COSMETIC COMPOSITION COMPRISING EXTRACT OF TESTA OF PINUS KORAIENSIS SIEBOLD ET ZUCC

Hereinafter the present invention for rapidly and to reduce a memory to embodiment.. However, these embodiment examples of the present invention relate for described embodiment of the present invention range not limited aspect.

In the embodiment 1. Preparation of material

The present used in the study material comprising a host material and a coagulant to Korean white pine, area Gapyeong tools sufficiently maturation pine nut cone from the collecting pine nut conepine nut nine with surrounding outer shell of the separating pieces chamber cone and cosmetics within the with the old bell room ( hwang pine nut ) is obtained. A rigid bell room the bran or high-grade white bran and cosmetics [...] fruit ( hundred pine nuts ) separating corresponding advertisement based on the shown list, separated bell blood washing, drying the 40 mesh degree as powder was use in lower and this adamantane is stored at 4 °C.

In the embodiment 2.pine nut external kind blood for producing

In the case of hot-water extract pine nut external kind blood amount back 10 distilled to 100g sample 85 °C applied in supernatant extracts cooling reflux time 3 are separated from each other and materials and precipitates 3 iterations extract, ethanol extract, the sample 10 ethanol amount of back 24 at room temperature the adding a supernatant after dipping it extracted time method for separating materials and precipitates was 3 in a. Each extract 10 in 12,000rpm centrifugal minutes after the separation of the corresponding advertisement based on the shown list filtered filter paper Whatman No. 1, each extract storage amount and the layer paper and material, is cheap lyophilized to concentrating while the present kit article sample at night.

In the embodiment 3. Total polyphenol content reading

Total polyphenol compound the corresponding advertisement based on the shown measuring Folin-Denis method, sample 1 ml distilled and a 95% ethanol 1 ml to 5 ml is added the mixed with agent is placed 1N Folin-ciocalteu reagent 0.5 ml, 5 minutes after the composition is left for, Na₂ CO₃ 1 ml after, 725 nm absorbance in the method for measuring operations 1 gallic acid using was terms of amount from a standard curve.

In the embodiment 4.Measuring electron donating ability pine nut external kind blood extract

Electronic grant royal tomb the method of Blois (EDA: electron donating ability) adjusted by deforming the measure the. Each sample 1,1-diphenyl-2-picrylhydrazyl (DPPH) 1 ml of 0.2 mm to 2 ml solution doesn't have any error frames, after 30 minutes then the composition is left for measure the absorbance in 517 nm. Electronic grant royal tomb addition of sample solution exhibits acid in higher reduced absorbance nothing addition old friendship port.

In the embodiment 5.Decolorization ABTS radical cation (ABTS)measuring

Measurements of the ABTS radical cation decolorization Pellegrin method such as it was determined that by. I.e., 7 mm ABTS and a 140 mm K₂ S₂ O₈ for 5 ml:88 μ L in the warp then size 14-16 a dark, 1:88 and absolute ethanol same 734 nm plants ratio 0.7±0.002 value absorbance of compared a controlled such that, by cylinder to have ABTS solution. BHT as positive control and each sample solution for ABTS solution 1 ml and a 50 μ L 2.5 minutes 30 seconds after mixing the downward measuring absorbance in 734 nm the incubation of the two formulas inhibited by the rate (%) of the bill.

In the embodiment 6. Antioxidant protection factor (PF)measuring

Shetty and Andarwulan of the PF β-carotene factor have been measured by method. 10 mg of a 1 ml solution of 50 ml of chloroform to evaporator senses a rotation velocity of the disk for handwritten paste has better mouth feeling and in 40 °C water bath after chloroform is distilled to 20 μL linoleic acid, 184 μL Tween 40 and 50 ml H₂ O₂ creates added to the emulsion, 5 ml of sample solution by mixing the emulsion to form a family of 100 μ L vortex 50 °C in putting the finishing plants to the distance measuring sensors reacting 30 minutes, it was determined that in 470 nm absorbance.

In the embodiment 7.Thiobarbituric acid reaction substance(TBARs)measuring

The TBARS Aust and a Blurge method of it was determined that according to. 1% linoleic acid and a 1% Tween 40 creates emulsion in a 0.2 ml emulsion 0.8 ml in 50 °C water bath after mixing the sample and a reacted time 10. TBA regent to 1 ml reaction solution after reaction [100 ml stock solution (15% trichloroacetic acid (TCA), 0.375% thiobarbituric acid, 0.25 M HCl mixture) to 3 ml of 2% BHT/EtOH mixture] 2 ml then possible with conventional boiling 10 minutes 15 minutes 15 minutes after to the distance measuring sensors 1000 rpm (vision, VS-5500N) in the separated at room temperature in 10 minutes after incubation the supernatant, and corresponding advertisement based on the shown measuring absorbance 532 nm, TBARS × 0.0154 numerical absorbance value is generated for reaction mixture 1 ml to a layer containing a filler in µm of 1, 1, 3, 3-tetraethoxy propane (TEP).

In the embodiment 8. Convergence for measuring effectiveness of

Astringent according to the assay method it was determined that such as Lee. Similar protein and skin using blood protein (hemoglobin), each container tube centrifugal separator and hemoglobin solution sample solution when blended with an succussion occurs in incorporated into 1:1 1,500rpm 3 in it was determined that absorbance in 576 nm after isolation of the centrifugal minutes. Addition of solution a sample assay Astringent army and showed acid in higher reduced absorbance nothing addition army.

In the embodiment 9. Whitening for measuring effectiveness of

(1) Tyrosinase inhibitory activity measuring

Tyrosinase inhibitory activity measurements it was determined that such as Yagi method according to. A substrate layer covering the both fasteners reaction 0.175M sodium phosphate buffer (pH 6.8) 0.5 ml 10 mm L-DOPA to 0.2 ml liquid and sample solution, a mixed solution prepared by the addition of 0.1 ml in 25 °C mushroom tyrosinase (110U/mL) 0.2 ml to 2 minutes produced in reaction measured at 475 nm for DOPA chrome. Tyrosinase inhibitory activity addition of solution a sample showed acid in higher reduced absorbance nothing addition old friendship port.

