TWI417127B - Extracts from peony's root cortex for application of skin whiteness and method of preparing the same - Google Patents

Description

Cortex peel extract and its manufacturing method and application of skin whitening

The present invention relates to a peony bark extract and a method for producing the same, and more particularly to a peony bark extract having an activity of inhibiting melanin production and a method for producing the same.

In recent years, due to the improvement of the standard of living of the people, modern people have paid more and more attention to their appearance. However, the depletion of the ozone layer has led to an increase in the demand for whitening, blemishes and freckle-related products, and the market size of cosmetic products has expanded year by year. In addition, modern people have a healthy concept, in order to avoid damage to the skin caused by chemical makeup products, the application of Chinese herbal medicines in health foods and beauty has been generally valued, especially the application of Chinese herbal extracts in cosmetics. To become a global trend is also the focus of the active development of the domestic cosmetics and biotechnology industry.

The color of the skin is related to the amount of melanin in the skin. Ultraviolet light can induce the production of melanin in the skin tissue, and the production of melanin is related to the activity of tyrosinase and the protein of tyrosinase. -1 (tyrosinase-related protein-1; TRP-1; DHICA oxidase) and TRP-2 (tyrosinase-related protein-2; TRP-2; DOPA chrometautomerase) are inextricably linked. TRP-2 can catalyze the conversion of dopachrome to DHICA (5,6-dihydroxy indole carboxylic acid), while TRP-1 can catalyze the oxidation of DHICA.

In recent years, many studies have been published on the study of tyrosinase activity at home and abroad, and the research on whitening is even more promising. However, many studies are looking for substances that can inhibit the activity of tyrosinase, or other ways to achieve whitening effect. The ingredients that have been confirmed to be effective in inhibiting tyrosinase activity are arbutin (AR) and tannin. (kojic acid; KA) and hydroquinone (HQ), among which tannic acid and arbutin are whitening ingredients that can be added by the Department of Health, and hydroquinone is classified as drug control. However, each has its side effects, such as citrate, which causes liver cancer (Tamotsu et al ., 2004). Hydroquinone is used in many countries to treat pigmentation and is used as a drug, but it is unstable and has a melanocyte Toxic and oxidizable. Applying to the skin may also cause allergies, redness and discomfort (Teruhisa et al ., 2003; Halder et al . 2004), so it is necessary to find a safe and effective whitening ingredient.

In view of the above, it is desirable to provide a botanical substance having an activity of inhibiting melanin production in order to provide a cosmetic composition and/or a food additive having whitening effect.

Accordingly, an aspect of the present invention provides a method for producing a peony bark extract, which comprises at least one extraction step and a gel chromatography step for obtaining melanin-producing activity from peony bark by at least one organic solution. The peony bark extract and the obtained peony bark extract can be further applied to cosmetic compositions and/or food additives.

Next, another aspect of the present invention provides a peony bark extract which is obtained by the above extraction step and gel chromatography step. Since the prepared peony bark extract has an inhibitory effect on melanin production, it can be applied to a cosmetic composition and/or a food additive.

Furthermore, another aspect of the present invention provides an application of a peony bark extract to skin whitening, which utilizes the above extraction step and a gel chromatography step to obtain a peony bark extract having an inhibitory effect on melanogenesis. Can be applied to skin whitening.

According to the above aspect of the invention, a method for producing a peony bark extract is proposed. In one embodiment, a first extraction step is first performed by extracting the peony bark material with a first organic solution to obtain a first extract. Next, a second extraction step is performed, which extracts the first extract by using a second organic solution to divide the first organic phase and the aqueous phase, wherein the first organic phase has a second extract and the aqueous phase has a third Extracts. Then, a third extraction step is performed, which extracts the second extract by using a third organic solution to divide the second organic phase and the third organic phase, wherein the second organic phase has a fourth extract, and the third organic The phase has a fifth extract. Thereafter, a gel chromatography step is performed to obtain a peony bark extract obtained from the fifth extract, wherein the peony bark extract is inversely thinned by using 8:2 by volume of methanol and water plus 10 drops of acetic acid as a developing solution. The layer chromatography analysis, according to formula (I), obtains a retention factor (Rf) value of, for example, 0.462 to 0.513:

According to an embodiment of the invention, the first organic solution may be, for example, an ethanol solution, and the second organic solution may be, for example, an ethyl acetate/water solution, and the third organic solution may be, for example, a n-hexane/methanol solution.

According to an embodiment of the invention, the first organic phase may be, for example, an ethyl acetate phase.

According to an embodiment of the invention, the second organic phase may be, for example, a methanol phase.

According to an embodiment of the present invention, the above-mentioned peony bark extract may include, but is not limited to, mudanpioside E, mudanpioside B, and suffruticoside E.

According to an embodiment of the present invention, the above-mentioned peony bark extract or the first extract has an activity of inhibiting melanin production.

According to another aspect of the invention, a peony bark extract is provided. In one embodiment, the peony bark extract can be obtained by the above extraction step and a gel chromatography step, and the peony bark extract is developed by adding 8 drops of acetic acid in a volume ratio of 8:2 methanol and water. The liquid is subjected to reverse phase thin layer chromatography analysis, and a retention factor (Rf) value of, for example, 0.462 to 0.513 is obtained according to the formula (I).

