Composition for wound treatment containing extract of <i>Stellera chamaejasme </i>or fraction thereof and method for treating wound of subject

Abstract

Provided are a composition including Stellera chamaejasme extract for treating wound, a method of treating wound of a subject, a cosmetic composition for wound improving, skin wrinkle improving, or skin anti-aging, and a method of cosmeticizing for wound improving, skin wrinkle improving, or skin anti-aging.

Claims

1. A method of improving skin wrinkles, comprising applying to the wrinkled skin a cosmetic composition, wherein the cosmetic composition is for improving skin wrinkles, said cosmetic composition comprises a fraction of a Stellera chamaejasme extract as an active ingredient, wherein the Stellera chamaejasme extract is extracted from an aerial part of Stellera chamaejasme, wherein the Stellera chamaejasme extract is extracted in water, acetone, a C.sub.1-C.sub.6 alcohol, or a combination thereof, wherein said improving skin wrinkles is by promoting expression of a collagen gene in a cell, and wherein the fraction is a hexane fraction, a butanol fraction, or a combination thereof, wherein the hexane fraction and the butanol fraction are obtained by suspending the Stellera chamaejasme extract in water and then subsequently fractionating a suspension by using hexane and butanol.

2. The method of claim 1, wherein the Stellera chamaejasme extract is extracted in 75% (v/v) to 100% (v/v) ethanol, 75% (v/v) to 100% (v/v) methanol, 75% (v/v) to 100% (v/v) butanol, or a combination thereof.

3. The method of claim 1, wherein an amount of the Stellera chamaejasme extract or the fraction thereof is in a range of 0.001 percent by weight (wt %) to 80 wt %, based on an amount of the cosmetic composition.

4. The method of claim 1, the cosmetic composition further comprises a cosmetically acceptable excipient or carrier.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows that an ethanol extract of an aerial part of Stellera chamaejasme promotes migration of keratinocyte;

(2) FIG. 2 shows that a fraction of an ethanol extract of an aerial part of Stellera chamaejasme promotes migration of keratinocyte;

(3) FIG. 3 shows that an ethanol extract of an aerial part of Stellera chamaejasme promotes expression of COL1A1 and COL3A1 genes in a fibroblast;

(4) FIG. 4 shows that various solvent extracts of an aerial part of Stellera chamaejasme promote expression of COL1A1 and COL3A1 genes in a fibroblast;

(5) FIG. 5 shows that butanol extracts of an aerial part of Stellera chamaejasme, the butanol extracts being extracted in butanols having different concentrations, promote expression of COL1A1 and COL3A1 genes in a fibroblast;

(6) FIG. 6 shows an effect of a presence of an ethanol extract of Stellera chamaejasme on an expression level of MMP-1;

(7) FIG. 7 shows measurement results of a degree of effects of an ethanol extract of Stellera chamaejasme on expression of Type I procollagen in a human fibroblast irradiated with UVB;

(8) FIG. 8 shows effects of a Stellera chamaejasme extract on epithelialization of a rift wound in an animal experiment;

(9) FIG. 9 shows effects of a Stellera chamaejasme extract on reduction of wounded area of a rift wound in an animal experiment;

(10) FIG. 10 shows that a Stellera chamaejasme extract promotes recovery of a rift wound in an animal experiment, tested by a hematoxylin-eosin (H&E) staining protocol;

(11) FIG. 11 shows that a stem extract of Stellera chamaejasme promotes migration of keratinocyte;

(12) FIG. 12 shows that a fraction of a stem extract of Stellera chamaejasme promotes migration of keratinocyte;

(13) FIG. 13 shows that a stem extract of Stellera chamaejasme promotes expression of COL1A1 and COL3A1 genes in a fibroblast;

(14) FIG. 14 shows that a fraction of a stem extract of Stellera chamaejasme promotes expression of COL1A1 and COL3A1 genes in a fibroblast;

(15) FIG. 15 shows clinical test results where a stem extract of Stellera chamaejasme promotes recovery of a rift wound in an animal experiment; and

(16) FIG. 16 shows quantitative test results where a stem extract of Stellera chamaejasme promotes recovery of a rift wound in an animal experiment.

BEST MODE

(17) Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the present invention is not intended to be limited by these Examples.

Example 1: Preparation of Stellera chamaejasme Extract and Fraction Thereof

(18) In Example 1, a Stellera chamaejasme extract was prepared from Stellera chamaejasme, and effects of the Stellera chamaejasme extract and a fraction thereof on wound treatment were tested.

(19) 1. Preparation of Stellera chamaejasme Extract and Fraction Thereof

(20) (1) Preparation of Ethanol Extract of Stellera chamaejasme

(21) Only an aerial part of Stellera chamaejasme grown in Ulaanbaatar, Mongolia was collected. The collected aerial part was washed with water and dried. The dried aerial part of Stellera chamaejasme was sliced into, by using a straw cutter, small segments having a size in a range of 2 cm to 3 cm. 100 g of the sliced aerial part of Stellera chamaejasme and IL of 95% (v/v) aqueous solution of ethanol were added to a glass Erlenmeyer flask, and then mixed together. The mixture was extracted while stirring at a rate of 150 revolutions per minute (rpm) at a room temperature of about 20° C. for 48 hours.

