KR20070119296A - Agent having effects of skin aging resister and skin whitening, manufactured using extract of pine tree as its main ingredient - Google Patents

Abstract

An agent comprising an extract of pine tree is provided to prevent skin aging and whiten the skin due to the antioxidation action of the pine tree extract, thereby providing a cheap skin anti-aging and whitening agent. A skin anti-aging and skin whitening agent comprises 1-10% of an extract of pine tree which is obtained by extracting whole pine tree, pine needles or the woody parts of the pine tree with water or an organic solvent such as ethanol and methanol. The agent is formulated in an orally taking form or a skin application form.

Description

Agent having effects of skin aging resister and skin whitening, manufactured using extract of pine tree as its main ingredient}

The present invention relates to an anti-aging and skin lightening agent using pine extract as an active ingredient, and more particularly, to an anti-aging and skin lightening agent using an extract extracted using an organic solvent for pine needles or pine bark. It is about.

Pine is an evergreen shrub that is wild all over Korea. Pine leaves, bark, and rosin are known to be able to cure various diseases from ancient times. Pine needles are known to have stomach diseases, hypertension, stroke, neuralgia, and asthma. It is said to be effective. Even folk remedies have been known to be effective for colds, especially cough colds, when pineapple decocted water, pine needle juice, or pine soaked in honey.

In recent years, the inventors pay attention to the fact that pine extracts contain many useful ingredients. Recently, various inventions using pine extracts have been introduced.

Patent No. 268401 relates to a method for improving palatability of pine extracts and to oral ingestion prepared by the method, which is characterized by relieving the rosin smell, bitter taste and astringent taste of pine extract to facilitate people's drinking. Patent No. 249128 relates to a pine extract-containing external wool agent, characterized in that the pine extract has a hair growth promoting effect, and Patent Publication No. 2005-51053 protects brain cells using pine needle extract as an active ingredient. Or relates to a composition for treating stroke and health supplements using the same, Patent Publication No. 1994-10987 relates to a shampoo and rinse agent with the addition of pine extract, the essential oil and chlorophyll extracted from pine are currently produced Added to shampoo and rinse agent to maintain cleanliness and hygiene of hair and scalp In addition, the use of the bark of the pine is another invention related to the natural antibacterial component isolated from the bark of pine (Registration No. 10-0280368), the invention related to a method for preparing a composition for treating oral diseases (Registration No. 10-0044435 ).

However, whether pine extract is effective in preventing skin aging and skin whitening is not known at all, and it has been verified through clinical trials.

The present inventors focused on the fact that the pine extract has an antioxidant effect, and proved through preclinical experiments that the pine extract has the effect of anti-aging and skin whitening, the present invention is the anti-aging and skin of the pine extract The invention relates to a new use as a whitening agent.

On the other hand, pine is native to all over Korea, but it is not a high value added species because its value as a timber is not high. Fresh pine needles can be processed and sold as functional foods, but pine needles or bark (bark), which have become deciduous, can only be used as firewood, and their use has been extremely limited. However, not only fresh pine needles, but also deciduous pine needles and pine bark and branches, and sawdust left after wood processing at sawmills contain antioxidants, which can be extracted from the pine as a whole. It can increase the utilization of the whole and further increase the added value of pine trees.

The inventors pay attention to the fact that the pine extract has an antioxidant activity, and through preclinical experiments, the pine extract has been found to have the effect of preventing skin aging and skin whitening, and an object of the present invention is to use pine extract as an active ingredient. It is to provide skin anti-aging and skin lightening agent.

On the other hand, the present inventors have found that it is possible to extract antioxidants from pine needles, pine bark or branches, and sawdust, etc., which have become the leaves of pine, and the other object of the present invention is the utility value and added value of pine To increase it.

The present invention is to extract each part of the pine using an organic solvent such as ethanol, and then to use the fraction to prepare the skin anti-aging and skin lightening agent. More specifically, the present invention, after extracting pine needles, pine bark (bark) or wood parts of pine with methanol or ethanol, using the extract for each site to investigate the effects on skin anti-aging and whitening, based on this It is to be able to provide anti-aging and skin lightening agents.