(2) cell line extract pine nut external kind blood whitening efficacy assay

1) cell survival rate measurements

(Is) cell culture

The present experiments using 1% penicillin/streptomycin (100U/mL) and 10% fetal bovine serum (FBS) of each culture of the 's modified eagle' dulbeco is added is s medium (DMEM) into its original media for, 37 °C, 5% CO₂ incubator to passaging adapts the good.

(Or) MTT assay-induced cell survivability measurements

Cell survival rate measurements it was determined that such as Carmichael method according to. Each cell line 96 well plate to a [melanoma (B16F10), fibroblast (CCD-986sk), macrophage (Raw 264.7) cell] 0.6-8×10³ cells/well 0.18 ml and the provide a way to make the connected, and can be prepared according concentration sample after added 0.02 ml 37 °C, 5% CO₂ incubator 24 in good time. The control group the sample and by adding distilled water of equal and manufacturing grey yarn having under same good. Herein prepared with 5 mg/mL concentration by adding 0.02 ml MTT solution grown on time 4 after each removing culture DMSO per well: 30 minutes at room temperature by the application of ethanol (1:1) 0.15 ml reaction then to it was determined that absorbance 550 nm in ELISA reader.

2) Cellular tyrosinase measuring inhibitory activity

Crammed Melanoma (B16F10) after each cells grown on culture dish, by etching are removed and then medium was then being washed with PBS. Lysis buffer to each dish (67 mm sodium phosphate buffer, 1% triton X-100,0.1 mm phenylmethyl sulfonyfluoride) a 100 μ L added black water slurry to ice after the impurities fixed to the of cells, ultra sonication the fibers and feeds them to a cells. 13,200rpm 1 same time after the composition is left for 20 minutes in an upper layer is gained from has solution enzyme solution. Same senses a rotation velocity of the disk 67 mm phosphate buffer (pH6.8) to 8.0 mm of 40uL 96-well plate L-DOPA 120uL and a sample solution after placed in a, 67 mm phosphate buffer (pH 6.8) to 37 °C 40uL senses a rotation velocity of the disk by the addition of enzyme solution in 30 minutes after grown on, generated amount of measured at 490 nm DOPA chrome.

Rate measurements in 3) Melanoma cell (B16F10) melanin biosynthesis,

Skin melanoma from cells in preference to Hosoi measurements such as melanin biosynthesis, it was determined that according to method. DMEM 100 mm culture dish to cultured B16F10 cells improves 1×10⁶ cell/dish culture nor post-incubation time can be brought and the 24 and can be prepared into sample concentration by adding 2 ml, 48 breathable process has been completed the washing (pH 7.4) buffer. Then 0.25M trypsin-EDTA solution of cells in a high sensitivity without using glutaraldehyde cells harvesting 1×10⁶ of 1 ml per cells are hydrophobicized by treatment with 5% TCA, 2,500rpm in signal separator separates the centrifugal times 2 is responsible for phosphorylation melanin ether then washing buffer: 2 by the application of ethanol (1:3) 1 ml ether 1 ml after the separation of the centrifugal times the creaning-drying to. Dried melanin 1N NaOH to 1 ml 80 °C applied in a 1 405 nm absorbance spectrophotometer reacting a metal salt of time it was determined that in. Melanin biosynthesis, addition of a sample solution showed acid in higher reduced absorbance nothing addition old friendship port.

Intracellular through 4) Western blot of proteins involved pigmentation determining expression of an

MITF, measuring be shaped various type tyrosinase expression of proteins in performed for all the following method. I.e., B16F10 behind grown on culture dish using sample handling 48 hours then PBS washed with centrifugation collecting and of cells, cell lysate using RIPA buffer have been prepared. Cells the amount of proteins contained in lysate factor have been measured by Bradford method. For 15 μ g protein is separated into 8% polyacrylamide containing PVDF membrane SDS-PAGE electroblot 120 V in 1 time in the embodiment. Ear PVDF membrane in 5% skim milk for 1 then 4 °C in primary antibody overnight in size.. Behind washing the three times TBST again harmless and environmentally favorable and has excellent secondary antibody TBST then the time response 1 which washes the three times by reacting X-ray film is subjected to an ECL kit (Amersham-Pharmacia). LAS 4000 X-ray film from an band was and quantifies using Image analysis device.

In the embodiment 10. Said method

(1) Elastase measuring inhibitory activity

Elastase inhibitory activity measurements it was determined that such as Cannell method according to. As substrate N-succinyl-(L-Ala)₃-p-nitroanilide using resulting from substrate 20 minutes in 37 °C p- nitroanilide wherein the formation of 405 nm measured at. I.e., concentration so that each test solution and can be prepared and taken vitro at 0.5 ml, 50 mm tris-HCl buffer (pH 8.6) senses a rotation velocity of the disk to porcine pancreas elastase (2.5U/mL) solution 0.5 ml 50 mm tris-HCl buffer (pH 8.6) senses a rotation velocity of the disk to after loading the substrates N-succinyl-(L-Ala)₃-p-nitroanilide (0.5 mg/mL) reacting by the addition of it was determined that 20 minutes. Addition of solution a sample inhibitory activity Elastase raw viscosity incorporatign primary partial coupling nothing addition old friendship absorbance reduction showed to.

(2) Collagenase measuring inhibitory activity

[...] Collagenase inhibitory activity measurements it was determined that such as sch method according to. 0.1M tris-HCl buffer (pH 7.5) to fasteners reaction i.e. 4 mm CaCl₂ by adding, 4-phenylazobenzyloxy carbonyl-Pro-Leu-Gly-Pro-D-Arg (0.3 mg/mL) a substrate layer covering the both 0.25 ml liquid and sample solution, a mixed solution prepared to 0.1 ml collagenase (mL/0.2 mg) at room temperature by adding 0.15 ml 20 minutes after the composition is left for extended storage and is easily carried on the reaction 6% citric acid 0.5 ml, by the addition of ethyl acetate 1.5 ml in 320 nm it was determined that absorbance. Addition of solution a sample inhibitory activity Collagenase raw viscosity incorporatign primary partial coupling nothing addition old friendship absorbance reduction showed to.

(3) cell line extract pine nut external kind blood wrinkle efficacy assay

1) cell survival rate measurements

Is) cell culture

The present experiments using 1% penicillin/streptomycin (100U/ml) and 10% fetal bovine serum (FBS) of each culture of the 's modified eagle' dulbeco is added is s medium (DMEM) into its original media for, 37 °C, 5% CO₂ incubator to passaging adapts the good.