(22) Next, the mixture was filtered through a filter paper (Whatman, NO. 2, 8 micrometers (μm)) to thereby obtain a filtrate. The obtained filtrate was concentrated under reduced pressure by using a rotary evaporator (N-1100 available from EYELA). In order to remove the remaining solvent, freeze drying was performed thereon using a freeze dryer (FDCF-12003 available from Operon) for 48 hours, thereby obtaining 3 g of dried Stellera chamaejasme extract from which the solvent was removed.

(23) (2) Preparation of Fraction of Ethanol Extract of Stellera chamaejasme

(24) (2.1) Fraction of Ethanol Extract

(25) 3 g of the dried ethanol extract of Stellera chamaejasme and 100 mL of water were added to an Erlenmeyer flask and then suspended. The suspension was poured into 250 mL of a separatory funnel, and 100 mL of n-hexane (extra pure grade, 100% (v/v)) was added thereto, followed by sufficient shaking. Then, the mixture was allowed to stand for 3 hours at room temperature. A hexane layer was only taken from the mixture using a separatory funnel. This process was repeated for three times, and the obtained hexane layer was concentrated under reduced pressure by using a rotary evaporator (N-1100 available from EYELA) to thereby obtain a hexane fraction.

(26) Also, in the same manner, 100% (v/v) of ethyl acetate was added to and mixed with the remaining water layer resulting from the hexane fraction separation process. Then, the same process was performed thereon to thereby obtain an ethyl acetate fraction.

(27) Also, in the same manner, water-saturated butanol was added to and mixed with the remaining water layer resulting from the ethyl acetate fraction separation process. Then, the same process was performed thereon to thereby obtain a butanol fraction. The remaining part of was used a water fraction

(28) The ethanol extract of Stellera chamaejasme and a fraction thereof were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 milligrams per milliliters (mg/mL) and used in the following experiment.

(29) (2.2) Extracts of Other Solvents and Fraction Thereof

(30) Extracts of other solvents and fractions thereof were obtained in substantially the same manner as in Section (2.1), except that, 100% (v/v) acetone, methanol at a given concentration, butanol were used instead of ethanol.

(31) 2. Increase of Migration of Human Keratinocyte

(32) It was tested whether the extract of Stellera chamaejasme or a fraction thereof increases migration of a human keratinocyte as follows.

(33) HaCaT human keratinocytes were added to each well of a 24-well microtiter plate containing 1 mL of HyClone Dulbecco's modified eagle's medium (DMEM) containing 2.5% (v/v) of fetal bovine serum such that 2×10.sup.5 cells were placed in each well. Next, the keratinocytes were cultured at a temperature of 37° C. and in 5% CO.sub.2 incubator until a confluency thereof reached 80%. A cell monolayer in the medium was “scratch-injured” with a p10 pipette tip.

(34) Subsequently, 200 μl of serum-free DMEM, and the Stellera chamaejasme extract or a fraction thereof were added to the scratched cell monolayer at a concentration of 5 μg/mL and 10 μg/mL in each medium. Then, the human keratinocytes were cultured under the same culture conditions for 24 hours. In a negative control, the same amount of DMSO, which was used to dissolve the extract, was treated, and in a positive control, a Centella asiatica extract (10 μg/mL) was used at a given concentration. Thereafter, the cell layer was stained using a crystal violet reagent, to test a degree of recovery of scratch wound. The Centella asiatica extract was purchased from Biospectrum (Korea).

(35) FIG. 1 shows that the ethanol extract of an aerial part of Stellera chamaejasme promotes migration of keratinocyte. FIG. 2 shows that the fraction of an ethanol extract of an aerial part of Stellera chamaejasme promotes migration of keratinocyte. In FIGS. 1, A, B, C, and D respectively represent DMSO (negative control), the 5 μg/mL Stellera chamaejasme extract, the 10 μg/mL Stellera chamaejasme extract, and 10 μg/mL Centella asiatica extract (positive control). In FIGS. 2, A, B, C, D, E, F, G, and H respectively represent DMSO (negative control), the hexane fraction of the 5 μg/mL ethanol extract of an aerial part of Stellera chamaejasme, the ethyl acetate fraction of the 5 μg/mL ethanol extract of an aerial part of Stellera chamaejasme, the butanol fraction of the 5 μg/mL ethanol extract of an aerial part of Stellera chamaejasme, the 5 μg/mL Centella asiatica extract, the hexane fraction of the 10 μg/mL ethanol extract of an aerial part of Stellera chamaejasme, the ethyl acetate fraction of the 10 μg/mL ethanol extract of an aerial part of Stellera chamaejasme, and the butanol fraction of the 10 μg/mL ethanol extract of an aerial part of Stellera chamaejasme.

(36) As shown in FIGS. 1 and 2, in the negative control conditions, the scratch injury of the treated cells remains relatively unhealed after 24 hours of the treatment. On the other hand, in the cells treated with the ethanol extract of an aerial part of Stellera chamaejasme, the scratch injury were healed due to proliferation and migration of wound edges, and thus after 24 hours, the scratched area reduced.