Various methods can be applied in extracting the extract of pine, but different methods do not change the properties of the extract extracted by each method. Therefore, the present invention is not to be limited by the extraction method of pine extract illustrated below.

Hereinafter, the present invention will be described based on Examples.

(Example 1)

The present embodiment relates to the production of pine extract used in the present invention, the pine needles (blue pine needles) of Korean red pine ( Pinus densiflora Siebold et Zuccarini) native to Korea, bark including twigs, and sawdust left after processing wood at the sawmill (Not dried) was dried in a well-ventilated shady place as a raw material.

Each of the pine needles, pine bark and sawdust was added to 10 g of 70% ethanol at a rate of 90 ml, and then heated at 90 ° C. for 1 hour, cooled to room temperature, and then centrifuged at 5,000 rpm / 15 minutes or filtered. ) The water and the extract were separated. After drying the solids and extracts (liquid phase), they were used as samples to investigate the effects on skin anti-aging and whitening.

In this example, the recovery rate of each fraction is as follows.

Table 1. Recovery of each fraction by ethanol extract (%)

(Example 2)

This example was performed to test the cytotoxicity of the extract extracted in Example 1.

In this example, the cell viability of the normal cell line was measured by partially modifying the colorimetric MTT assay of Charmichael et al. (1987) to confirm the effect of pine extract on normal cells. In this example, NIH3T3 (KCLB 21658, mouse embryo) and HFF (human foreskin fibroblast) were used as test cells. After fully cultured NIH3T3 and HFF cells were inoculated at a concentration of 1 10 ⁴ cells / 0.1 ml / well in a 96-well microplate and preincubated for 4 hours, cell viability of normal cells using MTT solution was expressed by the following equation. Measured.

% Cell viability against normal cells = B / A × 100

 In Equation 1), A means O.D. of well cultured only normal cells, and B means O.D. of experimental wells.

According to the results of this example, since all treatments showed a survival rate of 95% or more at a concentration of 0.01-10 mg / ml, the pine extract extracted by Example 1 was confirmed to have little toxicity to human cells.

(Example 3)

This example was performed to determine how the extract prepared in Example 1 causes the removal of nitrite.

Nitrite is not only contained in vegetables, but also used as a food additive as a coloring agent, preservative, and Clostridium botulinum growth inhibitor of meat products such as sausage and ham. Since nitrite is a self-toxic substance, when it is ingested at a certain concentration or more, it reacts with amines in food in the stomach or intestine to produce nitrosoamine, a carcinogen. In addition to the ability, it is widely used as an item for determining the degree of strong antioxidant activity. For this reason, the nitrite removal activity of the pine extract was measured in this example.

Nitrite scavenging activity was measured by Kang (1995, J. Food Sci. Technol., 27) and Kim (1987, Bull. Korean Fish. Soc., 20). . That is, 1 ml of 1 mM NaNO ₂ solution was completely dissolved in each pine extract in distilled water, and then 0.6 ml of the sample solution filtered using a 0.45 µm syringe filter (Advanced MFS. Inc.) was added thereto, and 0.1N HCl (pH 1.2) was added thereto. ) The pH of the reaction solution was adjusted to a final volume of 10 ml. After the reaction was conducted at 37 ° C. for 1 hour, 5 ml of 2% acetic acid was added to 1 ml of the reaction solution, followed by 0.4 ml of 0.5% sulfanilic acid and 0.5% naphylamine mixed solution prepared using 30% acetic acid as a solvent. Add and mix. After the reaction mixture was reacted for 15 minutes at room temperature, the absorbance was measured at 520 nm using a spectrophotometer (Phamacia biotech, Model Ultrospec 1000) to determine the amount of remaining nitrite. Nitrite scavenging activity was expressed as a percentage (%) as follows.