According to other aspects of the present invention, there is provided a cosmetic composition and/or food additive, characterized in that the cosmetic composition and/or food additive may comprise the first step obtained by the above extraction step and gel chromatography step. An extract or a peony bark extract, and the first extract or the peony bark extract has an activity of inhibiting melanin production.

Applying the peony bark extract of the present invention and a method for producing the same, which obtains a peony bark extract having an inhibitory effect on melanin production from peony bark by at least one extraction step and a gel chromatography step, and can remove the cells A toxic ingredient that can be further applied to cosmetic compositions and/or food additives.

As described above, the present invention provides a peony bark extract and a method for producing the same, which utilize at least one extraction step and a gel chromatography step to obtain a peony bark extract having an activity of inhibiting melanin production.

The paper referred to here, "Paeonia" means Paeonia Ranunculaceae (Ranunculaceae) (Paeonia) Peony (Paenoina Suffruticosa) root bark (root cortex). Mudanpi is a kind of Chinese herbal medicine. It is mainly used in traditional Chinese medicine to provide functions of cooling blood, clearing away heat, promoting blood circulation and dispersing phlegm. The peony bark used in the present invention is confirmed by Dr. Lin Hanqin, and the peony bark extract having the activity of inhibiting melanin production is provided by the method disclosed in the present invention.

As used herein, "at least one extraction step" refers to the extraction of peony bark using an organic solvent system of different polarity. In one example, the aforementioned peony bark is extracted with an ethanol solution, and then the peony bark extract is extracted with an ethyl acetate/aqueous phase and n-hexane/methanol. In another example, the aforementioned peony bark is extracted with an ethanol solution, and then extracted with an ethyl acetate/aqueous phase, n-hexane/methanol, and then subjected to a gel chromatography step to obtain A peony bark extract that inhibits melanin production activity. In still another example, the aforementioned peony bark extract is subjected to reverse phase thin layer chromatography analysis using a volume ratio of 8:2 methanol and water plus 10 drops of acetic acid as a developing solution, and obtained according to formula (I), for example. The retention factor (Rf) value of 0.462 to 0.513:

As used herein, "antimelanogenic activity" means that the aforementioned peony bark extract can effectively reduce the formation of melanin and thereby achieve skin whitening effect. In one example of the present invention, the aforementioned inhibition of melanin production activity means that the formation of dopaquinone and melanin is effectively reduced by inhibiting the activity of tyrosinase. In another example of the present invention, the melanin-producing activity of the aforementioned peony bark extract can be expressed as an effective reduction of dopaquinone by down-regulating the expression of related proteins in melanogenesis. ) and the formation of melanin.

In one embodiment, the peony bark extract of the present invention can be obtained by the following method. Please refer to FIG. 1 , which is a flow chart showing the process of the peony bark extract according to an embodiment of the present invention. First, a first extraction step is performed in which a crushed peony skin material 101 is immersed in 95 by using a pulverizer to break up about 18.0 kg of peony skin material 101 at room temperature or slightly below room temperature. Volume percent of ethanol solution. Next, a filtration step of filtering the above-mentioned 95% ethanol solution containing the pulverized peony material 101 by gauze, filter paper or other filtration means, and repeating four times, to obtain a filtrate. Then, after the ethanol solution is removed by a reduced pressure concentration method, a first extract (hereinafter referred to as Ps-1) 103 is obtained, wherein the first extract 103 is a crude extract of peony bark.

Thereafter, the first extract 103 is subjected to a partitioning step by using an ethyl acetate/water solution, whereby the first organic phase and the aqueous phase are obtained by layering, wherein the first organic phase is an ethyl acetate phase and has a second extract. (hereinafter referred to as Ps-2) 105, and the aqueous phase has a third extract (hereinafter referred to as Ps-3) 107.

Subsequently, the second extraction step 105 is subjected to a third extraction step using a n-hexane/methanol solution to separate the second organic phase and the third organic phase, wherein the second organic phase (for example, a hexane phase) There is a fourth extract (hereinafter referred to as Ps-4) 109, and a third organic phase (for example, a methanol phase) has a fifth extract (hereinafter referred to as Ps-5) 111, and wherein the methanol solution can be, for example, 90 volumes percentage.

Then, a gel chromatography step is performed in which the fifth extract 111 is subjected to, for example, a gel chromatography column to obtain a peony bark extract. Further, in the gel chromatography step, the fifth extract (methanol phase extract) 111 is passed through a gel chromatography column, for example, a Sephadex LH-20 gel chromatography column. After 100 parts by volume of methanol was extracted, the peony bark extract 113 was collected. The aforementioned peony bark extract 113 is analyzed by thin layer chromatography, and is divided into a first part, a second part and a third part according to the order of collection, wherein the first part is a peony bark extract (hereinafter referred to as Ps-6) 113. The obtained peony bark extract 113 is subjected to reverse phase thin layer chromatography analysis with a volume ratio of 8:2 methanol and water plus 10 drops of acetic acid as a developing solution, and a development resistance of, for example, 0.462 to 0.513 is obtained according to the formula (I). Hysteresis (Rf) value:

In one embodiment, the peony bark extract obtained above may include, but is not limited to, a monoterpene glycoside and a benzophenone glycoside, wherein the monoterpene glycoside may be, for example, a formula (II) and/or a formula. The structure shown in (III), and the benzophenone glycoside may be, for example, the structure of the formula (IV):

In the structures of the above formula (II), formula (III) and formula (IV), R ₁ may include, but is not limited to, H, and R ₂ and R ₃ may include, but are not limited to, hydroxyl (OH), methyl or ethyl. Base, G represents galloyl (G), and a is a positive integer from 1 to 3. In an example, the structure represented by the above formula (II), formula (III) or formula (IV) may include, but is not limited to, mudanpioside E, mudanpioside B, peony P (suffruticoside E) or any combination of the above.