(37) 3. Increase of Expression of COL1A1 and COL3A1 Genes in Fibroblast

(38) It was tested whether the Stellera chamaejasme extract promotes expression of collagen genes in a human fibroblast.

(39) Human dermal fibroblasts (HDF, available from American Type Culture Collection, USA) were added to each well of a 6-well microtiter plate containing 2 mL of HyClone DMEM containing 2.5% (v/v) of fetal bovine serum such that 2×10.sup.6 cells were placed in each well. Next, the human dermal fibroblasts were cultured under the same conditions until a confluency thereof reached 80%.

(40) The Stellera chamaejasme extract was added to the cultured cells as such at a concentration of 1 μg/mL, 5 μg/mL, and 10 μg/mL in each medium. Then, the cell culture was allowed to continue for 24 hours under the same conditions described above. In a negative control, the same amount of DMSO, which was used to dissolve the extract, was treated, and in a positive control, a Centella asiatica extract (available from Biospectrum, Korea) was used. After a lapse of 24 hours, expression of collagen genes was tested by reverse transcription polymerase chain reaction (RT-PCR) assay. In detail, after a lapse of 24 hours, the total RNAs were collected from the cells using a TRIzol reagent (available from Invitrogen, Carlsbad, Calif., USA), which was then reverse transcripted to perform RT-PCR as follows. First, in order to synthesize cDNAs, the RNAs were reverse transcripted using a reverse transcriptase. The RT-PCR was performed using specific primers shown below. The relative amount of mRNA expression of each gene was standardized based on an amount of β-actin.

(41) FIG. 3 shows that the ethanol extract of an aerial part of Stellera chamaejasme promotes expression of COL1A1 and COL3A1 genes in a fibroblast.

(42) As shown in FIG. 3, it was found that the ethanol extract of an aerial part of Stellera chamaejasme significantly activates expression of at least one gene selected from the group consisting of COL1A1 and COL3A1, which are collagen synthesis-related factors.

(43) FIG. 4 shows that various solvent extracts of an aerial part of Stellera chamaejasme promote expression of COL1A1 and COL3A1 genes in a fibroblast. In FIG. 4, Samples 1, 2, 3, 4, 5, 6, and 7 respectively represent an acetone extract, an acetone extract, a 100% ethanol extract, a 25% methanol extract, a 50% methanol extract, a 75% methanol extract, and a 100% methanol extract.

(44) FIG. 5 shows that butanol extracts of an aerial part of Stellera chamaejasme, the butanol extracts being extracted in butanols having different concentrations, promote expression of COL1A1 and COL3A1 genes in a fibroblast. In FIG. 5, Samples 1, 2, 3, and 4 respectively represent a 25% butanol extract, a 50% butanol extract, a 75% butanol extract, and a 100% butanol extract.

(45) As shown in FIGS. 4 and 5, it was found that the acetone, methanol, ethanol, and butanol extracts of an aerial part of Stellera chamaejasme significantly activates expression of at least one gene selected from the group consisting of COL1A1 and COL3A1, which are collagen synthesis related factors.

(46) TABLE-US-00001 TABLE 1 β-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

(47) 4. Measurement Experiment of Suppression of Collagenase-1 Secretion in Fibroblast that is Senescence-Induced by UV Irradiation

(48) The suppressing effects of the ethanol extract of Stellera chamaejasme on formation of collagenase-1 (Matrix Metalloproteinase-1, MMP-1) was tested.

(49) First, human fibroblasts were added to each well of 24-well microtiter plate containing DMEM medium containing 2.5% fetal bovine serum such that 1×10.sup.5 cells were placed in each well. the human fibroblasts were cultured until a confluency thereof reached 90%. Then, the ethanol extract of Stellera chamaejasme dissolved in a serum-free DMEM medium was added thereto at a concentration of 1 μg/mL, 5 μg/mL, and 10 μg/mL in each medium. Then, the cell culture was allowed to continue for 24 hours. Thereafter, irradiation with 15 mJ ultraviolet B (UVB) was performed by using a UV irradiator. Subsequently, the ethanol extract of Stellera chamaejasme dissolved in a serum-free DMEM medium was further added thereto at a concentration of 1 μg/mL, 5 μg/mL, and 10 μg/mL in each medium. After 24 hours, the cell culture medium was collected therefrom, which was then centrifuged to thereby obtain a supernatant only.

(50) A level of MMP-1 of the collected supernatant was measured by using ELISA kits: MMP-1 (QIA55, available from Merck&Co., USA). First, the supernatant was added to a 96-well plate, on which a mouse anti-MMP-1 antibody as a primary antibody was uniformly coated. Then, for 2 hours, incubation was performed thereon at room temperature to allow the formation of an antigen-antibody complex. After a lapse of 2 hours, an anti-MMP-1 antibody conjugated to a horseradish peroxidase, as a chromophore, was added to the 96-well plate. Then, for 1 hour, incubation was performed thereon. After a lapse of 1 hour, tetramethylbenzidine (as a chromogenic substrate) was added thereto. Then, for 30 minutes, incubation was performed at room temperature thereon to allow color formation. Subsequently, a terminating buffer was added thereto to stop the reaction. The color of the reaction solution was yellow, and a degree of yellow color varied depending on a degree of the reaction progression. The absorbance of the wells of the yellow-colored 96-well plate was measured at 450/540 nanometers (nm) using a plate reader.