Nitirite scavenging activity (%) = ( One - A-C  ) × 100 B

In the above formula, A is the absorbance of (NaNO ₂ + sample solution), B is the absorbance of NaNO ₂ , C is the absorbance of the sample itself.

As a result of this Example, it was confirmed that pine extract has a strong nitrite removal ability. That is, the ethanol extract of each part of the pine, it was confirmed that the activity is almost the same as BHT and ascolic acid known to have a nitrite removal ability, especially at low concentrations of pine needles showed higher activity than tocopherol. In addition, it was confirmed that there is a strong activity in the precipitate of the insoluble fraction, it was confirmed that each part of the pine is excellent in the nitrite removal ability. This indicates that pine tree has a strong antioxidant activity, in particular, it can inhibit the cell transformation caused by nitrite and the like and carcinogenesis.

In this example, the results of measuring the effect of each pine extract on nitrite removal are shown in Table 2.

Table 2. Effect on nitrite removal (unit:%)

(Example 4)

This example was performed to determine the effect of pine extract on the activity of superoxidase dismutase (SOD).

The O ₂ active oxygen (superoxide) ^- · is produced in vivo metabolic processes as a substance that affects the aging, the material is reactive and destructive when electron reduction that causes a very strong and highly toxic to cells and tissues. The SOD secreted to remove · O ₂ ^{- -} Due to these toxic radicals to cause disease and various types of adult disease, arthritis associated with aging, in the body O ₂ · the conversion of a H ₂ O ₂ and O ₂ Play a role. SOD-like substances are not enzymes, but they are low-molecular plant chemicals that function similar to SOD, protecting the body from free radicals by inhibiting the reactivity of free radicals.

For this reason, SOD activation is used as an important item to measure the antioxidant power, this embodiment evaluated the effect of pine extract on the antioxidant power as SOD activity.

SOD-like activity measured is hypoxanthin a xanthine oxidase (XOD) · O ₂ generated as oxidation ^by-of suppressing the generation of sikimeuroseo NBT ^- there is color development nitroblue tetrazolium (NBT) by is reduced to blue, SOD is · O ₂ It measured by the principle which suppresses a reduction.

That is, 5 ~ 5mM xanthine (pH7.4) 5µl, 10mM EDTA 5µl, 2mM NBT 5µl, 50mM phosphate buffer (pH7.8) 430 5 μl of xanthine oxidase (50 unit) was added sequentially and the final volume was 500 μl. Meanwhile, 200 μl of xanthine oxidase was added to the microtube and centrifuged at 13,000 rpm for 10 minutes. The supernatant was discarded, and 600 μl of 50 mM phosphate buffer was added and diluted. The absorbance change was measured at 560 nm for 1 minute, and the control was added with phosphate buffer instead of the sample solution. In order to determine the sample amount and concentration at which the reduction rate of NBT was inhibited by 50% before the experiment, a calibration curve was obtained by dividing the control O.D value by the O.D value by the sample solution as the R value, and the sample amount was obtained when R was 2. SOD like activity unit (SOD like activity unit) refers to the amount of 50% inhibition of absorbance change for 1 minute in the absence of the sample, SOD like activity was calculated according to the following equation.

SODlikeactivity (unit / g) = One  × Dilution factor × 1000  Sample amount (ml) when R is 2

As a result of this Example, it was shown that the same or higher activity as the positive control ascorbic acid. That is, at high concentrations, pine extract appeared to be at least 10 times more active than ascorbic acid, and it was confirmed that it can effectively decompose various active oxygen generated in the metabolic process of the human body. However, the concentration of less than 1 mg / ml showed less activity than ascorbic acid, and if you want to develop an antioxidant product using pine extract, it can be seen that it is desirable to plan the product at a concentration of at least 1 mg / ml. .

The effect of the pine extract measured in this Example on the SOD-like activity is shown in Table 3.