It is worth mentioning that the peony bark extract of the present invention is not divided according to the order of the polarity of the solvent, but is sequentially divided by the ethyl acetate phase and the methanol phase to extract the peony bark extract. . Since the division step is carried out by the method of the present invention, delamination is easy and foaming is not easy, so that the process time can be shortened. Secondly, the method of the present invention can find better and more effective ingredients in the peony bark, and can remove components which are toxic to cells.

In addition, the peony bark extract obtained by the present invention is exemplified by the following examples, which can down-regulate the melanocortin-1-receptor (MC1R) and the small eye deformity-related transcription factor. (microphthalamia-associated transcription factor; MITF), tyrosinase and protein expression of TRP-1, and effectively reduce the formation of extracellular melanin.

According to the statement, the most important factor causing skin pigmentation is the melanin synthesis in melanocytes due to exposure to ultraviolet light, which is the main key enzyme that catalyzes the synthesis of melanin. The peony bark extract and the first extract obtained by the present invention are exemplified by the following examples, and can effectively reduce the extracellular activity by down-regulating the protein expression of MC1R, MITF, tyrosinase and TRP-1. The formation of melanin is therefore applicable to skin whitening cosmetic compositions and/or food additives.

For example, when the peony bark extract and the first extract obtained by the present invention are applied to a cosmetic composition, the form may include, but not limited to, a liquid, an emulsion, an ointment, a powder, a whitening agent, a spotting agent, and a spotting agent. Or a cosmetic of any combination of the above. Further, when the peony bark extract obtained by the present invention is applied to a food additive, the form thereof may include, but is not limited to, a nutraceutical or a health food.

The following examples are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the present invention. Retouching.

Example 1: Preparation of Cortex Mouth Extract

This example produces a peony bark extract using the method of Figure 1. First, a first extraction step is carried out by using a pulverizer to pulverize about 18.0 kg of peony bark material at room temperature or slightly below room temperature, and then immersing the smashed peony bark material at 95% by volume. In the ethanol solution. Next, a filtration step of filtering the above-mentioned 95% ethanol solution containing the pulverized peony bark material by gauze, filter paper or other filtration means is repeated four times to obtain a filtrate. Then, after removing the ethanol solution by a reduced pressure concentration method, a first extract (Ps-1) 103 is obtained, wherein the first extract 103 is a crude extract of peony bark.

Thereafter, the first extract 103 is subjected to a second extraction step using an ethyl acetate/water solution to obtain a second extract (Ps-2) 105 and a third extract (Ps-3) 107, and the second extract 105 is an ethyl acetate extract.

Subsequently, the second extraction step 105 is subjected to a third extraction step using a n-hexane/methanol solution to separate the second organic phase from the third organic phase, wherein the second organic phase (eg, n-hexane phase) There is a fourth extract (Ps-4) 109, and a third organic phase (such as a methanol phase) has a fifth extract (Ps-5) 111, and the methanol solution can be, for example, 90 volume percent.

Then, a gel chromatography step is performed in which the fifth extract (Ps-5) 111 is obtained by, for example, a gel chromatography column to obtain a peony bark extract, wherein the gel chromatography step can utilize, for example, glucose condensation. After the gel chromatography column (Sephadex LH-20 gel chromatography column; Pharmacia Fine Chemicals AB Uppsala, Sweden) was carried out and washed with 100 volume percent of methanol, each part of the peony bark extract 113 was collected.

The peony bark extract 113 is analyzed by the reverse phase thin layer chromatography layer, and is divided into the first part, the second part and the third part according to the order of the collection, wherein the first part (Ps-6) is Peony peel extract 113. The obtained peony bark extract 113 is subjected to reverse phase thin layer chromatography analysis with a volume ratio of 8:2 methanol and water plus 10 drops of acetic acid as a developing solution, and a development resistance of, for example, 0.462 to 0.513 is obtained according to the formula (I). Hysteresis (Rf) value:

Example 2: Evaluation of cytotoxicity of peony bark extract

In this example, it was first tested whether the peony bark extract of Example 1 at different stages of the process was cytotoxic to skin cells or melanocytes. In this example, the Hs68 human skin fibroblast strain (BCRC number: 60038) and the B16 mouse melanoma cell line (ATCC number: CRL-6322) for testing, wherein the ATCC is a typical American species center (American Abbreviation for Type Culture Collection; ATCC). The above cell lines are cultured in a cell culture medium by a sterile technique, wherein the cell culture medium of the Hs68 cell line and the B16 cell line is Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island, NY). And 10% by volume of fetal bovine serum (Hazelton Product, Denver, PA, USA) and 1 volume percent of antibiotic (containing penicillin and streptomycin, penicillin-streptomycin). The above cells were seeded at 1.0 × 10 ⁴ cells per cell (cell density 1.0 × 10 ⁵ /mL, and the volume added per well was 100 μL), and then cultured at 37 ° C and maintained at a humidity of 5% carbon dioxide for at least 24 hours. . Thereafter, an intermediate extract having a concentration of 100 μg/mL (first extract (Ps-1), second extract (Ps-2), third extract (Ps-3), fourth extract (Ps-) 4), the fifth extract (Ps-5)), the peony bark extract (Part 1, Ps-6), the peony bark extract (Part 2, Ps-7), the peony bark extract (Part III, Ps-8), arbutin (Sigma Chemical Co, St. Louis, USA) or vitamin C (ascorbic acid, AA; ascorbic acid; Sigma Chemical Co, St. Louis, USA), added to the above cells, wherein arbutin and Vitamin C was used as a positive control group. After 72 hours of culture, 10 μL of 3-(4,5-dimethylazole-2)-2,5-diphenyltetrazolium bromide [3-(4,5-dimethylthiazol-2) was added to each well of cells. -yl)-2,5-diphenyltetrazolium bromide; MTT tetrazolium; 5 mg/mL], after 4 hours at 37 ° C, the supernatant was removed, and 100 μL of dimethyl sulfoxide (DMSO) was added. ;Sigma Chemical Co, St. Louis, USA) Dissolved MTT formazan (MTT formazan; product of MTT tetrazolium after reduction), after 10 minutes of shaking, measured its absorbance at a wavelength of 570 nm to calculate cell viability ( Cell viability), the results are shown in Table 1. The above-mentioned absorbance measurement can be performed according to, for example, the CellTiter 96 AQ manual (CellTiter 96 AQ, Promega, Madison. WI, USA), using, for example, a Multi-Detection Microplate Reader; Synergy ^TM 2, BioTek Instruments , Inc., USA).

Please refer to the first table, which shows the cell survival rate of the Hs68 cell line and the B16 cell line treated with the peony bark extract of different process stages for 72 hours according to an embodiment of the present invention. In the first table, the cell survival rate (%) of the untreated control group was taken as 100%, and the cell survival rate of the other treatment group was converted to a value obtained by comparison with the untreated control group. Each value is derived from at least 3 samples or more.

From the results of the first table, it is known that the first extract (Ps-1), the third extract (Ps-3), and the first extract (Ps-1) and the first extract of the peony bark extract in different extraction stages are compared with the cell survival rate of the control cells. Four extracts (Ps-4), peony bark extract (Part I, Ps-6), cell viability for Hs68 human skin fibroblasts and B16 mouse melanoma cell lines (101.1%, 100.4, respectively) %; 120.0%, 115.2%; 117.4%, 116.7%; 107.2%, 101.9%) were higher than the cell viability of the control cells (100.0%, 100.0%) and the cell survival rate of the arbutin-treated cells (80.1%, 74.7). %). Although arbutin has a good inhibitory activity against tyrosinase, the first extract (Ps-1), the third extract (Ps-3), and the fourth extract (Ps-) in the above extraction stage are compared with arbutin. 4) And the peony skin extract (Part 1; Ps-6) caused significantly less cytotoxicity to Hs68 cell line and B16 cell line than arbutin-treated cells. Further, according to observation by an optical microscope, the first extract (Ps-1), the third extract (Ps-3), the fifth extract (Ps-5), and the peony bark extract in the above extraction stage (Part 1; Ps-6) did not adversely affect the cell type and proliferation of Hs68 cell line and B16 cell line (results not shown).

Example 3: Evaluation of peony skin extract inhibiting tyrosinase activity

In this example, the B16 mouse melanoma cell line of Example 2 was used to further evaluate the activity of the peony skin extract to inhibit tyrosinase. In general, B16 cells were seeded at 1.0 x 10 ⁵ cells per well in a 24-well dish (1 mL volume per well) and then incubated for at least 24 hours at 37 ° C maintaining a humidity of 5% carbon dioxide. Thereafter, 1 μM α-melanocyte stimulating hormone (α-MSH; Sigma Chemical Co, St. Louis, USA) (control group) was used, or the α-MSH binding concentration was 50 μg/mL or 100. Μg/mL of intermediate extract (first extract, third extract, fourth extract, peony bark extract (part 1), peony bark extract (part 2), peony bark extract (third part) ), arbutin or vitamin C, for 72 hours at 37 ° C for B16 cells, and then the following determination.

1. Analysis of intracellular tyrosinase activity

After the B16 cells were treated for 72 hours at 37 ° C, the culture solution of each well was removed, and washed with a phosphate buffered saline (PBS) solution, for example, 900 μL of sodium phosphate buffer solution (sodium) was added. Phosphate buffer, 50 mM, containing 1% Triton X-100) (pH 6.8), frozen-thawed at -80 ° C for 30 minutes, then removed at 25 ° C for 25 minutes and at 37 Thaw at °C for 5 minutes to dissolve the cells. Thereafter, for example, 100 μL of L-dopa (L-DOPA) solution (10 mM) was added to each well of cells. After the treatment, and to the action of 37 ℃ 4 hours using a spectrophotometer, e.g. Synergy ^TM 2 multifunction machine microplate readings (Multi-Mode Microplate Reader; BioTek , USA), measured in the reaction mixture in each well at 475 nm The absorbance was compared with the control group (final concentration of DMSO 0.1%) and the positive control group (arbutin and vitamin C).