(51) FIG. 6 shows the effect of a presence of an ethanol extract of Stellera chamaejasme on an expression level of MMP-1. As shown in FIG. 6, it was found that the ethanol extract of Stellera chamaejasme has a significant suppressing effect on expression of a MMP-1 gene. As a positive control, epigallocatechin gallate (EGCG) was used.

(52) 5. Measurement Experiment of Promotion of Collagen Production in Fibroblast that is Senescence-Induced by UV Irradiation

(53) It was tested whether the ethanol extract of Stellera chamaejasme promotes expression of Type I procollagen (Type-1 procollagen).

(54) First, human fibroblasts were added to each well of 24-well microtiter plate containing DMEM medium containing 2.5% fetal bovine serum such that 1×10.sup.5 cells were placed in each well. the human fibroblasts were cultured until a confluency thereof reached 90%. Then, the ethanol extract of Stellera chamaejasme dissolved in a serum-free DMEM medium was added thereto at a concentration of 1 μg/mL, 5 μg/mL, and 10 μg/mL in each medium. Then, the cell culture was allowed to continue for 24 hours. Thereafter, irradiation with 15 mJ UVB was performed by using a UV irradiator. Subsequently, the Stellera chamaejasme extract dissolved in a serum-free DMEM medium was further added thereto at a concentration of 1 μg/mL, 5 μg/mL, and 10 μg/mL in each medium. Then, the cell culture was allowed to continue in the same manner. After a lapse of 24 hours, the cell culture medium was collected therefrom, which was then centrifuged to thereby obtain a supernatant only. Subsequently, a level of procollagen Type I C-peptide (PIP) in the obtained supernatant was measured by using a PIP EIA Kit (Cat. #MKI01, available from Takara Bio Inc., Japan). PIP is a peptide that is cleaved from procollagen during the process of synthesis of collagen from procollagen in vivo. A level of PIP is in proportion to a level of collagen. Thus, PIP is a peptide that is used as an index for testing a level of collagen in the art.

(55) In detail, a mouse anti-PIP monoclonal antibody conjugated to a horseradish peroxidase (POD) (also referred to as an “antibody-POD conjugate”) was added to a 96-well plate, on which a mouse anti-PIP monoclonal antibody was uniformly coated. Subsequently, the cell culture medium and a standard solution were each added to each well, and then the plate was allowed to stand at a temperature of 37° C. for 3 hours. The wells was then washed, and a substrate solution including hydrogen peroxide and tetramethylbenzidine was added to each well. Then, the wells was incubated for 15 minutes. A stop solution was next added to each well so as to stop the reaction. The absorbance of the wells was measured at 450 nm using a plate reader.

(56) FIG. 7 shows the measurement results of a degree of effects of an ethanol extract of Stellera chamaejasme on expression of Type I procollagen in a human fibroblast irradiated with UVB. As shown in FIG. 7, it was found that the ethanol extract of Stellera chamaejasme has a significant promoting effect on expression of Type I procollagen.

(57) 6. Improvement of Wound Healing During Animal Experiment

(58) It was tested whether the Stellera chamaejasme extract promotes wound healing in an animal experiment.

(59) (1) Laboratory Animals

(60) Sprague-Dawley (SD) rats (male, 7-week-old) were purchased from Koatech, Co., Ltd. A condition of a temperature of 25±1° C., a humidity of 50%±10%, and a photoperiod of 12 hours of light and 12 hours of darkness were maintained by automatic control. In addition, the rats were allowed to freely consume feeds of normal diet and water. All of the laboratory animals were allowed to have one week for adapting to the environment of an animal room, and then used in experiments. The dietary, the dosage, and the method of administration to experimental groups are shown in Table 2.

(61) TABLE-US-00002 TABLE 2 Experimental Dosage and administration group Testing material method G1 Vehicle — G2 Centella asiatica extract 1%(v/v), once a day G3 Centella asiatica extract 3%(v/v), once a day G4 Ethanol extract of Stellera 1%(v/v), once a day chamaejasme G5 Ethanol extract of Stellera 3%(v/v), once a day chamaejasme

(62) (2) Wound Induction and Treatment

(63) SD rats were fasted for 24 hours before putting the SD rats under anesthesia, and 100 mL of water were fed to the SD rats. 1 hour prior to wound induction in each of the experimental groups, 100% zoletil was mixed with 100% rompun at a ratio of 1:2 to prepare a mixture. Each of the experimental group was put under anesthesia by an intramuscular injection using the mixture at 0.1 cc/kg. After that, hairs on the dorsal surface of each of the rats were removed by using an electric depilator for all experimental groups. After applying disinfection of 10% povidone-iodine and 70% (v/v) ethanol to the dorsal surface, a full-thickness excisional wound (including the dermis layer) having a square size of 2 cm×2 cm was induced by cutting with sterilized surgical scissors.