Table 3. Effect of Pine Extracts on SOD-like Activity

(Example 5)

This example was performed to determine the effect of the electron donating ability (Electron donating ability) by the pine extract. Free radicals are produced by oxidation of epinephrine and normal metabolism by mitochondria, macrophages, or xantine oxidase or glutathione reductase in the cytoplasm, and various other physiological metabolisms. The electron donating action is an electron donating action that inhibits oxidation by donating electrons to these oxidative free radicals. Therefore, the good electron donating ability means that the oxidation inhibitory effect by the provided electrons is strong, which is widely applied as a very reliable method for measuring the antioxidant power.

In this example, the measurement of electron donating activity was performed by applying the method of Blois (1958, Nature, 181). In other words, 0.8 ml of 0.4 mM DPPH (1,1-diphenyl-2-pictyl-hydrazyl) solution dissolved in 99.9% ethanol was added to 0.2 ml of each sample, followed by shaking and mixing for 10 seconds, followed by reaction at room temperature for 10 minutes, using a spectrophotometer. Absorbance at 525 nm was measured. Electron donating activity is expressed as a percentage according to the following equation.

Electron donating ability (%) = ( One - A  ) × 100 B

In the above formula, A is the absorbance of the addition sphere, B is the absorbance of the non-addition sphere.

As a result of the measurement of the present example, it was confirmed that each pine extract had a strong electron donating ability at high concentration. In the case of nitrite scavenging activity or SOD-like activity, pine needles were found to have the strongest activity. In addition, at a low concentration of 0.1 mg / ml, the activity was shown to be stronger than ascorbic acid, and it was confirmed that the antioxidant activity by electron donation was very strong.

Antioxidant activity was also shown to be very strong in pine needles and wood, but it was confirmed that the activity was rapidly lowered at low concentrations, and it was confirmed that it was very feasible to commercialize at least 1 mg / ml.

The effect of pine extract on the electron donating ability measured by the present embodiment is shown in Table 4.

Table 4. Effect on electron donating ability (unit:%)

(Example 6)

This example was performed to measure the effect of inhibiting apoptosis caused by hydrogen peroxide mediated pine extract.

After dispensing a certain amount of hydrogen peroxide into the medium, an artificial oxidation reaction was induced, and then the effect of pine extract on the inhibition of oxidative death of normal cell lines was verified. The same cell line as in Example 2 was cultured in the same manner, and then the control group in which 10 μl of 1% hydrogen peroxide solution was dispensed in the preculture step, and the same amount of hydrogen peroxide solution and the treatment group in which the pine extract was dispensed in different concentrations. The effect on cellular injury was examined by dividing into (Experimental).

As a result, it was confirmed that only 28.9% of the cells survived by the hydrogen peroxide solution, which was severely injured by artificial oxidation. However, this oxidation was greatly inhibited by the bark and pine needles, and the survival rate of the cells was improved. Particularly, in the case of the shell, very high activity is confirmed in the sediment as well as the extract, which can be considered an invention of great value in terms of utilization of the existing resources. Pine needles and wood parts were found to be inferior in antioxidant activity at low concentrations, and pine needles and wood parts should be used at concentrations of 10 mg / ml or more.

The effect on the inhibition of oxidation by hydrogen peroxide of the pine extract, measured by this example is shown in Table 5.

Table 5. Effect on Oxidation Inhibition by Hydrogen Peroxide

(Unit:% survival)

(Example 7)

This example was performed to measure the melanin pigment production inhibitory effect of the pine extract.

In this example, each pine extract was added to the culture medium of rat melanoma cells (B16BL6) to investigate the melanin production inhibitory effect (whitening effect) at the cellular level (Lotan R .., Lotan D., 1980 Cancer Res., 40). In addition, the inhibition of melanin production was investigated by using cysteine, which is widely used in verification of medicines, as a positive control.

