KR20070082635A - Antiaging composition for external skin application containing the needles of red pine - Google Patents

Abstract

An anti-aging composition for external skin application containing the extract of red pine needles is provided to remove free radicals, protect cell membrane damage due to UV(ultraviolet), inhibit production of reactive oxygen species due to UV, restore expression decrease of superoxide dismutase and catalase due to UV, promote production of HSP70(heat shock protein 70) and suppress MMP-2(matrix metal protease-2) biosynthesis. The anti-aging composition for external skin application contains 0.001-10.0 wt.% of the extract of red pine needles which is prepared by extracting freeze-dried red pine needles with 70% ethanol at room temperature for 48 hours, and evaporating ethanol from the extract by using an evaporator, and has a formulation selected from solution, gel, emulsion, suspension, microemulsion, microcapsule, microgranule, dispersion, cream, skin, lotion, powder, ointment, spray or stick.

Description

Anti-aging composition for external skin application containing the needles of red pine}

1 is a graph measuring the effect of the red pine leaf extract used in the present invention on lactic acid dehydratase (LDH) production after UV irradiation.

Figure 2 is a graph measuring the effect of red pine leaf extract used in the present invention on the production of reactive oxygen species of HaCaT keratinocytes.

Figure 3 is a photograph measuring the effects of SOD and catalase protein expression in HaCaT keratinocytes by Western blot of red pine leaf extract used in the present invention.

Figure 4 is a photograph measuring the effect of HSP70 synthesis of red pine leaf extract used in the present invention.

Figure 5 is a graph measuring the effect of MMP-2 synthesis of red pine leaf extract used in the present invention.

The present invention relates to an anti dermal solvent composition for skin aging containing red pine leaf extract as an active ingredient. More specifically, the skin external preparation composition for preventing skin aging according to the present invention contains red pine leaf extract as an active ingredient, which has excellent free radical scavenging activity effect, protection effect of cell membrane damage by UV irradiation, and inhibition of generation of active oxygen species by UV irradiation. Effect, restoring the reduction of superoxide dismutase (SOD) and catalase expression by UV irradiation, protecting the cells by promoting HSP70 production, inhibiting MMP-2 biosynthesis by UV irradiation Ultimately, it prevents aging of the skin.

Skin tissues have various antioxidant systems and protective factors, such as heat shock protein (HSP), to protect the skin, but damage to skin cells occurs due to decreased activity caused by endogenous aging or photoaging. It is known that the biosynthesis of matrix metalloproteinases (MMPs), an enzyme that degrades skin tissue, increases collagen biosynthesis, decreases wrinkles, reduces elasticity, and induces skin aging. Therefore, antioxidants that can protect skin cells, substances that inhibit MMPs biosynthesis that break down collagen, which is a substrate substance that makes up skin, and substances that prevent damage to skin cells, have been found to alleviate skin aging. .

Among the substances that alleviate the skin aging, research and development of functional herbal materials has been steadily made to prevent or solve the skin aging phenomenon using herbal ingredients. The biggest feature of herbal skin care is not only to identify the cause of skin aging and improve systemic symptoms by looking at the whole body, but also to fundamentally improve the cause of skin problems and to prevent the recurrence. Emphasis is placed on. Pine leaves, which are native to various Korean medicines and used in folk medicine, are known to have effects on liver disease, genitourinary system diseases, gastrointestinal diseases, purifying system diseases, and skin diseases. Aroma, antimicrobial, antioxidant and melanin activity inhibitory effects of the vertebrae have been reported (Jung et , al , Antioxidant principles from the needles of red pine, Pinus densiflora. Phytother Res., 17 (9): 1064-1068, 2003). Studies on the pharmacological effects of pine needles have been reported on the effects of pine needle supplementation on lipid metabolism, antimicrobial activity, antimutagenicity, antioxidant activity, and antihypertensive effect in normal rats (Kang et al. al ., Korean J. Food Sci. Technol., 27 (6): 978-984, 1995). There is no systematic research on the effects of skin aging.

Thus, the present inventors have found the effect on the red pine leaf extract that can prevent skin aging while maintaining the health of the skin through the study on the skin aging effect of pine needles also called red pine, and completed the present invention.

Accordingly, it is an object of the present invention to provide a skin external preparation composition which exhibits an excellent anti-aging effect by containing the red pine leaf extract in herbal medicine components as an active ingredient.