(64) (3) Observation of Changes in Wound by Naked Eye

(65) Each of the Stellera chamaejasme extract (1% or 3% (v/v)) and the Centella asiatica extract (1% or 3% (v/v)), which is known as effective in wound healing, were applied to the wounded skin for two weeks once a day. The Centella asiatica extract is the same as those described in Section 2 of Examples 1. The wounded area was photographed at 1, 4, 8, 11, and 15 days after each test material was applied to the wound, and the wounded area was measured and analyzed using ImageJ (NIH, USA).

(66) (4) Histological Examination

(67) On the 14.sup.th day after application of each test material, all experimental groups were put under inhalation anesthesia using ether, and a part, to which the test material was administered, was fixed with 10% neutral-buffered formalin solution. For histopathologic examination, the fixed skin tissue was cut one by one with a length of 2 cm×2 cm square with respect to the major axis of the wounded surface so that the wounded surface was examined evenly. The cut tissue was embedded in paraffin according to a general tissue processing method, and a piece of the tissue was formed to a thickness in a range of about 3 μm to 4 μm and attached to a slide glass. The attached pieces were stained with hematoxylin-eosin (H&E), and the stained pieces were examined using an optical microscope (Olympus BX53, Japan). The interpretation of the wounded tissue (e.g., a cut) was score-evaluated according to the evaluation of Nisbet et al. (Tissue Eng Part A. 2010, September; 16 (9): 2833-42) and Barati et al (Diagn Pathol. 2013; 8:120). The criteria for the evaluation are shown in Table 3.

(68) TABLE-US-00003 TABLE 3 Histopatholo- Score gical parameter 0 1 2 3 Epithelial- Not Partially Completely Completely ization progressed progressed progressed, and maturely but immature progressed or thin

(69) FIG. 8 shows effects of the ethanol extract of Stellera chamaejasme on epithelialization of a rift wound in an animal experiment. In FIG. 8, G1, G2, G3, G4, and G5 in the x-axis represent the same experimental groups shown in Table 2, and the scores in the y-axis are measured values based on the epithelialization criteria shown in Table 3.

(70) As shown in FIG. 8, the Stellera chamaejasme extracts of the present invention shown as G4 and G5 significantly improved epithelialization, as compared with the control group, G1. In FIGS. 8 and 9, * indicates ANOVA by Scheffe's test (p<0.05).

(71) FIG. 9 shows effects of the ethanol extracts of Stellera chamaejasme on reduction of wounded area of a rift wound in an animal experiment. In FIG. 9, the wounded area represents the results of the measured wounded area at 1, 4, 8, 11, and 15 days after applying Stellera chamaejasme extract to the wound. In FIG. 9, G1, G2, G3, G4, and G5 in the x-axis represent the same experimental groups shown in Table 2, and the y-axis represents the wounded area. As shown in FIG. 9, the Stellera chamaejasme extracts of the present invention shown as G4 and G5 significantly decreased the wounded area, as compared with the control group, G1.

(72) FIG. 10 shows that the ethanol extracts of Stellera chamaejasme promote recovery of a rift wound in an animal experiment, tested by a hematoxylin-eosin (H&E) staining protocol. As shown in FIG. 10, it was found that the Stellera chamaejasme extract of the present invention promotes re-epithelialization of a wound, as compared with the control group. As shown in FIG. 8, when H&E tissue staining is used, two kinds of layers may be found; the upper pink layer is the epidermis and the lower pink layer is the dermis. Upon comparing the control group with the group treated with the sample, it can be seen that the group treated with the sample recovered in light of the thickness and cracking of the epidermis, i.e., the upper layer.

(73) According to the results described above, it was found that a Stellera chamaejasme extract promotes re-epithelialization of a rift wound in an animal experiment and significantly promotes recovery of the wound. The above results are based on the use of an ethanol extract of an aerial part of Stellera chamaejasme; however, a hexane, ethyl acetate, or butanol fraction of the ethanol extract of an aerial part of Stellera chamaejasme; and an acetone, methanol, or butanol extract of an aerial part of Stellera chamaejasme, or a hexane, ethyl acetate, or butanol fraction thereof were also found to have similar results.

(74) Further, according to the above described Example, under ultraviolet (UV) irradiation, it was found that a Stellera chamaejasme extract reduces expression of MMP-1, and promotes expression of collagen in a human fibroblast. That is, under UV irradiation, the Stellera chamaejasme extract reduces expression of collagenase and promotes expression of collagen in a human fibroblast. Thus, the Stellera chamaejasme extract may promote elasticity of a tissue containing human fibroblasts, for example, by promoting collagen synthesis in skin. Thus, the Stellera chamaejasme extract is effective for improving tissues including human fibroblasts, for example, the Stellera chamaejasme extract is effective for skin wrinkle improving or skin anti-aging.

Example 2: Preparation of Stem Extract of Stellera chamaejasme and Fraction Thereof

(75) In Example 1, a stem extract of Stellera chamaejasme and a fraction thereof were prepared from Stellera chamaejasme, and effects of the stem extract of Stellera chamaejasme and the fraction thereof on wound treatment were tested.