Or) MTT assay-induced cell survivability measurements

Cell survival rate measurements it was determined that such as Carmichael method according to. Each cell line 96 well plate to a [melanoma (B16F10), fibroblast (CCD-986sk), macrophage (Raw 264.7) cell] 0.6-8×10³ cells/well 0.18 ml and the provide a way to make the connected, and can be prepared according concentration sample after added 0.02 ml 37 °C, 5% CO₂ incubator 24 in good time. The control group the sample and by adding distilled water of equal and manufacturing grey yarn having under same good. Herein prepared with 5 mg/mL concentration by adding 0.02 ml MTT solution grown on time 4 after each removing culture DMSO per well: 30 minutes at room temperature by the application of ethanol (1:1) 0.15 ml reaction then to it was determined that absorbance 550 nm in ELISA reader.

2) regeneration effect skin extract pine nut external kind blood

Is) measuring said method using ELISA kit

Measuring kit biosynthesis i) Procollagen type I

Cells 5x10⁴ cells/well improve efficiency of vaccination 12 well plate after concentration, the sample is added to each well CO₂ incubator in good time 48. Thus demonstrated that despite is collected and culture of cells, the third to eo experiments. Cell culture degree of biosynthesis marble and the method using amount of procollagen type-IC peptide (PIP) EIA kit it was determined that propeptide.

Measuring kit active & TIMP-1 ii) MMP-1

Cells 5x10⁴ cells/well improve efficiency of vaccination 12 well plate after concentration, the sample is added to each well CO₂ incubator in good time 48. The MMP-1 and a TIMP-1 10ng/mL a TNF-α to glucose level by increasing activity of added at a concentration of. Culture of cells measurements of the Amersham matrix metallproteinase-1 MMP-1 the discharge connector, Human, was by using Biotrak ELISA system (GE Healthcare).

Or) through Western blot of proteins involved wrinkle determining expression of an intracellular

MMP-1 using Western blot, MMP-2, it was determined that amount of protein ProCOL1A2. CCD-986sk behind grown on culture dish using sample handling 48 hours then PBS washed with centrifugation collecting and of cells, cell lysate using RIPA buffer have been prepared. Cells the amount of proteins contained in lysate factor have been measured by Bradford method. Protein 20 μ g of 10% polyacrylamide containing PVDF membrane is separated into SDS-PAGE electroblot 120 V in 1 time in the embodiment. Ear PVDF membrane in 5% skim milk for 1 then 4 °C in primary antibody overnight in size.. Behind washing the three times TBST again harmless and environmentally favorable and has excellent secondary antibody TBST then the time response 1 which washes the three times ECL kit (Amersham-Pharmacia) X-ray film by reacting the exposed to. LAS4000 X-ray film from an band was and quantifies using Image analysis device.

In the embodiment 11. Waste [...] (PinuskoraiensisZucc Sieboldet)testa(testa) phenolfor producing from

In a region which does fat Gapyeong tools a cultivated in a harvested from Korean white pine, pine nut cone[...]bell room not utilized peeled off and to harvest the seed forest products waste pine nut external kind blood the effluent is extracted as the extractive solvent (testa). Sample efficiently extracts than an effective ingredient of alternatives in order to establish a metal harmful to human body is not water as solvent into its original selected and extracted the with ethanol, ethanol concentration by elution of the physiologically active components in phenolic compound it was determined that content on the main target. Result to the computer of the also showed to 1.

50% ethanol as solvent in Figure 1 such as a phenolic compound relationship with the derma when applied in patternability, and that is highest factions, a dilution rate in inflow of 40-50%, which have been found increased concentration of extraction solvent increases lower yield is the release of an phenolic compound sequences as signal.

Method for inducing optimum conditions corresponding advertisement based on the shown list identifying amount in the release of an phenolic compound, showed to result to the computer of the to table 1. Table 1 cd1a. mL/4.92±0.08 mg extract water such as, in content of 50% ethanol extract showed 5.89±0.06 mg/mL.

In the embodiment 12.Antioxidant activity of extracts ofpine nut external kind blood

(1) eraseDPPH extract pine nut external kind blood measuring ability

Oxidative free radical pine nut external kind blood water and ethanol extract of reducible substance using disproportionation reaction DPPH analysis as measured by corresponding advertisement based on the shown list, showed to result to the computer of the also 2.

Also as demonstrated 2 phenolic compound can not be discharged to the maximum in the AC electrolysis precursor and CaO precursor ability erase DPPH 50% ethanol extract, the obtained extract with extract and ethanol is excellent the effect of.

A longitude implying Phenolic change potency erase according to DPPH concentration, showed to result to the computer of the also 3.

Also added as demonstrated 3 content of phenolic it is unavoidable that there Te independent DPPH it is found that the increasing whereby, the dipped erase has been, 150 μg/mL 87.25% extract water at concentrations degree of, high 97.10% ethanol extract pine nut external kind blood that it represents a deterrent to a water soluble extract antioxidant it has been discovered surprisingly good force. As antioxidant BHT fiber number is extract ethanol in addition synthetic job form a gate oxide as compared to a cosmetic anti aging that it represents a deterrent includes a storage location of a data error with respect to the been determining re.

(2) extract pine nut external kind blood measuring decolorizationABTS radical cation

Extract relative antioxidant measurements chain breacking antioxidant and a hydrogen-donating antioxidant can measure both, applied to both organic phase and aqueous phase capable of is measured with ABTS radical cation decolorization. Use of standard material the system relatively compares extract to generated by reaction with potassium persulfate ABTS⁺ free radical antioxidant in the extract radical is removed by reflowing the solder and index of unique use to a concave depigmenting is resolution radar Image.

Experiments showed to 4 having a.

Also as demonstrated 4, been representing deterrent of 29.93% in the case of hot-water extract, 42.15% in 50% ethanol extract, the ethanol extract been found disclosed deterrent in.

Added antioxidant force longitude implying according to content of phenolic compound, result to the computer of the also showed to 5.

In Figure 5 such as a Te increased concentration of phenolics added independent ABTS it is confirmed that the compressed inhibitors has been, 50-200 μg/mL phenolics concentration of compressor is compared is high in comparison with the thermal antioxidant BHT job been force, low ion conductor 50 μg/mL phenolics 92.13% extract water, ethanol extract showed surprisingly good 90.04% of antioxidant force. Thus pine nut external kind blood extract, the water-soluble substance to low ion conductor as well as excellent antioxidant additive increased concentration of Te dependent antioxidant sequences as signal strength.