Please refer to FIG. 2A, which is a bar graph for evaluating the relative ratio of peony skin extracts in different extraction stages to inhibiting intracellular tyrosinase activity according to an embodiment of the present invention, wherein the horizontal axis is in the control group. , the first extract (Ps-1), the third extract (Ps-3), the fourth extract (Ps-4), the peony bark extract (Part I, Ps-6), and the positive control group (arbutin and Vitamin C), the vertical axis is the relative ratio of intracellular tyrosinase activity, and the long strip filled with diagonal lines indicates the first extract (Ps-1) and the third extract (Ps-3) at a concentration of 50 μg/mL. ), the fourth extract (Ps-4), the peony bark extract (Part I, Ps-6), arbutin and vitamin C were treated. Filling the long line of the cross line means using the first extract (Ps-1), the third extract (Ps-3), the fourth extract (Ps-4), and the peony bark extract at a concentration of 100 μg/mL. (Part 1, Ps-6) and the positive control group (arbutin and vitamin C) were processed.

As can be seen from the results of Figure 2A, the first extract (Ps-1) and the peony bark extract (Part I, Ps-6) were effective in inhibiting tyrosinase activity in B16 cells compared to the control group. , wherein the first extract (Ps-1) inhibits tyrosinase activity from 0% (50 μg/mL of the first extract) to 79.6% (100 μg/mL of the first extract), and The inhibition of tyrosinase activity by peony bark extract (Ph-6) was 31.1% (50 μg/mL of peony bark extract, part 1) to 65% (100 μg/mL of peony bark extract). (Part 1), the degree of inhibition and arbutin (50 μg/mL arbutin inhibited tyrosinase activity by 35.8%, and 10 μg/mL of arbutin inhibited tyrosinase activity by 62.5%) As well as vitamin C (the inhibition rate of tyrosinase activity of vitamin C at 50 μg/mL was 64.6%, and the inhibition rate of tyrosinase activity by 10 μg/mL of vitamin C was 87.0%). Therefore, the above results confirmed that the first extract of Example 1 and the peony bark extract were effective in inhibiting tyrosinase activity in melanocytes.

2. Analysis of intracellular dopaquinone content

After the B16 cells were treated for 72 hours at 37 ° C, the culture solution of each well was removed and rinsed with PBS solution, for example, 20 μL of sodium phosphate buffer solution (50 mM, containing 0.5% Triton X-100). (pH 6.9), frozen-thawed, frozen at -80 ° C for 30 minutes, then taken out and thawed at 25 ° C for 25 minutes and thawed at 37 ° C for 5 minutes to dissolve the cells. Next, 190 μL of the pre-warmed and freshly prepared substrate solution was added to each well of the cells, and the substrate solution may include 6.3 mM 3-methyl-2-benzothiazolinone hydrazone. ; MBTH) and a 1.1 mM dopa solution (formulated in 48 mM sodium phosphate buffer, pH 7.1) and added 4 volume percent (% v/v) of N,N' -dimethylformamide ( N , N'- dimethylformamide). After the above treatment and 4 hours at 37 ° C, the absorbance of the reaction mixture of each well at 508 nm was measured by a spectrophotometer, such as the above-mentioned multifunctional microplate reader, and compared with the control group (final concentration of DMSO). Comparing with 0.1%) and the positive control group (arbutin and vitamin C), the results are shown in Figure 2B. Since intracellular tyrosinase reacts with L-dopa solution to form dopaquinone, followed by cyclization to form DOPAchrome, while DOPAchrome and MBTH produce a pink product, thus having a strong absorbance at 508 nm.

Please refer to FIG. 2B, which is a bar graph for evaluating the relative ratio of the extract of the peony bark of different extraction stages to inhibiting the activity of intracellular dopa oxidase (DOPA oxidase) according to another embodiment of the present invention, wherein the horizontal axis In order, the control group, the first extract (Ps-1), the third extract (Ps-3), the fourth extract (Ps-4), the peony bark extract (Part I, Ps-6), and arbutin And vitamin C, the vertical axis is the relative ratio of intracellular dopa oxidase activity, and the long strip filled with oblique lines indicates the first extract (Ps-1) and the third extract (Ps-3) at a concentration of 50 μg/mL. ), the fourth extract (Ps-4), the peony bark extract (Part I, Ps-6), arbutin and vitamin C were treated. Filling the long line of the cross line means using the first extract (Ps-1), the third extract (Ps-3), the fourth extract (Ps-4), and the peony bark extract at a concentration of 100 μg/mL. (Part 1 , Ps-6), Arbutin and Vitamin C are processed.

As can be seen from the results of Fig. 2B, the first extract (Ps-1) and the peony bark extract (Part I, Ps-6) were effective in inhibiting dopa oxidase activity in B16 cells compared to the control group. , wherein the first extract (Ps-1) has an inhibition rate of dopa oxidase activity of 0% (50 μg/mL of the first extract) to 31.6% (100 μg/mL of the first extract), and The inhibition of dopa oxidase activity by peony bark extract (Ps-6) was 22.9% (50 μg/mL of peony bark extract, part 1) to 36.4% (100 μg/mL of peony bark extract). Things, the first part). Therefore, the above results confirmed that the first extract of Example 1 and the peony bark extract were effective for inhibiting dopa oxidase activity in melanocytes.

