as cuts, incisions, abrasions, lacerations, fractures, contusions, burns, or amputations. &Lt; / RTI &gt; delete [Claim 2] The method according to claim 1, wherein the P. primrose extract is a fraction of the P. primrose extract, which is obtained by suspending the P. primrose extract in water and sequentially fractionating the first fraction with hexane, ethyl acetate and butanol, , Ethyl acetate fraction, butanol fraction, or a combination thereof. delete 3. The composition of claim 1, wherein the root extract is from 0.001% to 80% by weight of the composition. The composition of claim 1, further comprising a pharmaceutically acceptable excipient or carrier. The composition according to claim 1, which promotes expression of a collagen gene in a cell. The composition according to claim 1, wherein expression of cytokine gene and chemokine gene in the cell is inhibited. The composition according to claim 1, which promotes expression of a differentiation factor gene of keratinocyte. The composition according to claim 1, which promotes migration of keratinocytes to the wound. The composition according to claim 1, wherein the wound is a cutaneous epidermis, dermis, or epidermis and dermis of the skin is lost. The composition of claim 1, for use in the proliferative, mature, or proliferative and maturation phases of the wound. A method of treating a wound in a subject comprising administering to the subject an amount of a composition of any one of claims 1, 3 and 5 to 12 effective to treat a wound, wherein said subject is a non-human mammal Way.

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