(3) measuring antioxidant protection factor (PF) extract pine nut external kind blood

Linoleic acid emulsion for β-carotene added to to lipid-soluble substance of extract pine nut external kind blood measuring antioxidant also results showed to 6.

1.1 PF in Figure 6 and a water extract indicative of the degree as compared to ethanol extract 1.1-1.2 PF between bulb by concentration degree of antioxidant BHT example, abnormal prion protein is extracted force antioxidant, which is similar to a, it has been determined that force.

Added antioxidant force longitude implying according to content of phenolic compound, result to the computer of the also showed to 7.

Added in Figure 7 such as a independent Te increased concentration of phenolics tubes is PF was capable of confirming the. 50-200 μg/mL phenolics of alcohol extract and water in each extract 1.15-1.76 PF 0.88-1.46 PF of BHT job contrast to 1.09-1.52 PF and a relatively high compared to PF for lipid-soluble substance may exhibit high antioxidant, it was discovered.

(4) measuring TBARS pine nut external kind blood extract

Lipid-soluble substance to determining the antioxidant extract pine nut external kind blood to corresponding advertisement based on the shown list compared to data of the measured TBARS, result to the computer of the also showed to 8.

In Figure 8 and a water extract and each 41.59% and 50% ethanol extract 66.94% of deterrent has been confirmed.

Confirming force antioxidant content of phenolic compound added according to corresponding advertisement based on the shown list, result to the computer of the also showed to 9.

In Figure 9 such as a Te increased concentration of phenolics added independent TBARS inhibition and increases the, 50 μg/mL phenolics low ion conductor showed force high antioxidant or more 90%. Comparison with a BHT job contrast the obtained extract with and ethanol extract both job compared section before 50-200 μg/mL phenolics concentration of antioxidant more robust than BHT showed force.

In the embodiment 13.pine nut external kind blood extractconverging effect

Flavonoids principles of an effect of the clip base and the cover skin protein polymer article incorporating the skin and shrinks in the negative. Email widow, a web page or convergence has and wrinkling to a basic meaning or shrink the senses that it is implemented, the astringent agent may be material, such as mucosal skin and the tip, and serves to to shrink, cell gap and lymph secretion of mucus in the lungs intercepts gap. which inhibit a. In addition the astringent agent may be bound with protein CR6 and the properties of hemoglobin generally portion of the sacrificial layer is patterned with extract protein depending on the degree-convergent effect can be determine from the degree. Such an effect the external surface of a mucous membrane of a skin by content or hardly soluble in for forming films of or protecting a results, or tissue of the densely. reducing permeability.

50-200 μg/mL phenolics concentration of extract pine nut external kind blood the convergence showed to 10 having a measured by.

In Figure 10 is in the obtained extract with pine nut external kind blood such as a convergence but effect of less than 20%, in extract ethanol at a concentration of 200 μg/mL phenolics of tannic acid job contrast increase slant detector detects the increase slant one which is similar to 62.4% 66.3% converging provided are pore-shrinking for cosmetic been determining re a data error with respect to the material.

In the embodiment 14.Result for measuring effectiveness of of whitening extractpine nut external kind blood

Tyrosinase derived from (1) Mushroom confirming inhibitory activity

A collector hue of the skin the melanin the receptor of the endothelial of the base layer a pigment cell in melanocyte called is biosynthesis in melanosome. Melanin in synthesizing starting amino acid is a tyrosine. Melanin Tyrosine L-3 by tyrosinase in a cell, 4-dihydroxyl phenylalanine (DOPA), DOPA quinone is oxidized. DOPA chrome is DOPAquinone then, 5,6-dihydroxyindole, is indole-5,6-quinone, then by polymerization to indole-5,6quinone melanin constitution: by an known. Melanin formation the skin in addition tyrosinase constitution: which play a significant role, out to oxidize the melanosome in tyrosine hydroxylase for making tyrosine to DOPA, DOPA oxidase for making DOPA quinone by oxidizing DOPA acting as an are important in the synthesis of melanin polymerization functions, enzyme.

Human skin melanin (melanin) or carotene (carotene) such as distribution of and vessels, wherein the pigment, of the terminal is determined, such as by thickness of stratum corneum, this most important determining a are skin melanin with light to the topically having the function, movable in X and Y or synthesized, melasma melanin formation of abnormalities in the surface, causing hyper-pigmentation freckle and is especially. Thus a substrate and melanin synthesis of tyrosine for decomposing an aromatic and for inhibiting tyrosinase, inhibit synthesis of melanin as for lightening skin coating may be replaced with bearings for the.. Antioxidant activity and in an intimate relationship to a vitamin C tyrosinase inhibition effect with regard to recent, kojic acid, arbutin, such as hydroquinone natural and synthetic whitening agent application in a skin-lightening cosmetic the studied development of wet liquid to flow down.

The present and in research 50, 100, 150, 200 μg/mL concentration of extract pine nut external kind blood the tyrosinase inhibition test showed to 11 also result to the computer of the corresponding advertisement based on the shown.

In the case of the obtained extract with pine nut external kind blood such as a in Figure 11, a relatively high effect of inhibiting 26.4-49.9%, the obtained extract with even in a lower portion of the 50% ethanol extract of a comparatively high-tyrosinase inhibitors 21.5-47.5% showed efficacy. The obtained extract with and ethanol added both extract phenolic compounds increased concentration of a massage cream, an essence, tend to effect is intensified whitening increases, rolled, both rollers are rolled in low concentration 100 μg/mL in cosmetic of whitening which captures an Image of the job is slightly larger than the kojic acid levels of whitening been excellent, in at a high concentration of 150 μg/mL or more similar and a kojic acid to show inhibitory effect on the levels of tyrosinase, a generally excellent whitening effect was judges that the.

(2) Melanoma cell (B16F10) confirming survival rates of

The retrieval MTT tests employing 96 well plate using ELISA reader result, and for many sample can be read out simple cell toxicity and cell proliferation is used widely in retrieval, dehydrogenation of mitochondrial metabolism processes of cells in enzymatic action yellow soluble MTT tetrazolium MTT formazan in a water-insoluble wavelength of 405-430 nanometer purple for reducing the is method.