3. Analysis of extracellular melanin content

This assessment measures the amount of extracellular melanin. Briefly, B16 cells were seeded at 3.0 x 10 ⁵ cells per well in a 6-well plate (3 mL volume per well) and then incubated for at least 24 hours at 37 ° C maintaining a humidity of 5% carbon dioxide. Thereafter, dimethyl hydrazine (DMSO; Sigma Chemical Co, St. Louis, USA) (control), or α-MSH combined with an intermediate extract of 50 μg/mL or 100 μg/mL (first extract, Second extract, third extract, fourth extract), peony bark extract (part 1), peony bark extract (part 2), peony bark extract (part 3), arbutin or vitamin C ( AA), etc., to B16 cells for 72 hours at 37 ° C, wherein the cell culture medium DMEM does not contain phenol red. After the B16 cells were treated for 72 hours at 37 ° C, 100 μL of the cell culture solution per well was added to a 96-well plate, and the cell culture solution of each well was measured by a spectrophotometer, such as the above-mentioned multifunctional microplate reader. The absorbance at 405 nm was compared with the control group (final concentration of DMSO 0.1%) and the positive control group (arbutin and vitamin C).

Please refer to FIG. 2C, which is a bar graph for evaluating the relative ratio of the peony bark extracts for inhibiting melanin formation in different extraction stages according to still another embodiment of the present invention, wherein the horizontal axis is the control group and the first extraction. (Ps-1), third extract (Ps-3), fourth extract (Ps-4), peony bark extract (Part 1, Ps-6), arbutin and vitamin C, and the vertical axis is the inhibition of melanin The relative ratio of formation, the long strip filled with diagonal lines indicates the use of the first extract (Ps-1), the third extract (Ps-3), the fourth extract (Ps-4), and the peony at a concentration of 50 μg/mL. Skin extract (Part 1 , Ps-6), arbutin and vitamin C were processed. Filling the long line of the cross line means using the first extract (Ps-1), the third extract (Ps-3), the fourth extract (Ps-4), and the peony bark extract at a concentration of 100 μg/mL. (Part 1 , Ps-6), Arbutin and Vitamin C are processed.

From the results of Figure 2C, it can be seen that compared with the control group, the first extract (Ps-1) and the peony bark extract (Part I, Ps-6) can effectively reduce the formation of extracellular melanin in B16 cells, among which peony The skin extract (Part I, Ps-6) has a better inhibitory effect than the first extract (Ps-1). Therefore, the above results confirmed that the peony bark extract of Example 1 was effective in reducing the formation of extracellular melanin.

4. Assessment of related protein expression during melanogenesis

In this evaluation, the B16 cell line was seeded at 1.0 × 10 ⁴ cells per cell in a 96-well plate (cell density 1.0 × 10 ⁵ /mL, volume added per well was 100 μL), and 1 μM α-MSH was used. (control group), or the above intermediate extract, α-MSH combined with concentration of 100 μg/mL, peony bark extract (Part 1), peony bark extract (Part 2), peony bark extract (Part 3) and The positive control group (arbutin and vitamin C) was treated. After the B16 cells were treated for 72 hours at 37 ° C, the culture solution of each well was removed and rinsed twice with PBS solution, after which the cells were fixed with 4% paraformaldehyde per well. The permeabilization solution (containing 0.1% Triton X-100 in 0.1% sodium citrate) was used to increase the permeability of the cells. Subsequently, each well of B16 cells was blocked by adding 1% bovine serum albumin solution for one hour. Then, each well of B16 cells was reacted with 40 μL of freshly configured mouse IgG primary antibody for 45 minutes at 4 ° C, wherein the mouse IgG antibody used may include an anti-MC1R IgG antibody, an anti-MITF IgG antibody, and an anti-tyramine. Acidase IgG antibody, anti-TRP-1 IgG antibody, anti-TRP-2 IgG antibody (all of the above antibodies are purchased from Santa Cruz, CA), the antibody dilution ratio can be, for example, 1:300, and is soluble in 0.5% BSA. PBS (PBS-BSA) solution and 0.1% sodium azide (Sigma-Aldrich). Thereafter, B16 cells were further reacted with fluorescein isothiocyanate (FITC) secondary antibody (anti-mouse IgG-FITC) (purchased from Santa Cruz, CA) for 1 hour at 4 °C. The dilution ratio of the IgG-FITC antibody may be, for example, 1:500. The nucleus is then stained with a DNA stain (eg, 0.1 μg/mL Hoechst 33342 stain, Promega, USA).

Subsequently, each protein (excitation wavelength: 495 nm, divergent wavelength: 525 nm) and the nucleus (excitation wavelength: 346 nm, divergent wavelength:) are detected by an instrument such as the above-described multi-function microdisk reader. The fluorescence intensity at 460 nm) was quantified, and the percent fluorescence intensity (%) was calculated from FITC/Hoechst 33342. Each value is derived from at least 3 samples or more.