In order to achieve the above object, the skin external preparation composition for preventing skin aging of the present invention is characterized by containing a red pine leaf extract as an active ingredient.

Hereinafter, the present invention will be described in more detail.

Red pine (赤松) used in the present invention uses the leaves of pine and pine (pinus densiflora Siebold et Zuccarini) and is also called pine needles (松葉), bitter taste, but the nature is warm and not poisonous, since the heart (() and parenteral ( It has been used in the treatment of symptoms such as wind, wind, sensation, daefeng nachang, and inverse spear.

The red pine leaf extract used in the present invention is accurately weighed 50 g of lyophilized red pine leaf, extracted with 70% ethanol for 48 hours at room temperature, and then completely extracted from the ethanol used for extraction using an evaporator. Concentrate by evaporation and use as sample without further manipulation.

The external preparation for skin composition of the present invention contains the above red pine leaf extract in an amount of 0.001 to 10% by weight based on the total weight of the composition. This is because a pronounced effect cannot be expected at less than 0.001% by weight, and if it exceeds 10% by weight, no apparent effect increases with increasing content.

The external preparation composition for skin according to the present invention contains a cosmetically and dermatologically acceptable medium and / or base. These are all formulations suitable for topical application, for example emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase and / or Or in the form of a nonionic vesicle dispersant, or in the form of a cream, skin, lotion, powder, ointment, spray or cone stick. These compositions can be prepared according to conventional methods in the art. In addition, the topical skin composition according to the present invention may be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The external preparation composition for skin according to the present invention is a fatty substance, an organic solvent, a dissolving agent, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a blowing agent, a fragrance, a surfactant, water, an ionic or nonionic emulsifier, Cosmetics or skin such as fillers, metal-ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any other ingredients commonly used in cosmetics And auxiliaries commonly used in the scientific field. These adjuvants are introduced in amounts generally used in the cosmetic or dermatological fields.

Hereinafter, although an Example and a test example are given and this invention is demonstrated in detail, this invention is not limited only to these examples.

Reference Example Preparation of Red Pine Leaf Extract

Accurately weigh 50 g of the lyophilized red pine leaves, extract them with 70% ethanol for 48 hours at room temperature, and then completely evaporate the ethanol used for extraction using an evaporator to concentrate the red pine leaf extract. Prepared.

Test Example 1 Measurement of Free Radical Scavenging Activity

The method used to measure the antioxidant power was 1,1-diphenyl-2-pycryl-hydrazyl (DPPH) method and red pine leaf prepared in the reference example The extract was diluted in ethanol at different concentrations (0, 50, 100, 200, 300, 400 µg / mL), and 10 µl each was put into a 96-well plate, and the total volume of DPPH prepared with 5 mM ethanol solution was adjusted to 200 µl. After leaving at 37 ° C. for 30 minutes, absorbance was measured using a 520 nm ELISA reader (DI biotech, Korea) to determine free radical scavenging activity according to Equation 1 below. At this time, the vitamin C was used as a positive control, known to have excellent antioxidant power, and the results are shown in Table 1 below.

% Radical scavenging activity = [(absorbance of control-absorbance of sample) / absorbance of control] X 100

density Radical Scavenging Activity (%) of Red Pine Leaf Vitamin C radical scavenging activity (%) 0 µg / mL 0 0 50 μg / mL 10 30 100 µg / mL 35 90 200 µg / mL 60 91 300 µg / mL 79 90 400 μg / mL 80 90

From the results of Table 1, it was confirmed that the red pine leaf extract has a weaker efficacy than vitamin C, which is known as a powerful antioxidant, but has a radical scavenging activity at a concentration of about 100 µg / mL or more.

Test Example 2 Measurement of Cell Membrane Damage Protection

Dr. German Cancer Research Center, Heidelberg, Germany Human keratinocytes HaCaT cell line from Fysenig was added 100 μl of 1 × 10 ⁵ cell / mL cells into each well of a 96-well plate for 24 hours in a 37 ° C., 5% CO ₂ incubator. Were incubated and attached. After incubation, the red pine leaf extract of the reference example was replaced with a culture solution containing 0.625, 1.25, 2.5, and 5 μg / mL, and cultured for 3 hours, and then the culture solution was removed, and 50 μl of phosphate buffer solution was added to each well. . After irradiating UV 30mJ / cm ² using an ultraviolet B lamp, phosphate buffered saline was removed, and 200 µl of the cell culture solution containing the red pine leaf extract of the above concentration was added to each well and incubated for 24 hours. After 24 hours, an appropriate amount of the culture supernatant was taken, and the amount of lactate dehydrogenase (LDH), an indicator of cell damage, was measured using a cytotox 96 non-radioactive cytotoxicity assay kit. Indicated. At this time, CTL is a control group not treated with the red leaf extract.