Prolonged periods of ischemia by pine nut external kind blood melanoma B16F10 cells by MTT assay rate at a concentration of 1, 5, 10, 25 μg/mL corresponding advertisement based on the shown list processing, result to the computer of the also showed to 12.

As seen from in Figure 12, water and alcohol is pine nut external kind blood mL/25 μg extract each concentration of precursor and CaO precursor rate cytotoxicity of 37% and 46% 1, 5, 10 μg/mL 90% close cell survival in it has been confirmed that exhibits.

Tyrosinase (3) Melanoma cell (B16F10) in confirming inhibitory activity

it is sour with the mote but Oh sacrifice in tyrosine upon exposure to light skin melanin formation that are most significant to acting enzyme tyrosinase activity as by inhibition of melanin at night, a performed for a freckles, freckles, a second insulating layer is formed on incidence of such as erythema age are known as HMG-COa reductase.

The Tyrosinase cu²⁺ a one of the clip base and the copper enzyme? Plant, microorganism and human distributed by winding the polyphenol oxidase to melanin synthetic process such as to limit the rate at which in melanin synthesis main control is enzyme representing the steps.

pine nut external kind blood extract is added to this concentration of 1, 5, 10 μg/mL L-DOPA produced wherein the formation of measured by having a showed to 13. Also as demonstrated 13, also 12 and a similarly pine nut external kind blood the obtained extract with acid and an alcohol extract mL/10 μg each at concentrations showed rate inhibitors of 11% and 12%.

In confirming (4) Melanoma cell (B16F10) melanin biosynthesis,

Human skin colour the most important determining which causes a public melanin parakeratosis sunlight or photoaging of the skin are inhibited as well as, aft, freckles, with pigmentation symptoms partial such as age spots cause serves to (hyperpigmentation). The receptor of the endothelial melanin peripheral portion of the through a for the decolouration of resinous melanocyte base layer are transferred in keratinocyte, . of the fluctuation width of the Q stratum corneum of skin. Skin black present in the skin a melanocyte is UV exposure lamp external moisture is high, a resistance value is formed generation of melanin. Won material and material, capable of to confirm the Tyrosinase enzyme activity of melanin forming enzyme a main search is inhibitors of color cathode is recent enzyme inhibition activity of tyrosinase in melanocyte raw material whitening with associated enzyme expression are provided in an environment the electromotive disturb the delivery system signal for study carried out at a and produce.

B16F 10 in a cell to melanin content is 1, 5, 10 μg/mL pine nut external kind blood the obtained extract with acid and an alcohol extract concentration of treated result showed to 14 also.

Concentration processing extract such as a in Figure 14 because of the decrease in melanin biosynthesis the control by from being impeded as much as possible. In addition 1,5 μg/mL of low concentration of cleaning inhibitors but not high force inhibiting biosynthesis of melanin that is found aspects of the purpose: to provide whitening effect if height a purity of extract using functional cosmetics material industrializing was it judges that may be.

(5) extract pine nut external kind blood melanin effect small interfering RNA capable of inhibiting expression tyrosinase and which are involved in the synthesis

Extract pine nut external kind blood pro-collagen wrinkle that it represents a effects excellent in proteins involved and mRNA expression amount has been confirmed. Tyrosinase enzyme relates to synthetic Melanin be achieved, wherein an influence on B16F10 mouse melanoma cells to see the concentration by 1-10 μg/mL after treating the concentration of 48 time behind the western blotting tyrosinase protein expression is confirmed. The various conditions of cells although expression other things, an difference β-actin in house keeping gene for positive control at night.

Result to the computer of the also showed to 15.

Also 15 as demonstrated, B16F10 untreated extract each group but is increased protein expression of transcription factor, in group a B16F10 pine nut external kind blood extract and untreated, where the expression of the tyrosinase protein concentration dependent fashion than 10 μg/mL decreases in concentration of 30% or more water and an alcohol extract showed efficacy inhibiting expression of lectin-like. Thus expression of mushroom tyrosinase pine nut external kind blood extract concentration dependent fashion for a difference from an the reduced extract pine nut external kind blood according to tyrosinase expression of melanin formation by low-resolution step effective in inhibiting could confirm it is.

Effect small interfering RNA capable of inhibiting expression (6) Melanin microphthalmia-associated transcription factor (MITF) and which are involved in the synthesis

Melanin biosynthesis signal material for transmitting signal handle a great variety of delivery system of wet liquid to flow down are involved. Melanin has some intracellular crf2r signal transduction pathways are synthesized through, one from the synthesis of melanin path is PKA (protein kinase A)/ cAMP (cyclic adenosine monophosphate) as primary path at melanin when exposed is skin UV cAMP of cells signaling downstream is increased and the first-in first-out PKA material, via CREB (cAMP response element binding protein) (microphthalmia-associatedtranscription factor) expression of MITF enhance to. Melanin synthetic process cells and important in the MITF modulators of transcription to. to facilitate transfer of tyrosinase.

Whitening effect of extracts of pine nut external kind blood order to check the expression of MITF associated with melanin synthesis may be achieved, wherein an influence to see the B16F10 mouse melanoma 1-10 μg/mL by concentration cells after treating the concentration of 48 time behind the western blotting MITF protein expression is confirmed. The various conditions of cells although expression other things, an difference β-actin in house keeping gene for positive control at night. Result to the computer of the also showed to 16.

In Figure 16 such as a B16F10 untreated extract each group but is increased protein expression of transcription factor, in group a B16F10 pine nut external kind blood extract and untreated expression of MITF protein concentration dependent fashion than decreases 10 μg/mL inhibitors water extract concentration of about 40%, about 30% the to show inhibitory effect on alcohol extract, the obtained extract with acid and an alcohol extract both inhibiting expression of MITF a modulators of transcription, is efficacious in the has been confirmed. Thus expression of MITF pine nut external kind blood extract concentration dependent fashion for a difference from an the reduced extract pine nut external kind blood according to effective in inhibiting melanin formation could confirm it is. This extract pine nut external kind blood MITF and in research before verification whitening effect through small interfering RNA capable of inhibiting expression of tyrosinase protein expression sequences as signal by an interval not.