(76) 1. Preparation of Stem Extract of Stellera chamaejasme and Fraction Thereof

(77) (1) Preparation of Stem Extract of Stellera chamaejasme

(78) Only a stem of an aerial part of Stellera chamaejasme grown in Ulaanbaatar, Mongolia was collected. The collected stem was washed with water and dried. The dried stem of Stellera chamaejasme was sliced into, by using a straw cutter, small segments having a size in a range of 2 cm to 3 cm. 100 g of the sliced stem of Stellera chamaejasme and IL of ethanol were added to a glass Erlenmeyer flask, and then mixed together. The mixture was extracted while stirring at a rate of 150 rpm at a room temperature of about 20° C. for 48 hours.

(79) Next, the mixture was filtered through a filter paper (Whatman, NO. 2, 8 μm) to thereby obtain a filtrate. The obtained filtrate was concentrated under reduced pressure by using a rotary evaporator (N-1100 available from EYELA). In order to remove the remaining solvent, freeze drying was performed thereon using a freeze dryer (FDCF-12003 available from Operon) for 48 hours, thereby obtaining 3 g of dried stem Stellera chamaejasme extract from which the solvent was removed.

(80) (2) Preparation of Fraction of Stem Extract of Stellera chamaejasme

(81) 3 g of the dried stem Stellera chamaejasme extract and 100 mL of water were added to an Erlenmeyer flask and then suspended. The suspension was poured into 250 mL of a separatory funnel, and 100 mL of n-hexane (extra pure grade, 95% or greater) was added thereto, followed by sufficient shaking. Then, the mixture was allowed to stand for 3 hours at room temperature. A hexane layer was only taken from the mixture using a separatory funnel. This process was repeated for three times, and a hexane fraction was obtained and concentrated under reduced pressure by using a rotary evaporator (N-1100 available from EYELA) to thereby obtain a resulting hexane fraction.

(82) Also, in the same manner, ethyl acetate was added to and mixed with the water fraction from which the hexane fraction was removed. Then, the same process was performed thereon to thereby obtain an ethyl acetate fraction.

(83) Also, in the same manner, water-saturated butanol was added to and mixed with the water fraction from which the ethyl acetate fraction was removed. Then, the same process was performed thereon to thereby obtain a butanol fraction.

(84) The extract of Stellera chamaejasme and a fraction thereof were dissolved in DMSO at a concentration of 20 mg/mL and used in the following experiment.

(85) 2. Increase of Migration of Human Keratinocyte

(86) It was tested whether the stem extract of Stellera chamaejasme or a fraction thereof increases migration of a human keratinocyte as follows.

(87) HaCaT human keratinocytes were added to each well of a 24-well microtiter plate containing 1 mL of HyClone DMEM containing 2.5% (v/v) of fetal bovine serum such that 2×10.sup.5 cells were placed in each well. Next, the keratinocytes were cultured at a temperature of 37° C. and in 5% CO.sub.2 incubator until a confluency thereof reached 80%. A cell monolayer in the medium was “scratch-injured” with a p10 pipette tip.

(88) Subsequently, 200 μl of serum-free DMEM, and the Stellera chamaejasme extract or a fraction thereof were added to the scratched cell monolayer at a concentration of 5 μg/mL and 10 μg/mL in each medium. Then, the human keratinocytes were cultured under the same culture conditions for 24 hours. In a negative control, the same amount of DMSO, which was used to dissolve the extract, was treated, and in a positive control, a GW501516 (available from Sigma-Aldrich, USA), i.e., a peroxisome proliferator activated receptor δ (PPARδ) activator, or a Centella asiatica extract was used at a given concentration. Thereafter, the cell layer was stained using a crystal violet reagent, to test a degree of recovery of scratch wound. The Centella asiatica extract was purchased from Biospectrum (Korea).

(89) FIG. 11 shows that the stem extract of Stellera chamaejasme promotes migration of keratinocyte.

(90) FIG. 12 shows that the fraction of a stem extract of Stellera chamaejasme promotes migration of keratinocyte.

(91) As shown in FIGS. 11 and 12, in the negative control conditions, the scratch injury of the treated cells remains relatively unhealed after 24 hours of the treatment. On the other hand, in the cells treated with the stem extract of an aerial part of Stellera chamaejasme or a fraction thereof, the scratch injury were healed due to proliferation and migration of wound edges, and thus after 24 hours, the scratched area reduced. In FIG. 11, GW represents GW501516.

(92) 3. Increase of Expression of COL1A1 and COL3A1 Genes in Fibroblast

(93) It was tested whether the stem extract of Stellera chamaejasme or a fraction thereof promotes expression of collagen genes in a human fibroblast.

(94) HDF (available from American Type Culture Collection, USA) were added to each well of a 6-well microtiter plate containing 2 mL of HyClone DMEM containing 2.5% (v/v) of fetal bovine serum such that 2×10.sup.6 cells were placed in each well. Next, the human dermal fibroblasts were cultured under the same conditions until a confluency thereof reached 80%.