From FIG. 1, in the group treated with the red pine leaf extract at different concentrations and irradiated with ultraviolet B, the increase in activity in the cell culture medium of LDH by ultraviolet B irradiation showed a concentration-dependent decrease. In particular, when treated with 5 ㎍ / mL red pine leaf extract was observed to inhibit LDH to the control level not irradiated with ultraviolet B. From these results, it can be judged that the red pine leaf extract effectively inhibits cell membrane damage of HaCaT cells by ultraviolet B irradiation.

Test Example 3 Determination of Reactive Oxygen Species Production

2.0 x 10 ⁵ in each well of a 96-well black plate for fluorescence measurements 100 μl of human keratinocytes HaCaT cells were injected into cells / mL, and cultured for 24 hours in a 37 ° C., 5% CO ₂ incubator, and then treated with the red pine leaf extract of the reference example. Concentrations of the extract were 0.625, 1.25, 2.5, and 5 µg / mL. Incubate the test sample for 24 hours, wash with HEPES-buffered control salt solution (HCSS) to remove the remaining medium, and prepare 2 ', 7'-dichlorodihydro-fluorescein diacetate (Dichlorodihydro-) prepared at 20 μM in HCSS. 100 μl of fluorescein diacetate (DCFH-DA) was added, incubated in 37 ° C., 5% CO ₂ incubator for 20 minutes, and washed with HCSS. Then, 100 μl of HCSS treated for each concentration of the red pine leaf extract was added and irradiated with ultraviolet B (30mJ / cm ² ), and the fluorescence intensity after 3 hours was measured with a fluorometer (Ex = 485 nm, Em = 530 nm). Was measured and the results are shown in Figure 2 below. The measurement result showed the average value after 6 repeated measurements, and CTL was a control group without the red leaf extract.

As shown in FIG. 2, after 3 hours of UV irradiation, intracellular reactive oxygen species was increased by 182.1% in the group irradiated with ultraviolet B as compared to the control group not irradiated with ultraviolet ray. This increase was observed to inhibit the generation of reactive oxygen species in a concentration-dependent manner when the red pine leaf extract was treated by concentration. From these results, it can be judged that the red pine leaf extract is excellent in inhibiting the generation of active oxygen species in the cell by ultraviolet irradiation.

[Test Example 4] Superoxide dismutase (SOD) and catalase expression reduction recovery measurement

2 mL of 1 × 10 ⁵ cells / mL human keratinocytes HaCaT cells were added to a 6-well plate and incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator, and the red pine leaf extract of the reference example was treated at a concentration of 5 μg / mL. It was. Next, after incubation for 24 hours, the remaining medium was removed by washing with phosphate buffered saline solution, 50 μl of phosphate buffered saline was added and irradiated with ultraviolet ray B. After phosphate buffered saline was removed, the red pine leaf extract was treated and cultured for 48 hours. . After culturing for 48 hours, cells were collected and lysed (Lysis buffer: 250 mM NaCl, 25 mM Tris-HCl pH 7.5, 5 mM EDTA pH8.0, 1% NP-40, 0.1M PMSF, 1M DTT, protease inhibitor cocktail.DW) After crushing at 4 ℃ to quantify the protein amount in BCA solution. Samples were prepared by mixing the same amount of protein and the red leaf extract buffer, and then separated by sodium dodesyl sulfate (SDS) polyacrylamide gel electrophoresis. Acrylamide gel containing protein isolated for Western blot analysis was transferred to nitrocellulose membrane by electroblotting, followed by 5% skim milk. ) Was washed twice with TBS-T (0.1% Tween 20 in TBS). After washing, the SOD and the catalase primary antibody (1: 2000) were treated and left at 4 ° C. for 12 hours, and then reacted at room temperature for 1 hour using a secondary antibody (1: 2000) suitable for the primary antibody. After washing with TBS-T, an enhanced chemiluminescence (ECL) solution was applied, and the expression patterns of SOD and catalase proteins were compared by photosensitive X-ray film in the dark. In order to confirm that the protein has been transferred in the same amount, the above procedure was repeated using actin as the primary antibody. The results are shown in FIG. 3, and the measurement results show the average values after four repeated measurements. In Figure 3, 1 is an untreated group, 2 is UVB 30mJ / cm ² , 3 is UVB + red pine extract 5 ㎍ / mL, 4 is UVB + red pine extract 1 ㎍ / mL, 5 is the red pine extract alone 5 ㎍ / mL It is processed.

