KR20010106886A - Extracts of Zanthoxylum piperitum seed coat and process for preparation thereof - Google Patents

Abstract

The present invention relates to vinewood seed bark extract and a method for producing the same, vinegar seed bark extract of the present invention prepared by stirring extraction of vinegar seed bark as an extraction solvent exhibits skin whitening effect by inhibiting tyrosinase enzyme activity. It is excellent in inhibiting lipogenase enzyme activity and free radical scavenging ability to prevent skin aging.

Description

Extract of bark tree seed bark and its preparation method {Extracts of Zanthoxylum piperitum seed coat and process for preparation

The present invention relates to vinegar seed bark extract and a method for manufacturing the same, more specifically, thyrosinase enzyme activity inhibitory activity, lipoxygenase enzyme activity inhibitory activity and free radical scavenging ability excellent skin whitening and skin aging inhibitory effect It relates to a seed shell extract and a method for producing the same.

The phenomenon of darkening of the skin by ultraviolet rays is a natural bioreaction performed by skin tissues to prevent damage to the skin by ultraviolet rays. The product of such a bioreaction is a pigment called melanin. The main function of melanin is to protect skin cells by absorbing ultraviolet rays harmful to the human body and removing free radicals, which are free radicals produced by ultraviolet rays.

Skin aging is also a natural phenomenon that occurs with decreasing adaptability to the external environment with age. Recently, free radical theory has attracted attention as a mechanism of aging, which was first claimed by D. Harman in the mid-1950s, and is currently being widely studied in medicine, pharmacy and dermatology. Free radicals can be classified into reactive oxygen species and organic free radicals, which are caused by UV rays, environmental pollution, drug metabolism, and in vivo abnormal reactions. The resulting free radicals can damage lipids, proteins, sugars and nucleic acids in the cellular components and cause cell membrane destruction, which can lead to cell death, cell mutations, deactivation of enzymes, and various skin diseases such as skin cancer. . These results ultimately promote skin aging. In addition, the produced free radicals can peroxidize lipids in vivo, and both free radicals and lipid peroxides can cause skin inflammatory reactions. Continued skin inflammation also provides a cause of skin aging, the path of skin inflammation is known to be the main cause of the products produced by the activity of the two enzymes below.

Lipoxygenase and cyclooxygenase are the major enzymes involved in the skin inflammation pathway, especially lipoxygenase, which is an important enzyme involved in the early stages of the biochemical reaction of inflammation. The enzyme uses Arachidonic acid, an important unsaturated fatty acid in mammals, to produce an intermediate mediator of inflammation called leucotriene through oxidative metabolism. Cyclooxygenase enzymes are also directly involved in the inflammatory response through the production of lipid oxides called prostaglandins. Therefore, if it is possible to inhibit the activity of the enzymes it is possible to induce the inhibition of mediators of inflammation, and consequently can inhibit the progression of skin aging.

Conventionally, raw materials such as kojic acid, hydroquinone and vitamin C have been added to cosmetics to prevent skin diseases such as skin blackening, blemishes and freckles. These raw materials are used to produce melanin pigment, which is a major component of skin blackening. It is functional raw materials that inhibit the activity, that is, inhibit the activity of tyrosinase enzymes involved in the main stage of melanin synthesis or reduce the melanin pigment produced. In addition, various antioxidants, including vitamins C, E, and butylhydroxytoluene (BHT), have been formulated in cosmetics to remove free radicals and inhibit the production of lipid peroxides, which are a major cause of skin aging. However, the use of such skin whitening agents and antioxidants is gradually decreasing due to problems of skin safety of raw materials themselves and the stability of raw materials such as oxidation and discoloration in cosmetic formulations.

Recently, in order to overcome the above disadvantages of raw materials with skin whitening and skin anti-aging effects, efforts have been made to find natural products having excellent skin safety and raw material stability in natural plants of nature and having efficacy over conventional whitening agents and antioxidants. have. Currently, many natural plant extracts with excellent skin whitening and skin aging inhibitory ability have been developed and are widely used in cosmetics.

However, to date, natural plant extracts having both skin whitening and anti-aging effects are not common and their effects are not excellent. In addition, the extraction method by heat extraction or deposition was also used in the extraction method, so the contents of the active ingredients in the extract were low due to the destruction of the active ingredients in the extraction process or by passive extraction.