In addition, immunofluorescence and flow cytometer (for example, FACScan flow cytometer, such as Elite ESP, Beckman Coulter, Brea, CA), fluorescent microscope and WinMDI software analysis, such as Nikon Corporation of Japan, can also be used. A fluorescent microscope of the model TE2000-U was fabricated to observe the fluorescence distribution of each protein and cell nucleus. For the analysis of flow cytometry, 1×10 ⁵ cell/ml cells were cultured in a cell culture dish for 24 hours, and the above intermediate extract, peony bark extract (Part 1), and peony bark extract (Part 2) were added. ), the peony bark extract (the third part) and the positive control group (arbutin and vitamin C) were reacted for 72 hours, then the cells were beaten with trypsin (1 ×) in a 37 ° C, 5% CO ₂ incubator. The supernatant (collected together) was placed in a 15 ml centrifuge tube, centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. Then, the cells were fixed by adding 0.4% of paraformaldehyde. After reacting at 4 ° C for at least 30 minutes, the permeabilization solution (containing 0.1% Triton X-100 in 0.1% sodium citrate) was used to increase the permeability of the cells. Subsequently, after centrifugation at 4 ° C, 1200 rpm for 5 minutes, the supernatant was removed. Then, each tube of cells is added to the above-mentioned one-stage antibody, and after reacting for 3 hours at room temperature, centrifugation at 1200 rpm for 5 minutes, the supernatant is decanted, and the above secondary antibody is added to each tube at room temperature. After 2 hours in the dark, the assay was performed using a flow cytometer.

Please refer to FIG. 3A and FIG. 3B, respectively, for evaluating the first extract (Ps-1; FIG. 3A) and the peony bark extract according to an embodiment of the present invention (Part I, Ps-6; FIG. 3B) a bar graph showing the expression of related proteins in the formation of melanin, wherein the horizontal axis is the fluorescence intensity of DNA content, MC1R, MITF, tyrosinase (TYR), TRP-2, and TRP-1, and the vertical axis is Fluorescence intensity of protein (%). Blank bars indicate control cells. The strips filled with diagonal lines indicate cells treated with a first extract (Ps-1; Figure 3A) at a concentration of 100 μg/mL or an extract of Cortex Moutan (Part 1, Ps-6; Figure 3B).

From the results of Fig. 3A and Fig. 3B, it can be seen that the DNA content of the first extract (Ps-1) and the peony bark extract (Part I, Ps-6) did not change significantly compared to the control group, There was no significant effect on the proliferation of B16 cells. Secondly, the above results also confirmed that the first extract (Ps-1) of Example 1 and the peony bark extract (Part I, Ps-6) can regulate MC1R, MITF, tyrosinase and TRP- by down-regulation. The protein expression of 1 reduces the formation of melanin.

Example 4: Analysis of the components of the peony bark extract

This example further analyzes the high performance liquid chromatography (HPLC) fingerprint profile of the peony bark extract obtained in Example 1. In this embodiment, the HPLC system used may be a high performance liquid chromatography/diode array detector system such as the Agilent 1100 series HPLC-DAD system (Agilent Technologies, Inc. CA, USA). ). The chromatographic conditions are exemplified as follows: the chromatography column can be, for example, an Agilent Eclipse XDB-C18 column (5 μm, 2.1 mm × 150 mm) or otherwise equivalent. The mobile phase consisted of methanol (solvent A) and reagent water (solvent B) containing 0.1% formic acid. Solvent gradient program setting: 10% to 80% methanol is injected from 0 minutes to 35 minutes, 80% to 100% methanol is injected from 35 minutes to 42 minutes, and 100% methanol is injected from 42 minutes to 65 minutes. The flow rate can be controlled, for example, at 0.59 mL/min. The chromatographic temperature can be maintained at 25 °C. The sample of the peony bark extract can be first dissolved in methanol and filtered through a 0.45 μm filter, and then subjected to shaking extraction using an ultrasonic oscillator (for example, Branson 5210E-MTH ultrasonic oscillator, Branson Ultrasonics Corporation, CT, USA). An HPLC fingerprint analysis was performed in which the sample volume injected into the HPLC-DAD system was, for example, 10 μL. The wavelength of the detected UV spectrum can be, for example, 200 nm to 400 nm. In addition, a technique using liquid chromatography-tandem mass spectrometry (LC-MS-MS), such as an Esquire HCT LC-MS-MS system with an electrospray ionization source (Bruker Daltinics, MA, USA), mass spectrometric analysis of the peony bark extract. The results obtained are shown in Figure 4.

Referring to Figure 4, there is shown an HPLC fingerprint of a peony bark extract according to an embodiment of the present invention, wherein the fourth image is obtained by analyzing 18 batches of peony bark extract, and the vertical axis is the absorbance unit (absorbance unit; au ), the horizontal axis represents the retention time (unit: minute). As can be seen from the results of FIG. 4, the HPLC fingerprint of the peony bark extract obtained in Example 1 has 18 peaks, wherein the third peak is an internal standard, such as peony saponin E, for use in performing. Quantitative analysis. After analysis, in one illustration, the peony bark extract contains at least 20% by weight to 30% by weight of puerarin E, 5 to 15% by weight of puerarin B, and 5 to 10% by weight of peony phenol. In another illustration, the peony bark extract comprises at least 26.4 weight percent puerarin E, 9.2 weight percent peony saponin B, and 6.2 weight percent peony phenol.

It should be noted that the present invention is exemplified by specific process conditions, specific cell strains, specific analytical methods or specific instruments, and illustrates the application of the peony bark extract of the present invention and a method for producing the same, but any of the technical fields of the present invention. It is to be understood by those skilled in the art that the present invention is not limited thereto, and that the peony bark extract of the present invention can be subjected to other process conditions, other cell strains, other analytical methods or instruments without departing from the spirit and scope of the present invention. In addition, when the peony bark extract and the first extract obtained by the present invention are applied to a cosmetic composition, the form thereof may include, but not limited to, a liquid, an emulsion, an ointment, a powder, a whitening agent, a spotting agent, a freckle agent, or the like. Any combination of cosmetics. Furthermore, when the peony bark extract obtained by the present invention is applied to a food additive, its form may include, but is not limited to, a nutraceutical or a health food.