The culture medium of B16BL6 was added to 50 ppm of cysteine and incubated for 3 days, followed by trypsin treatment to recover cells from the culture vessel, which was centrifuged and the melanin released from the culture was extracted. 1 ml of 1 N sodium hydroxide was added to the solution, heated for 10 minutes to dissolve melanin, and absorbance was measured at 400 nm. The absorbance was calculated by converting the absorbance per 10 ⁶ cells (converted from the number of cells in the initial culture), and this was calculated as the melanin production relative to the absorbance of the control group to express the inhibition of pigment production in%.

As a result, cysteine was found to inhibit melanin pigment production by about 32%. However, this level was about the same as the sediment (10 mg / ml) in each pine area. In the case of pine extract by the organic solvent, it was confirmed that the inhibition of melanin pigment production by more than 80% at 10mg / ml has a strong whitening action. In the case of the extract, even at a concentration of 0.1 mg / ml, there is a strong pigment inhibitory ability than the concentration of 50 ppm cysteine, it was confirmed that the development of a whitening product using the pine extract is very high.

The melanin pigment production inhibitory effect of the pine extract measured by this Example is shown in Table 6.

Table 6. Effect on melanin production inhibition

(Example 8)

This example was performed to measure the ability to inhibit erythema production by ultraviolet irradiation of pine extract.

In this example, the effect of pine extract on erythema production by UV irradiation was investigated using brownish guinea pigs. Brownish guinea pigs were purchased from Korean experimental animals, and the starting weight of the males before and after 400 g was used for 5 rats of each treatment group. The guinea pig feed was used as a free intake method, and after irradiating the minimum amount of UV rays three times a day, the regression of erythema over time was measured using a colorimeter (Minolta, Japan). Value) was calculated as a value subtracting the brightness value before initiation. Ascolic acid 500mg / kg, cysteine 200mg / kg, gamma-linoleic acid 200mg / kg was added to the control group as a control agent, the treatment was added to the formulation with 100mg / kg pine extract each It was supposed to be paid.

As a result, all of the pine extracts showed better healing ability than the control group. That is, in the case of the control, it was found that the healing of erythema proceeded very slowly over time, but the pine needles and bark extract were found to have excellent healing ability. This means that the skin that has been blackened by the ultraviolet rays is quickly recovered again, which means that it takes a very short time for the skin to be restored to normal even if the skin is damaged by the ultraviolet rays. In addition, this is very effective in maintaining skin whitening, it can be said that it is very reasonable to apply to the product for whitening based on this.

As measured in the present embodiment, the effect of inhibiting erythema production by ultraviolet irradiation of pine extract is shown in Table 7.

Table 7. Effect of UV Irradiation on Suppression of Erythema Formation

As can be seen from the above examples, the pine extract, which is the main component of the present invention, is harmless to the human body, and the component can be absorbed by the human body by the method of ingestion or direct application to the human skin. Therefore, the present invention can be prepared in any form for oral ingestion or skin application.

The present invention relates to the use of the pine extract, which focuses on the antioxidant activity of the pine extract, and relates to providing an anti-aging and skin lightening agent using the pine extract as an active ingredient, and the present invention provides a low price. In addition, it is very useful in terms of preventing skin aging and providing a whitening agent, as well as increasing the utility value and added value of pine.

Claims (7)

Skin anti-aging and skin whitening agent with pine extract as an active ingredient. The skin anti-aging and skin lightening agent according to claim 1, wherein the pine extract uses whole pine, pine needles, bark, or pine wood, respectively. The skin anti-aging and skin lightening agent according to claim 1, wherein the pine extract is extracted using water or an organic solvent, and the extracted fraction is used. The skin anti-aging and skin lightening agent according to claim 3, wherein the organic solvent is ethanol or methanol. The skin anti-aging and skin whitening agent according to any one of claims 1 to 4, wherein the pine extract is used as a 1-10% solution. The skin anti-aging and skin lightening agent according to claim 1, which is prepared in a form in which the human is directly ingested by mouth. The skin anti-aging and skin lightening agent according to claim 1, which is prepared in a form applied to human skin.