In the embodiment 15.pine nut external kind blood extractmeasurement results said method

(1) Elastase confirming inhibitory activity

Reduced resilient, and improve a pleated skin, pigmentation in the form a representative aging of and the like, at most representative aging of. having wrinkles directly the nozzle in one. Granulocyte opening neutral of a human body present in the elastase intradermal skin elasticity a substrate main maintaining the elastin protein is enzyme for decomposing an aromatic, elastase acts on the skin in tissue elastin is decomposed to thereby by the raw or resilience of thebellow skin as thus loses and, the folds and the roughened part is narrow promoting the aging of the skin. Thus of inhibiting the activity of elastase inhibiting skin aging by is even would be able.

The another important Elastase a substrate protein collagen the technique to resolve the non-specific hydrolase is, elastase inhibitor for treating a skin wrinkle using to improve the, presents a, current ursolic acid elastase inhibitors. used and the like. In addition Cannell for decomposing elastin body of, or the like, that has enzyme granules leukocytes that tissue or more one of the enzyme in the activity of the direct tissue destruction a extremely high is skin-wrinkling, and elasticity believed that 31. Thus elastase inhibitory activity by measuring wrinkle generated. A a substrate Elastase N-succinyl-(Ala) - 3-p-nitroanilide nitroanilide (NA) from decomposition using conventional pipe joint wherein a defect occurs while measuring the absorbance change in color of the elastase activity it checks. A are utilised various forms of elastin vivo or method using a substrate with a synthesized instead of measuring checks if the jump is.

A inhibitors Elastase ursolic acid and the like which is using elastase inhibitors, ursolic acid of the water or oil such as poorly soluble the solvent has physical properties durable structure having a latticed structure formulated generally employs to reduce the possibility of the pulse to.

Concentration of extract pine nut external kind blood 50-200 μg/mL phenolics of adjusted to corresponding advertisement based on the shown measuring the elastase inhibitory activity, showed to result to the computer of the also 17.

In Figure 17 such as a 50 μg/mL phenolis pine nut external kind blood extract at concentrations addition of 200 μg/mL to stores a concentration dependent fashion in elastase inhibitor it is unavoidable that there section confirms with corresponding advertisement based on the shown list, 28.47% at concentrations addition of 50% ethanol 200 μg/mL phenolics can be identifying inhibitors of force.

(2) Collagenase inhibitory activity

Extracellular matrix (extracellular matrix) collagen component matter of the skin fibroblast created at a main substrate proteins. About 30% of the total weight of protein vivo in addition that occupy the same tissue during 3 robust as constitution: a spiral structure. Collagen the skin, gun (tendon), bone and of the teeth, the substance which provide the greater part of the organic material, in particular bone and skin the dermis of gun content WIPO. Collagen at chief function of skin mechanical rigidity, includes a silane coupling agent easily coupled tissue drag and pressurization of connective tissue, bears of cell adhesion and are, such as differentiation for separating and composing cells. is known. Such collagen the age and irradiation with ultra-violet light the reduced by photoaging by, and to corrugating in skin is associated constitution: know. The trypsin in addition collagen such is divided by the barrier rib is subjected to the action degrading enzyme of the invention to the, water is decomposed by collagenase that report is.

50-200 μg/mL phenolics concentration of extract pine nut external kind blood the collagenase corresponding advertisement based on the shown measuring inhibitory activity, showed to result to the computer of the also 18.

In Figure 18 such as a in the obtained extract with pine nut external kind blood 3.04-7.39% of collagenase been identified inhibiting effect and water solubility leading to, ethanol extract in 3.53-51.69% of the inhibition effect is identified as alcohol, compared to the obtained extract with extract, which display inhibitory effects on collagenase more life. Skin extract pine nut external kind blood discarded these results of inhibiting the activity of collagenase in collagen by separator layer and prevents decomposition of the corrugations for improving functional cosmetics material was a decision is made that the data error with respect to the element.

(3) Fibroblast cell (CCD-986sk) confirming survival rates of

MTT measurement results 1) CCD986sk fibroblst cells

The retrieval MTT tests employing 96 well plate using ELISA reader result, and for many sample can be read out simple cell toxicity and cell proliferation is used widely in retrieval, dehydrogenation of mitochondrial metabolism processes of cells in enzymatic action yellow soluble MTT tetrazolium MTT formazan in a water-insoluble wavelength of 405-430 nanometer purple for reducing the is method.

pine nut external kind blood extract to measuring a rate cell survival CCD-986sk cells treated concentration of 1, 5, 10, 25 μg/mL also result showed to 19.

Also as demonstrated 19, the obtained extract with acid and an alcohol extract both 25% at concentrations of 25 μg/mL degree of cell toxicity but viability high concentration of 1, 5, 10 μg/mL it has been confirmed that exhibits.

Procollagen type I (4) ELISA kit in fibroblast (CCD-986sk) kit biosynthesis using measurement results

Most skin dermis of collagen is present in the layer, the paper side weft yarns to occupy about total dry weight skin 70-80% is output from the speaker as sound, while extracellular matrix the stroke of the make connected to the inner skin. However natural aging according to inner product, such as a reduction in cell activation due to reduced biosynthesis of collagen, various adverse ambient by stress increase and sun light with an increase in oxygen species such as deactivation by high external factor decomposed by accelerated the skin having wrinkles generated while substrate is destroyed. Collagen in addition an important role in wound healing is responsible for initial, promote synthesis of collagen in epithelial damage while wound to quickly. recovery without scar. Collagen synthesis promoting material been disclosed to date during most representative notably a major components of centella asiatica asiaticoside, asiatic acid, as to the aromatic hydrocarbon madecasic acid.

CCD-986sk pine nut external kind blood in a cell extract water and alcohol is in concentration pro-collagen synthesis of 1, 5, 10 μg/mL having a measured by showed to 20.

1-10 μg/mL in Figure 20 such as a concentration of extract independent the concentration precursor and CaO precursor biosynthesis of pro-collagen, in particular 10 μg/mL biosynthesis of each concentration of 62% and 50% concentration same example, abnormal prion protein is extracted characteristic ratio epigallocatechin-3-Ogallate (EGCG) and below a temperature at which material compared with a relatively good pro-collagen biosynthesis but exhibiting was capable of confirming the.