From the results of FIG. 3 below, after irradiation of UVB to HaCaT cells and observing the protein expression of SOD and catalase after 48 hours, the expression of SOD and catalase was remarkably decreased, and the red pine leaf extract 5㎍ Treatment with / mL was found to effectively inhibit the reduction of antioxidant enzyme expression by ultraviolet light. From these results, the red pine leaf extract has not only anti-oxidant effect on its own but also excellent protection against damage to the antioxidant enzyme system in the cell against oxidative stress induced internally or externally, ultimately protecting skin cells. It can be determined that there is a function.

Test Example 5 Cell Protection Measurement

HSP70, a protein induced in response to stress, is known to protect cells and individuals against various stresses and to prevent cell death. In addition, HSP70 protein, which is overexpressed in advance, is known to protect cells from stress and various toxicity. In order to measure cell protection by promoting HSP70 production, 2 mL of 1 × 10 ⁵ cell / mL normal fibroblasts were added to a 6-well plate and cultured for 24 hours at 37 ° C. in a 5% CO ₂ incubator. Then, the red pine leaf extract of the reference example was treated with 0.25, 0.5, 1 μg / mL. After the test sample was treated and cultured for 24 hours, the cells were collected, crushed at 4 ° C. with a cell crushing solution, and the protein amount was quantified with a BCA solution. Samples were prepared by mixing the same amount of protein and red pine leaf extract buffer, and then separated by sodium dodesyl sulfate (SDS) polyacrylamide gel electrophoresis. Acrylamide gels containing proteins isolated for Western blot analysis were transferred to nitrocellulose membranes by electroblotting and stained with Ponceus S solution. It was confirmed that the same amount was transferred. Protein transfer membranes were incubated for 1 hour at room temperature with TBS-T (0.1% Tween 20 in TBS) containing 5% skim milk to block nonspecific proteins and TBS-T Washed twice. After washing, the HSP70 primary antibody (1: 1000) was treated and left at 4 ° C. overnight, and then reacted at room temperature for 1 hour using a secondary antibody (1: 2000) suitable for the primary antibody. After washing with TBS-T, an enhanced chemiluminescence (ECL) solution was applied to the X-ray film in the dark, and the expression patterns of HSP70 proteins were analyzed. The results are shown in FIG. 4. In Figure 4, 1 is an untreated group, 2 is a red pine extract 0.25㎍ / mL, 3 is a red pine extract 0.5μg / mL, 4 is treated with a red pine extract 1μg / mL.

From the results of FIG. 4, it was found that the HSP70 protein production was much induced when the red pine leaf extract was treated with 1 μg / mL, and HSP70 protein production was induced depending on the treatment concentration. Therefore, the red pine leaf extract was able to determine the expression of HSP70 protein to a certain level or more can be shown to protect the cells against the stress given later.

Test Example 6 Measurement of MMP-2 Biosynthesis Inhibition

When UV rays are irradiated to cultured skin cells and human skin, the expression of various matrix metalloproteinases (MMPs) increases, and the increased MMPs break down collagen constituting the skin and form skin wrinkles. depending on, 1X10 in 6 well plates to a method for increasing the lean collagen the amount of skin aging as a method of evaluating the aging degree of aged cells by ultraviolet test the effects of ever Pine extract for inhibiting ECM damage ⁵ 2 mL of normal fibroblasts (cell / mL) were added thereto, followed by incubation for 24 hours at 37 ° C. in a 5% CO ₂ incubator, followed by cell attachment. The red pine leaf extract of the reference example was 0.25, 0.5, 1 μg / mL. Treated. Then irradiated with ultraviolet B 30mJ / cm ² in the same manner as in Test Example 5, and then treated with red pine leaf extract was incubated for 24 hours. After incubation, the cell culture solution was collected, a sample was prepared by mixing the cell culture solution and the red pine leaf extract buffer, and then introduced into a gelatin-containing zymogram gel and separated by electrophoresis. The gramogram gel containing the separated protein for MMP-2 confirmation was incubated for 30 minutes at room temperature with renaturing buffer (2.7% Triton X-100) in accordance with the instructions of the reagents, and then developed (developing buffer) (50mM Tris Base, 40mM 6N HCl, 200mM NaCl, 5mM CaCl ₂ 2H ₂ O, BriJ 35 0.02%), and then incubated for 1 hour at room temperature, and then changed to fresh developing buffer and reacted overnight at 37 ℃. After completion of the reaction, the gel was stained to compare the degree of biosynthesis. In order to confirm that there is no difference between samples, cells were collected and crushed at 4 ° C. with a cell crushing solution, and then transferred to a nitrocellulose membrane by electroblotting, followed by staining with Ponceus S solution. Confirmed. The results are shown in FIG. 5.