In order to solve the above problems, the present inventors attempt various extraction methods while searching various natural plant extracts of nature in order to efficiently extract plant extracts containing a large amount of active ingredients of plants and excellent skin whitening and anti-aging ability. As a result, the bark seed bark extract obtained through the stirring extraction method contains a large amount of active ingredients, and this extract not only inhibits tyrosinase enzyme activity but also inhibits lipid peroxidation by free radical scavenging and lipoxygenase enzyme. Confirmation of excellent and completed the present invention.

Chopi tree used in the present invention belongs to the Unhyangdae of the plant classification and is a native species mainly in the Yasan in the sub-central region of Korea. The thorns are large and the leaves grow to face each other.

Accordingly, it is an object of the present invention to provide an extract of the bark of the bark tree seed having a skin whitening and skin aging inhibitory effect.

Another object of the present invention is to provide a method for producing the seed bark seed bark extract by stirring extraction.

The present inventors add pulverized bark tree seed bark to the extraction solvent to obtain an extract, and investigate the tyrosinase enzyme activity inhibition rate, free radical scavenging rate and lipid peroxidation rate of the bark tree bark seed bark extract of the present invention The present invention was completed by confirming that the extract is effective in skin whitening and skin aging.

According to one aspect of the present invention, there is provided a method for producing a bark seed extract comprising the following processes:

Adding dried pulverized bark seed bark to 90 ethanol extraction solvent and stirring to prepare a bark seed bark extract;

Adding dried and ground chopped bark seed bark to 90 ethanol extraction solvent and immersing to prepare the bark seed bark extract;

Adding dried pulverized bark seed bark to 90 ethanol extraction solvent and heating to obtain bark seed bark extract;

Investigating the tyrosinase enzyme inhibition rate and DPPH free radical scavenging rate of the three bark seed bark extracts obtained in the above step and comparing the values;

Adding dried pulverized bark seed bark to an extraction solvent including water and stirring to obtain an extract;

Investigating the tyrosinase enzyme inhibition rate of the present invention bark seed bark extract obtained in the above step and comparing the value with arbutin, green tea leaf extract powder, golden root extract powder;

Investigating DPPH free radical scavenging rate of the bark seed bark extract of the present invention and comparing the value with butylhydroxytoluene (BHT), green tea leaf extract powder, golden root extract powder; And

Investigating the lipid peroxidation inhibition rate of the extract of the bark of the present invention bark and comparing the value with butylhydroxytoluene (BHT), green tea leaf extract powder and golden root extract powder.

When explaining in detail the method for producing the bark seed bark extract according to the present invention.

In the first step, the seed bark seedlings are washed with dry water and then pulverized, and the group consisting of distilled water, ethanol, methanol, propanol, butanol, acetone, 1.3-butylene glycol, and ethyl acetate as extract solvents for the dry weight of the ground powder. After adding at least one solvent selected from the amount of 5 to 20 times by weight, and stirring at 100 to 500rpm with a magnetic stirrer (Magnetic Stirrer) for 24 hours at room temperature to extract the active ingredients. The extract was centrifuged at 3000 to 7000 rpm for 10 minutes to extract only the supernatant and filtered using a 0.4 to 100 μm filter to obtain an extract filtrate of step 1. In the second step, the extract was concentrated under reduced pressure in a reduced pressure evaporator (Heidolph LABOROTA 4002) to obtain a dry extract having excellent skin whitening and antioxidant effects.

The bark seed bark extract prepared according to the present invention has not only an excellent inhibitory effect on tyrosinase enzyme activity but also shows a free radical scavenging effect and a strong lipid peroxidation inhibitory effect, and thus has an excellent skin whitening and skin aging inhibitory effect.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples, but the present invention is not limited to the Examples and Experimental Examples.

Example 1: Preparation of the bark seed bark extract by stirring

The seed bark seedlings were washed with clean water and crushed to dryness. Then, 200 g of 90 ethanol was added to 10 g of the dry weight of the pulverized product, and stirred at 400 rpm for 4 hours at room temperature. The extract was centrifuged (5,000 rpm, 10 minutes). The solution was taken only and filtered using a filter (Gelman, 0.45um) to obtain a seed bark extract of the bark.

Comparative Example 1: Preparation of the bark seed bark extract by immersion

The seed bark seedlings were washed with clean water and dried and pulverized. Then, 200 g of 90 ethanol was added to 10 g of the dry weight of the ground product, and the resultant was deposited at room temperature for 4 days. The extract was centrifuged (5,000 rpm, 10 minutes) to take only the supernatant and filtered using a filter (Gelman, 0.45um) to obtain the seed bark extract.