It can be seen from the above embodiments of the present invention that the peony bark extract of the present invention and the method for producing the same have the advantages that the peony bark extract can be effectively obtained by using at least one extraction step and the gel chromatography step to provide inhibition of melanin production activity. It also removes components that are toxic to cells. Since the partitioning step is carried out by the method of the present invention, delamination is easy, foaming is not easy, and the processing time is shortened, so that the obtained peony bark extract can be applied to a cosmetic composition and/or a food additive.

While the invention has been described above in terms of several embodiments, it is not intended to limit the scope of the invention, and the invention may be practiced in various embodiments without departing from the spirit and scope of the invention. The scope of protection of the present invention is defined by the scope of the appended claims.

101. . . Peony skin material

103. . . First extract

105. . . Second extract

107. . . Third extract

109. . . Fourth extract

111. . . Fifth extract

113. . . Peony peel extract

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing the process of extracting a peony bark according to an embodiment of the present invention.

Figure 2A is a bar graph showing the relative ratio of peony bark extracts at different extraction stages to inhibition of intracellular tyrosinase activity, in accordance with one embodiment of the present invention.

Figure 2B is a bar graph showing the relative ratio of peony bark extracts at different extraction stages to inhibition of intracellular dopa oxidase activity in accordance with another embodiment of the present invention.

Figure 2C is a bar graph showing the relative ratio of peony bark extracts for inhibiting melanin formation at different stages of extraction in accordance with yet another embodiment of the present invention.

Figure 3A is a bar graph depicting the performance of related proteins in the first extract upon melanin production, in accordance with an embodiment of the present invention.

Figure 3B is a bar graph showing the expression of related proteins in the production of melanin by the peony bark extract according to another embodiment of the present invention.

Figure 4 is a graph showing the HPLC fingerprint of a peony bark extract according to an embodiment of the present invention.

101. . . Peony skin material

103. . . First extract

105. . . Second extract

107. . . Third extract

109. . . Fourth extract

111. . . Fifth extract

113. . . Peony peel extract

Claims (8)

A method for producing a peony bark extract, comprising: performing a first extraction step of extracting a peony bark material by using a first organic solution to obtain a first extract, wherein the first organic solution is an ethanol solution And performing a second extraction step of extracting the first extract by using a second organic solution to divide a first organic phase and an aqueous phase, wherein the second organic solution is an ethyl acetate/water solution, The first organic phase is an ethyl acetate phase, the first organic phase has a second extract, and the aqueous phase has a third extract; and a third extraction step is performed, which is extracted by a third organic solution. a second extract to divide a second organic phase and a third organic phase, wherein the third organic solution is a n-hexane/methanol solution, and the second organic phase has a fourth extract, the third organic phase a methanol phase, and the third organic phase has a fifth extract; and performing a gel chromatography step to obtain a peony bark extract having anti-melanogenic activity from the fifth extract, wherein The paeonol extract contains at least 20% by weight to 30% by weight of mudanpioside E, 5 to 15% by weight of peponin B, and 5 to 10% by weight of peony phenol E (suffruticoside E) And the peony bark extract is subjected to a reverse phase thin layer chromatography analysis with a volume ratio of 8:2 methanol and water plus 10 drops of acetic acid as a developing solution, and 0.462 is obtained according to formula (I). One of the 0.513 retention factor (Rf) values: The method for producing a peony bark extract according to claim 1, wherein the first organic solution is a 95 volume percent ethanol solution, and the second organic solution is a 3:1 volume ratio ethyl acetate/water solution. And the third organic solution is a 1:1 molar ratio of hexane/90 volume percent methanol solution. The method for producing a peony bark extract according to the first aspect of the invention, wherein the gel chromatography step is carried out by using a Sephadex LH-20 gel chromatography column. The method for producing a peony bark extract according to the above aspect of the invention, wherein the first extract has an activity of inhibiting melanin production. A peony bark extract, characterized in that a peony bark material is obtained according to any one of the first to fourth aspects of the patent application, a first extract or the peony bark extract, and the peony bark extract A reverse phase thin layer chromatography analysis was carried out with a volume ratio of 8:2 methanol and water as a developing solution, and a development retardation (Rf) value of 0.462 to 0.513 was obtained according to the formula (I): Wherein the peony bark extract or the first extract has an activity of inhibiting melanin production. The peony bark extract according to claim 5, wherein the peony bark extract comprises at least 20% by weight to 30% by weight of puerarin E, 5 to 15% by weight of peony saponin B and 5 Percent by weight to 10% by weight of peony P. A cosmetic composition characterized in that the cosmetic composition comprises at least one of a peony bark extract or a first extract according to any one of the methods of claim 1 to 4, and the peony bark extract Or the first extract has an activity of inhibiting melanin production, wherein the cosmetic composition is a cosmetic of a liquid, an emulsion, an ointment, a powder, a whitening agent, a spotting agent, and a freckle agent. A food additive, characterized in that the food additive comprises at least one peony bark extract or a first extract obtained according to any one of the methods of claim 1 to 4, and the peony bark extract The substance or the first extract has an activity of inhibiting melanin production, wherein the food additive is a nutrient or health food having an activity of inhibiting melanogenesis.