Fibroblast (CCD-986sk) procollagenase protein (5) Western blotting analysis using small interfering RNA capable of inhibiting expression in effect

In the biosynthesis of a collagen on skin wrinkles and reduced, adverse ambient in any number of ways such as by external factor is collagen decomposition of accelerated the skin while generated substrate is destroyed. In the biosynthesis of collagen in addition an important role in wound healing is responsible for initial, promote synthesis of collagen in epithelial damage of a wound to quickly restores..

Thus in the biosynthesis of collagen extract pine nut external kind blood enzyme relates to be achieved, wherein an influence on protein expression of the enzyme of procollagenase to see the western blotting CCD-986sk procollagenase protein expression in a cell is confirmed. The various conditions of cells although expression other things, an difference in house keeping gene GAPDH a positive control at night. Result to the computer of the also showed to 21.

Also as demonstrated 21, procollagenase UV-B unit in the vacuum control group materials in the normal expression of a significantly reduced it was observed that. A population of cells CCD-986sk pine nut external kind blood extract and each transcription factor of protein expression in which the amount of an added concentration dependent fashion extract corresponding advertisement based on the shown list increases, the obtained extract with in effect not only has hardly of a concentration processing mL/10 μg of extracts of alcohol but substantially in a degree expression close to normal group sequences as signal recovered. Thus pine nut external kind blood alcohol extract the damaged procollagenase collagen at to activate to thereby promote synthesis of one selected from the group consisting the corrugated improved, promote recovery in epithelial damage the for performing security on excellent effect can be provided to.

(6) fibroblast (CCD-986sk) (MMP-1) proteins involved of pleats confirming expression amount mRNA and

The component comprising the non-connective tissue skin cells during the skin collagen 90% of a dry weight of three categories, proteins that occupy the same degree. Thus the degradation of collagen connective tissue elasticity and direct such as wrinkle formation. affecting the. Associated with aging in addition the MAPK to affected the most pt clients belong, using a covering materials are c-fos factors affected of p38. Such factors capable of meeting ardent needs expression of MMPs is energized of an. Among MMPs of edema produced in the body the MMP-1 protease specifically acting on collagen MMP-1 as collagen with the display status of the activity for reducing the degradation of heat that serve to maintain the of elasticity of tissue the skin which can prevent wrinkle formation it is also known.

Determining the activity of MMP-1 CCD-986sk in a cell also results showed to 22.

pine nut external kind blood such as water and alcohol is also 22 to both extract low-resolution concentration dependent fashion that exhibits was capable of confirming the. The obtained extract with in addition relatively MMP-1 extract alcohol compared to expression of inhibiting effect and water solubility leading to better effects to determining presence has been confirmed.

(7) Western blotting analysis using small interfering RNA capable of inhibiting expression of protein in fibroblast (CCD-986sk) intestitial collagenase (MMP-1) 92kD gelatinase (MMP-9) and effect

Skin intradermal cell extracellular matrix protein (extracellular matrix) reconstruction of which the biological reaction is provided, these matrix protein are skin structure of plays the role are important in maintaining the and resiliences make. Double in particular MMPs is matrix protein a to dissolve intestitial collagenase (MMP-1) which play a significant role, 72kD gelatinase (MMP-2), 92kD gelatinase (MMP-9), neutrophil collagenase (MMP-8), such as one or more of a collagenase (MMP-13) 26 are already known MMPs. Excess expression of MMPs in skin exposed to ultra-violet light are increased ;, increased skin MMPs a collagen to decompose to matrix protein deficiency such as order for a bit rate to become generation of wrinkles with skin aging by is.

Skin connective tissue during that occupy the same 90% collagen MMP-1 as protease specifically acting on collagen with the display status of the activity for reducing the degradation of heat that serve to maintain the of elasticity of tissue the skin can prevent wrinkle formation. During enzyme in addition MMPs 72kD gelatinase (MMP-2) MMP-9 in 92kD gelatinase on such as cell-to-cell layer dermis also present at a material causing decomposition of gelatin gelatinase activity of enzymes inducing also involved in generating of the pleats is a major factor. Therefore MMP-1 MMP-9 and with the display status of the activity of collagen for reducing the degradation of heat that serve to maintain the of elasticity of tissue the skin which can prevent wrinkle formation it is also known. MMP-9 and MMP-1 extract pine nut external kind blood thus be achieved, wherein an influence on protein expression in a cell to see the western blotting CCD-986sk MMP-1 and MMP-9 protein expression is confirmed. The various conditions of cells although expression other things, an difference β-actin in house keeping gene for positive control at night. Result to the computer of the also showed to 23.

As present in Figure 23 UV-B MMP-1 unit in the vacuum control group in the expression of normal materials the markedly increased showed. A population of cells CCD-986sk pine nut external kind blood extract and each transcription factor of protein expression in which the amount of an added concentration dependent fashion extract corresponding advertisement based on the shown list reduced, compared to the obtained extract with 10-40pts.wt.of further the efficacy of the extract alcohol, alcohol extract concentration of 10 μg/mL rolled, both rollers are rolled in functional cosmetics be used in inhibition of than EGCG is better can be of confirming the. Thus waste pine nut external kind blood MMP-1 extract protein inhibiting the expression of collagenase inhibition of flow as a result improved of the pleats by introducing one selected from the group consisting, promote recovery in epithelial damage the is for performing security on could confirm it.

MMP-9 control group out also in the case of the normal expression of protein materials the markedly increased showed.

Furthermore, a population of cells CCD-986sk pine nut external kind blood extract and 24 is as demonstrated, each transcription factor of protein expression in which the amount of an added extract corresponding advertisement based on the shown list reduced concentration dependent fashion, water extract concentration of 10 μg/mL rolled, both rollers are rolled in functional cosmetics in EGCG be used and a been represents inhibitors, alcohol extract, the EGCG job contrast a more superior deterrent indicative was capable of confirming the. Thus inhibiting the expression of protein MMP-9 extract pine nut external kind blood flow as a result of improved effect can be confirm that the user.

(8) TIMP-1 active kit measurement results

MMP activation such as α 2-macroglobulin RECK and number as well as non-specific inhibitors specifically inhibiting by tissue inhibitors of metallo-proteinase can be. 1:1 in 1:2:3 a chemical TIMPs specific binding to a specific inhibitors is number. The TIMP-1 MMP-1 in tissue of the. associated inhibitors. Determining the activity of TIMP-1 CCD-986sk be unit in the vacuum UV-B cells mL/1, 5, 10 μg extract concentration of 48 after the processing by good time. Culture of cells takes an TIMP-1 into its original determining the activity of, showed to result to the computer of the also 25.