Comparative Example 2: Preparation of the bark seed bark extract by heating

The seed bark seedlings were washed with clean water and dried and pulverized. Then, 200 g of 90 ethanol was added to 10 g of the dry weight of the ground product, and extracted by heating at 80 ° C. for 4 hours. The extract was centrifuged (5,000 rpm, 10 minutes) to take only the supernatant and filtered using a filter (Gelman, 0.45um) to obtain the seed bark extract.

Experimental Example 1: Comparison of the inhibition rate of tyrosinase enzymes of the bark seed bark extract according to the extraction method

In this experimental example, the tyrosinase enzyme inhibition rate was measured for the seed bark seed extract obtained in Example 1 and Comparative Examples 1 and 2. All of the reagents except for the above samples were manufactured by Sigma Co., Ltd. First, 1.15 mL of 0.1 M potassium-phosphate buffer solution (pH6.8) was placed in a test tube, and 0.1 mL of a 3 mM tyrosine solution dissolved in distilled water and 0.2 containing a sample. mL sample solution was added (final concentration of each sample in the reaction solution was 2 (v / v)). In each test tube, 0.05mL of 2000unit / mL tyrosinase enzyme solution dissolved in 0.05M potassium-phosphate buffer solution was added and reacted in a 37 ° C constant temperature water bath for 10 minutes. At this time, only 0.2 mL of distilled water was added to the control A instead of the sample. In control B, 0.05 mL of distilled water was added instead of the tyrosinase enzyme solution. After the reaction, the absorbance at 475 nm was measured with a photospectrometer. The tyrosinase enzyme inhibition rate of each sample was obtained by substituting the absorbance measurements into the following formula:

As a result, as shown in Table 1, the bark seed bark extract (Example 1) obtained through the stirring extraction method was much higher than the tyrosinase enzyme of Comparative Examples 1 and 2 obtained through the deposition or heat extraction at the experimental concentration (2). Inhibitory effect was shown. These results show that the stirring extraction method can extract a large amount of active ingredients from the bark of bark tree seeds more effectively than the deposition or heat extraction methods.

Inhibition Rate of Tyrosinase Enzyme of Extracts from Bark Tree Seed Bark sample Tyrosinase Enzyme Inhibition Rate () Example 1 28.0 Comparative Example 1 8.9 Comparative Example 2 3.2

Experimental Example 2: Comparison of DPPH Free Radical Scavenging Ratios of Extracts from Bark Tree Seeds by Extraction Methods

In this Experimental Example, DPPH free radical scavenging rate was measured for the seed bark seed extracts obtained in Example 1 and Comparative Examples 1 and 2. This experiment was carried out with a slight modification of the experimental method published by Fujita et al in the 1998 journal Yakugaku Zasshi. That is, 1 mL of 0.1 mM DPPH (1,1-diphenyl-2-picrylhydrazyl, Sigma) solution dissolved in methanol was added to a test tube, and 0.1 mL of each sample dissolved in 2 (v / v) concentration in ethanol was added. 1 mL of ethanol was added thereto, followed by reaction for 30 minutes in a 37 ° C. constant temperature water bath (final concentration of each sample in the reaction solution was 0.1 (v / v)). In this case, 1 mL of ethanol without a sample was added thereto. After the reaction, the absorbance at 516 nm was measured with a photospectrometer. The free radical scavenging ratio of each sample was calculated by substituting the absorbance measurement value in the following formula.

As a result, as shown in Table 2, the bark seed bark extract (Example 1) obtained through the stirring extraction method was much higher free radicals than those of Comparative Examples 1 and 2 obtained through the deposition or heat extraction at the experimental concentration (0.1). The scavenging effect was shown. These results also show that the stirring extraction method extracts a large amount of active ingredients from the bark of bark seed without destroying or heating extraction more effectively. In the above, the stirring extraction method was able to effectively extract effective ingredients within a faster time than the deposition extraction method, and compared to the heating extraction method, it was possible to prevent the destruction of the active ingredients by heat.

Free Radical Scavenging Rate of Extracts from Bark Tree Seed Bark sample Radical scavenging rate () Example 1 86.0 Comparative Example 1 70.6 Comparative Example 2 48.7

Example 2: Preparation of the present invention bark seed bark extract prepared by the stirring method

The seed bark seedlings were washed with clean water, dried and pulverized. Then, 400 g of 90 ethanol was added to 20 g of the dry weight of the ground product, and stirred (400 rpm) in a magnetic stirrer at room temperature for 24 hours. The extract was centrifuged (5,000 rpm, 10 minutes) and the supernatant (352 g) was collected and filtered using a filter (Gelman, 0.45 um) and concentrated under reduced pressure at 40 ° C. in a reduced pressure evaporator to 2.5 g (dry weight). The bark of the seed bark extract was obtained.