(95) The stem extract of Stellera chamaejasme was added to the cultured cells as such at a concentration of 1 μg/mL, 5 μg/mL, and 10 μg/mL in each medium. The fraction of the stem extract of Stellera chamaejasme was added thereto at a concentration of 15 μg/mL and 10 μg/mL in each medium. Then, the cell culture was allowed to continue for 24 hours under the same conditions described above. In a negative control, the same amount of DMSO, which was used to dissolve the extract, was treated, and in a positive control, a Centella asiatica extract (available from Biospectrum, Korea) was used. After a lapse of 24 hours, expression of collagen genes was tested by RT-PCR assay. In detail, after a lapse of 24 hours, the total RNAs were collected from the cells using a TRIzol reagent (available from Invitrogen, Carlsbad, Calif., USA), which was then reverse transcripted to perform RT-PCR as follows. First, in order to synthesize cDNAs, the RNAs were reverse transcripted using a reverse transcriptase. The RT-PCR was performed using specific primers shown in Table 1. The relative amount of mRNA expression of each gene was standardized based on an amount of β-actin.

(96) FIG. 13 shows that the stem extract of Stellera chamaejasme promotes expression of COL1A1 and COL3A1 genes in a fibroblast.

(97) FIG. 14 shows that the fraction of a stem extract of Stellera chamaejasme promotes expression of COL1A1 and COL3A1 genes in a fibroblast.

(98) As shown in FIGS. 13 and 14, it was found that the stem extract of Stellera chamaejasme or a fraction thereof significantly activates expression of at least one gene selected from the group consisting of COL1A1 and COL3A1, which are collagen synthesis-related factors.

(99) 4. Improvement of Wound Healing During Animal Experiment

(100) It was tested whether the stem extract of Stellera chamaejasme promotes wound healing in an animal experiment.

(101) (1) Laboratory Animals

(102) SD rats (male, 7-week-old) were purchased from Koatech, Co., Ltd. A condition of a temperature of 25±1° C., a humidity of 50%±+10%, and a photoperiod of 12 hours of light and 12 hours of darkness were maintained by automatic control. In addition, the rats were allowed to freely consume feeds of normal diet and water. All of the laboratory animals were allowed to have one week for adapting to the environment of an animal room, and then used in experiments. The dietary, the dosage, and the method of administration to experimental groups are shown in Table 4.

(103) TABLE-US-00004 TABLE 4 Constitution of experimental group, dosage of testing material, and administration method Experimental Dosage and administration group Testing material method G1 Not progressed — G2 Vehicle — G3 Stellera chamaejasme extract 3%(v/v), once a day G4 Centella asiatica extract 3%(v/v), once a day

(104) (2) Wound Induction and Treatment

(105) SD rats were fasted for 24 hours before putting the SD rats under anesthesia, and 100 mL of water were fed to the SD rats. 1 hour prior to wound induction in each of the experimental groups, 100% zoletil was mixed with 100% rompun at a ratio of 1:2 to prepare a mixture. Each of the experimental group was put under anesthesia by an intramuscular injection using the mixture at 0.1 cc/kg. After that, hairs on the dorsal surface of each of the rats were removed by using an electric depilator for all experimental groups. After applying disinfection of 10% povidone-iodine and 70% ethanol to the dorsal surface, a full-thickness excisional wound (including the dermis layer) having a square size of 2 cm×2 cm was induced by cutting with sterilized surgical scissors.

(106) (3) Observation of Changes in Wound by Naked Eye

(107) Each of the stem extract of Stellera chamaejasme (3% (v/v)) and the Centella asiatica extract (3% (v/v)), which is known as effective in wound healing, were applied to the wounded skin once a day. The Centella asiatica extract is the same as those described in Section 2 of Examples 1. The wounded area was photographed at 1, 4, 8, 11, and 14 days after each test material was applied to the wound, and the wounded area was measured and analyzed using ImageJ (NIH, USA).

(108) (4) Histological Examination

(109) On the 14.sup.th day after application of each test material, all experimental groups were put under inhalation anesthesia using ether, and a part, to which the test material was administered, was fixed with 10% neutral-buffered formalin solution. For histopathologic examination, the fixed skin tissue was cut one by one with a length of 2 cm×2 cm square with respect to the longitudinal direction of the wounded surface so that the wounded surface was examined evenly. The cut tissue was embedded in paraffin according to a general tissue processing method, and a piece of the tissue was formed to a thickness in a range of about 3 μm to 4 μm and attached to a slide glass. The attached pieces were stained with hematoxylin-eosin (H&E), and the stained pieces were examined using an optical microscope (Olympus BX53, Japan). The interpretation of the wounded tissue was score-evaluated according to an evaluation method known in the art (Nisbet et al. (Tissue Eng Part A. 2010, September; 16 (9): 2833-42) and Barati et al (Diagn Pathol. 2013; 8:120)). The criteria for the evaluation are shown in Table 5. The interpretation results are shown in Table 6.

(110) As shown in Table 6, it can be seen that the stem extract of Stellera chamaejasme has significant effects on formation of fibrous tissue, angiogenesis, and epithelialization.

(111) FIG. 15 shows clinical test results where the stem extract of Stellera chamaejasme promotes recovery of a rift wound in an animal experiment. FIG. 16 shows quantitative test results where the stem extract of Stellera chamaejasme promotes recovery of a rift wound in an animal experiment. As shown in FIGS. 15 and 16, it was found that the Stellera chamaejasme extract promotes re-epithelialization of a wounded part, and significantly facilitates recovery of a dehiscenced wound.