As can be seen in FIG. 5, MMP-2 biosynthesis was inhibited when 1 μg / mL of red pine leaf extract was treated, depending on the treatment concentration. It was confirmed that it was suppressed. From this, the red leaf extract may be able to block the occurrence of wrinkles, a skin aging symptom, by inhibiting MMP-2 biosynthesis of cells and ECM damage caused by UV rays.

[Examples 1-3 and Comparative Example 1]

To the composition of Table 2, Examples 1 to 3 and Comparative Example 1 were prepared in the form of a cream. In the manufacturing method, each oil phase and the water phase were completely dissolved at 70 ° C., and emulsified at 7,000 rpm for 5 minutes to prepare a cream formulation.

Component (content; wt%) Example 1 Example 2 Example 3 Comparative Example 1 Paid Beads wax 2 2 2 2 Stearyl alcohol 5 5 5 5 Stearic acid 8 8 8 8 Squalene 10 10 10 10 Propylene Glycol Monostearate 3 3 3 3 Polyoxyethylene ether One One One One Preservatives, Antioxidants dose dose dose dose Awards glycerin 4 4 4 4 Propylene glycol 8 8 8 8 Triethylamine One One One One Sodium Polyacrylate 0.2 0.2 0.2 0.2 Purified water To 100 To 100 To 100 To 100 Red Leaf Extract 0.001 One 10 _

Test Example 7 Skin Wrinkle Improvement Effect

The skin wrinkle improvement effect of Example 2 and Comparative Example 1 was evaluated in humans. Twenty five test subjects (30-50 years old) were applied to the right side of the face with the cream formulation prepared in Example 2 and the left side of the face with the cream formulation prepared in Comparative Example 1 twice a day for 6 consecutive weeks. It was. After 6 weeks, the wrinkles around the face were collected with a silicone resin copy (replica) before and after 6 weeks of use of the product, and the skin fine wrinkle device and skin image analyzer were used to compare the condition of the eye wrinkles. The results are shown in Table 3 below.

Before use 6 weeks after use Remarks Example 2 20.8 18.4 Average fine wrinkle depth 11.53% Wrinkle Reduction (%) Comparative Example 1 20.7 20.2 Average fine wrinkle depth 2.41% Wrinkle Reduction (%)

As can be seen in Table 3, when using Example 2 containing the red leaf extract, it was found that the effect of improving the fine wrinkles on the skin around the eyes of the experimenter was excellent.

As described above, the skin external preparation composition for preventing skin aging of the present invention contains red pine leaf extract and has an effect of inhibiting photoaging due to ultraviolet irradiation, and secretes a modulator to resist excessive endogenous or exogenous stress in cells. In addition to the effects of preventing cell damage that may occur in the future, it is excellent in inhibiting the activity of MMPs, which is an anti-aging indicator of skin. Therefore, the external preparation composition for skin containing the red pine leaf extract according to the present invention exhibits an effect of suppressing aging of the skin by alleviating wrinkles of the skin.

Claims (6)

A skin external preparation composition for preventing skin aging, which comprises red pine leaf extract as an active ingredient. According to claim 1, wherein the composition is an external composition for skin, characterized in that to protect the cell membrane damage by ultraviolet irradiation. The composition for external application for skin according to claim 1, wherein the composition protects cells by promoting the production of HSP70 protein. The composition for external application for skin according to claim 1, wherein the composition inhibits the biosynthesis of MMP-2 by ultraviolet irradiation. According to claim 1, wherein the composition for external application composition, characterized in that to improve the skin wrinkles. The composition for external application for skin according to any one of claims 1 to 5, wherein the active ingredient is contained in an amount of 0.001-10% by weight based on the total weight of the composition.

Images