Experimental Example 1: Comparative study on the inhibition rate of tyrosinase enzyme between the cosmetic component and the extract of bark of the present invention

In this experimental example, the tyrosinase enzyme inhibition rate was measured with respect to the bark seed bark extract obtained in Example 2. As a comparative example, Sigma's whitening agent Arbutin, Bioland's Green tea leaf powder, and Scutellaria root powder are widely used as cosmetic ingredients. It was. All of the reagents except for the above samples were manufactured by Sigma Co., Ltd. First, 1.15 mL of 0.1 M potassium-phosphate buffer solution (pH6.8) was placed in a test tube, and 0.1 mL of a 3 mM tyrosine solution dissolved in distilled water and 0.2 containing a sample. mL sample solution was added (final concentrations of each sample in the reaction solution were 50, 100, 150 μg / mL). In each test tube, 0.05mL of 2000unit / mL tyrosinase enzyme solution dissolved in 0.05M potassium-phosphate buffer solution was added and reacted in a 37 ° C constant temperature water bath for 10 minutes. At this time, only 0.2 mL of distilled water was added to the control A instead of the sample. In control B, 0.05 mL of distilled water was added instead of the tyrosinase enzyme solution. After the reaction was completed, the absorbance at wavelength 475 nm was measured with a photoelectric spectrometer. The tyrosinase enzyme inhibition rate of each sample was calculated by substituting the absorbance measurement value in the following formula.

As a result, as shown in Table 3, the bark seed bark extract obtained through the stirring extraction method showed a slightly lower inhibitory effect of tyrosinase enzyme than the whitening agent arbutin (Arbutin) but higher than the green tea leaf extract powder in the experimental concentration range. Compared with the golden root extract powder, the whitening effect of the bark seed bark extract was found to be superior, and it was confirmed that there was no whitening effect of the golden root extract powder in the above experimental concentration range.

Inhibition Rate of Tyrosinase Enzyme of Extracts from Bark Tree Seed Bark and Other Whitening Agents sample Final concentration of the sample in the reaction solution (µl / mL) Tyrosinase Enzyme Inhibition Rate () Extract obtained in Example 2 50 14.6 100 27.5 150 31.4 Arbutin 50 22.4 100 36.4 150 39.4 Green Tea Leaf Extract Powder 50 13.8 100 22.3 150 25.1 Golden Root Extract Powder 50 0.0 100 0.0 150 0.0

Experimental Example 2: Comparison of DPPH Freera Decal Scavenging Rate of Cosmetic Ingredients and Extracts of Bark Tree Seeds of the Present Invention

In this experimental example, DPPH free radical scavenging rate of the bark seed bark extract of Example 2 was measured. Sigma's antioxidant butylhydroxytoluene (BHT), Bioland's green tea leaf powder, and Scutellaria root powder This experiment was carried out with a slight modification of the experimental method published by Fujita et al in the 1998 journal [Yakugaku Zasshi]. That is, 1 mL of 0.1 mM DPPH (1,1-diphenyl-2-picrylhydrazyl, Sigma) solution dissolved in methanol was placed in a test tube, and 0.1 mL and 0.2 mL of each sample dissolved in 0.1 mg / mL in ethanol. 1 mL of ethanol was added thereto, followed by reaction in a constant temperature water bath at 37 ° C. for 30 minutes (final concentration of each sample in the reaction solution was 5 μg / mL and 10 μg / mL). In this case, 1 mL of ethanol without a sample was added thereto. After the reaction, the absorbance at 516 nm was measured with a photospectrometer. The free radical scavenging ratio of each sample was calculated by substituting the absorbance measurement value in the following formula.

As a result, as shown in Table 4, the bark seed bark extract obtained through the stirring extraction method showed a slightly lower free radical scavenging effect than the green tea leaf extract powder at the final reaction concentrations of 5 ㎍ / mL and 10 ㎍ / mL, respectively. It showed higher free radical scavenging effect than synthetic antioxidant butylhydroxytoluene (BHT), which is widely used as a food and cosmetic ingredient as an antioxidant. And the elimination rate of free radicals was much higher than that of golden root extract powder. These results suggest that the excellent free radical scavenging effect of the bark seed bark extract can prevent damage and destruction of skin tissues caused by free radicals.