(112) TABLE-US-00005 TABLE 5 Histopatholo- Score gical parameter 0 1 2 3 Inflammation Not Mild Moderate Marked progressed Formation of Not occurred Few A number of Majority of fibrous tissue or at least fibroblasts fibroblasts fibroblasts level of fibroblasts were formed Angiogenesis Not 5 or less 6 to 10 blood More than progressed blood vessels vessels per 10 blood per high HPF vessels power field per HPF (HPF) Epithelial- Not Partially Focal Completed ization progressed progressed defective

(113) TABLE-US-00006 TABLE 6 Histopathological parameter Experimental Formation of Epithelial- group Inflammation fibrous tissue Angiogenesis ization 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 *indicates P < 0.05, which represents statistical significance with G1.

Preparation Example 1: Preparation of a Pharmaceutical Preparation

(114) 1. Preparation of Soft Capsule

(115) A soft capsule was prepared by mixing 150 mg of an ethanol stem extract of Stellera chamaejasme or a fraction thereof, 2 mg of palm oil, 8 mg of hydrogenated palm oil, 4 mg of yellow wax, and 6 mg of lecithin and filling the soft capsule up to 400 mg per capsule according to a conventional method.

(116) 2. Preparation of Tablet

(117) 150 mg of an ethanol stem extract of Stellera chamaejasme or a fraction thereof, 100 mg of glucose, 50 mg of red ginseng extract, 96 mg of starch, and 4 mg of magnesium stearate were mixed, and 40 mg of 30% ethanol was added thereto to form granules. The granules were then dried at a temperature of 60° C., and tableted by using a tablet machine.

(118) 3. Preparation of Granules

(119) 150 mg of an ethanol stem extract of Stellera chamaejasme or a fraction thereof, 100 mg of glucose, 50 mg of red ginseng extract, and 600 mg of starch were mixed, and 100 mg of 30% ethanol was added thereto to form granules. The granules were then dried at a temperature of 60° C., and then filled in a pack. The final weight of the content was 1 g.

Preparation Example 2: Preparation of a Cosmetic Composition

(120) In Preparation Example 2, an ethanol stem extract of Stellera chamaejasme was used.

(121) 1. Preparation of Moisturizers (Skin Lotion)

(122) TABLE-US-00007 TABLE 7 Compounding component Amount (wt %) Stellera chamaejasme extract or a fraction thereof 0.1 Glycerin 3.0 Butylene glycol 2.0 Propylene glycol 2.0 Carboxy vinyl polymer 0.1 PEG-12 nonylphenyl ether 0.2 Polysorbate 80 0.4 Ethanol 10.0 Triethanolamine 0.1 Preservatives, pigments, perfumes Proper amount Distilled water The remaining part

(123) 2. Preparation of Nutrition Lotion (Milk Lotion)

(124) TABLE-US-00008 TABLE 8 Compounding component Amount (wt %) Stellera chamaejasme extract or a fraction thereof 0.1 Glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxy vinyl polymer 0.1 Wax 4.0 Polysorbate 60 1.5 Caprylic/capric triglyceride 5.0 Squalane 5.0 Sorbitan sesquioleate 1.5 Liquid paraffin 0.5 Cetearyl alcohol 1.0 Triethanolamine 0.2 Preservatives, pigments, perfumes Proper amount Distilled water The remaining part

(125) 3. Preparation of Nutrition Cream

(126) TABLE-US-00009 TABLE 9 Compounding component Amount (wt %) Stellera chamaejasme extract or a fraction thereof 0.1 Glycerin 3.0 Butylene glycol 3.0 Liquid paraffin 7.0 Betaglucan 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 Preservatives, pigments, perfumes Proper amount Distilled water The remaining part

(127) 4. Preparation of Massage Cream

(128) TABLE-US-00010 TABLE 10 Compounding component Amount (wt %) Stellera chamaejasme extract or a fraction thereof 0.1 Glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 45.0 Betaglucan 7.0 Carbomer 0.1 Caprylic/capric triglyceride 3.0 Wax 4.0 Cetearyl glucoside 1.5 Sorbitan sesquioleate 0.9 Vaseline 3.0 Parafin 1.5 Preservatives, pigments, perfumes Proper amount Distilled water The remaining part

(129) 5. Preparation of Pack

(130) TABLE-US-00011 TABLE 11 Compounding component Amount (wt %) Stellera chamaejasme extract or a fraction thereof 0.1 Glycerin 4.0 Polyvinyl alcohol 15.0 Hyaluronic acid extract 5.0 Betaglucan 7.0 Allantoin 0.1 Nonyl phenyl ether 0.4 Polysorbate 60 1.2 Ethanol preservative 6.0 proper amount Preservatives, pigments, perfumes Proper amount Distilled water The remaining part

(131) 6. Preparation of Ointment

(132) TABLE-US-00012 TABLE 12 Compounding component Amount (wt %) Stellera chamaejasme extract or a fraction thereof 0.1 Glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 15.0 Betaglucan 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 Preservatives, pigments, perfumes Proper amount Distilled water The remaining part