In Figure 25 such as a 10 μg/mL 74% in the case of the obtained extract with pine nut external kind blood at concentrations for treating, alcohol extract, the 75% of a comparatively high expression rate has been confirmed. The results indicate that by extract pine nut external kind blood TIMP-1 the activity of the normal by slow to similar group MMP-1 is suppressed the activity of the collagen for reducing the degradation of heat that serve to maintain the of elasticity of tissue the skin wrinkle formation may prevent been determined.

Formulation example 1.Cosmetic preparationfor manufacturing

Belonging to dicksoniaceae manufacturing

In the embodiment 2 of pine nut external kind blood extract 0.1 weight %, 5.2 weight % 1,3-butylene glycol, 1.5 weight % one alcohol oleate, ethanol 3.2 weight %, as polysorbate 20 3.2 weight %, benzophenone -9 2.0% weight, carboxylic luck cost Neel Polymer 1.0 weight %, 3.5 weight % glycerin, aroma of trace, trace preservative and residual recombinant collagen for regular method belonging to dicksoniaceae in have been prepared.

Milk lotion manufacturing

In the embodiment 2 of 0.1 weight % pine nut external kind blood extract, glycerin 5.1 weight %, 4.2 weight % propylene glycol, tocopheryl acetate 3.0 weight %, flow paraffin 4.6 weight %, triethanolamin 1.0 weight %, 3.1% weight [...], macadamia nut oil 2.5 weight %, as polysorbate 60 1.6 weight %, 1.6% weight [...], 0.6 weight % methylparaben profile, carboxylic luck cost Neel Polymer 1.5 weight %, aroma of trace, trace preservative, residual amounts of milk in method for regular recombinant collagen have been prepared lotion.

Nutritional cream manufacturing

In the embodiment 2 of 0.5 weight % pine nut external kind blood extract, glycerin 4.0 weight %, 3.5 weight % petrolatum, triethanolamin 2.1 weight %, % weight 5.3 paraffin flow, 3.0 weight % [...], lycopene 2.6 weight %, tocopheryl acetate 5.4 weight %, as polysorbate 60 3.2 weight %, carboxylic luck cost Neel Polymer 1.0 weight %, 3.1% weight [...], aroma of trace, trace preservative and residual recombinant collagen for regular method in cosmetic nutrient cream have been prepared.

Manufacturing massage cream

In the embodiment 2 of 0.5 weight % pine nut external kind blood extract, glycerin 4.0 weight %, 3.5 weight % petrolatum, triethanolamin 0.5 weight %, flow paraffin 24.0 weight %, 3.0 weight % [...], lycopene 2.1 weight %, tocopheryl acetate 0.1 weight %, as polysorbate 60 2.4% weight, carboxylic luck cost Neel Polymer 1.0 weight %, 2.3% weight [...], aroma of trace, trace preservative and residual recombinant collagen massage cream for regular method in manufacturing processes and the cost of production.

Formulations 2.e.g. The preparation for the food

For making health food containing

In the embodiment 2 of pine nut external kind blood extract 100 mg, vitamin mixtures dosage, vitamin A 70g acetate, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2g, vitamin C 10 mg, biotin 10g, a nicotinic amide 1.7 mg, folic acid 50g, Pantothenic acid calcium 0.5 mg, inorganic mixture dosage, sulfate 1.75 mg iron number 1,0.82 mg zinc oxide, 25.3 mg magnesium carbonate, potassium phosphate 15 mg number 1, number 2 55 mg calcium phosphate, citric acid potassium 90 mg, 24.8 mg chloride and magnesium chloride 100 mg calcium carbonate then mixing, than that of conventional high-for making the granules according to method, the food product contains all have been prepared. The, relatively ratio composition of mixtures vitamins and minerals said health food components--time but hearts mixing to exemplify a preferred embodiment, modified optionally ratio blends. a non-intrusive may embodiment.

Healthy drink manufacturing

Conventional health drink manufacturing method in the embodiment 2 according to extract pine nut external kind blood of 100 mg, vitamin C 15g, vitamin E 100g (powder), lactic acid iron 19.75g, zinc oxide 3.5g, a nicotinic amide 3.5g, vitamin A 0.2g, vitamin B1 0.25g, vitamin B2 0.3g and quantitative of then water, stirring 85 °C about 1 time should be heated to reach a solution made in the sterilized to filter out in hermetically sealing obtained container 2L rehrigeration custody then sterilization of the health drink have been prepared. The, symbol relatively composition, said beverage components--time but hearts mixing to exemplify a preferred embodiment or layer demand, demand country, using local uses and the like, according to preference in people modified optionally ratio blends. a non-intrusive may embodiment.

Formulations e.g. 3. The preparation for the pharmaceutical

Masked powders that are to be manufacturing

In the embodiment 2 of 20 mg extract pine nut external kind blood, lactose -100 mg 10 mg [...] and mixing, followed by filling the gap and welded into an airtight foil been produced with at it buys sacrifice in conventional method.

Manufacturing purification

In the embodiment 2 of 10 mg extract pine nut external kind blood, maize starch 100 mg, lactose -100 mg 2 mg magnesium stearic acid and after mixing in conventional method for preparing the tablets was by granule with a sugar alcohol.

Capsule number manufacturing

In the embodiment 2 of 10 mg extract pine nut external kind blood, crystalline cellulose 3 mg, 0.2 mg magnesium stearate and 14.8 mg lactose than that of conventional high-mixing method in gelatin capsule is filled with the capsules have been prepared agent.

Manufacturing injection

1 per ampoule pine nut external kind blood extract of 10 mg (2 ml) in the embodiment 2,180 mg mannitol, 2,974 mg and distilled sterilization for injection Na₂ HPO₄? 2H₂ O 26 mg been produced with at injection in a conventional method.

Liquid manufacturing

Purification water of 20 mg extract pine nut external kind blood in the embodiment 2, isomerization per 5g is applied to 10g and mannitol by applying a vegetable lemon direction then dissolving the components is mixed. Then the entire applied further recombinant collagen is adjusted in the range of 100 ml brown bottle after filling, sterilizable the method of preparing the normally.