Free Radical Scavenging Rate of Bark Seed Bark Extract and Various Antioxidants sample Final concentration of the sample in the reaction solution (㎍ / mL) Radical scavenging rate () SC ₅₀ (μg / mL) Extract of Example 2 5 48.4 5.2 10 87.4 BHT 5 47.8 5.5 10 70.4 Green Tea Leaf Extract Powder 5 67.4 <5 10 92.5 Golden Root Extract Powder 5 13.8 > 10 10 25.5 Note: SC ₅₀ is the scavenging activity (Scavenging activity): sample concentration required to erase 50 of the generated free radicals (㎍ / mL)

Experimental Example 3: Comparative study on the inhibition of lipid peroxidation between the cosmetic components and the extract of bark of the present invention

In this experimental example, the inhibition rate against lipid peroxidation induced by bioenzyme lipoxygenase was measured for the seed bark extract of the bark of Example 2. Sigma's antioxidant butylhydroxytoluene (BHT), Bioland's green tea leaf powder, and Scutellaria root powder ) And this experiment was conducted with Jay. J. Baumann et al. Conducted a slightly modified test method published in the 1980 journal Prostaglandins. In other words, 1.2 mL of 50 mM sodium-phosphate buffer (pH 7.0) was placed in a test tube, and 0.1 mL of 0.15 (w / v) sample solution dissolved in 50 mM sodium-phosphate buffer solution and 6.6 mM arachidonic acid (Sigma) 0.1 mL was added. In each test tube, add 0.1 mL of Soybean Lipoxygenase (Sigma) enzyme dissolved in 50mM sodium-phosphate buffer solution (finally 4000 units of enzyme per 1 mL of reaction solution) and 10 minutes in a 25 ℃ thermostatic water bath. (The final concentration of the sample in each reaction solution was 100 μg / mL). In this case, 0.1 mL of 50 mM sodium-phosphate buffer was added to the control instead of the sample solution.

After the reaction, 1 mL of 20 (w / v) trichloroacetic acid (TCA) solution and 2 mL of 0.67 (w / v) Thiobarbituric acid solution were added thereto, and the mixture was boiled in boiling water for 10 minutes. After the bath was cooled down to room temperature, the absorbance at 535 nm was measured with a photospectrometer. The lipid peroxidation inhibition rate of each sample was calculated by substituting the absorbance measurement value in the following formula.

As a result, as shown in Table 5, the bark seed bark extract obtained through the stirring extraction method showed 1.6 times, 1.4 times, 1.3 times higher lipid peroxidation inhibition rate than BHT, green tea extract powder, and golden root extract powder, respectively. Therefore, the superior lipid peroxidation inhibitory ability of the seed bark seed bark extract was found to be an excellent antioxidant that can increase the skin's defense against inflammation caused by UV and environmental pollution.

Inhibition of Lipid Peroxidation by Extracts from Bark Tree Seed Bark and Various Antioxidants sample Lipid peroxidation inhibition rate () Extract of Example 2 79.3 BHT 51.1 Green Tea Leaf Extract Powder 57.6 Golden Root Extract Powder 59.8

As can be seen from the results of Experimental Examples 4 and 5, the excellent free radical scavenging ability and lipid peroxidation inhibiting ability of the bark seed bark extract indicate that the bark bark seed bark extract of the present invention has excellent skin aging inhibitory ability. Showed

As described through the above examples and experimental examples, the present invention bark tree seed bark extract prepared by stirring extraction from the bark tree seed bark inhibits tyrosinase enzyme activity and shows skin whitening effect and at the same time inhibits lipoxygenase enzyme activity. And excellent free radical scavenging ability to prevent skin aging is expected to be very useful in the cosmetic industry.

Claims (3)

In the method of extracting the active ingredient from the material by a first step of filtering the supernatant obtained by adding a solvent to the pulverized product pulverized the plant material and stirring and centrifugation and a second step of concentration under reduced pressure, The plant material is the bark seed bark, the solvent is a mixture of one or more selected from the group consisting of distilled water, ethanol, methanol, propanol, butanol, acetone, 1,3-butylene glycol and ethyl acetate. To 20 times the weight ratio, the stirring is carried out at 100 to 500rpm and the centrifugal separation is carried out at 3000 to 7000rpm, the filtration process is carried out using a 0.4 to 100㎛ filter bark seed bark Method of Preparation of Extracts. The bark seed bark extract prepared by the method according to claim 1. The method of claim 2, the bark seed bark extract, characterized in that the skin whitening and anti-aging effect.