TWI480047B - Extract of peony root cortex, method of preparing the same and application thereof - Google Patents

Description

Cortex peel extract, its manufacturing method and application

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 a plurality of biological activities which combine DNA repair, inhibition of melanin production and oxidation resistance, 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.

Radiation and ultraviolet radiation on the organism can cause photodamage, causing edema, erythema, sunburn, apoptosis, and suppression of immune responses, causing photodamage of DNA in skin cells. DNA photodamage is the primary signaling molecule in the initiation of cell repair systems. When DNA produces endogenous DNA double strand breaks (DSB) or exogenous DNA single strand breaks (SSB), it changes the DNA structure and leads to a cascade of physiological mechanisms that allow cells to The cycle is stagnant and the DNA repair mechanism is initiated.

The above-mentioned endogenous DNA double-strand break (DSB) mainly comes from methylating species (MS) and reactive oxygen species (ROS) formed in normal physiological reactions of cells, which trigger free radicals to attack cells and produce cells. Toxicity, inability to repair, damage to DNA double-strand breaks, erroneous repair, triggering gene mutations and carcinogenicity. Repair is mainly performed by a mechanism of non-homologous end joining (NHEJ) and homologous recombination (HR).

The above-mentioned exogenous DNA single-strand break (SSB) is mostly caused by environmental external ultraviolet, radiation, air, chemical substances and other external factors, which will induce DNA damage, affect cell cycle arrest, DNA transcription or translation to produce mutation or breakage damage. , by template switching, translation synthesis (TLS), mismatch repair (MMR), base-excision repair (BER), nucleotide removal repair (nucleotide- Excision repair; NER) to repair damage to various DNA pairing errors, mutations or single strand breaks.

Secondly, ultraviolet light can induce skin tissue to produce melanin and darken the skin color, while melanin production is combined with tyrosinase activity and 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 whitening ingredient that is safe and effective in inhibiting melanin production.

In addition, skin aging and disease are also closely related to free radicals. In addition to the intrinsic aging caused by ageing, the skin is still adversely affected by the environment, accelerating or premature aging. These causes of exogenous aging, such as long-term exposure to sunlight or high-energy radiation, poor diet, tissue damage, microbial infections, excessive stress, excessive fatigue, or excessive oil intake, also increase free radicals and melanin. Production or loss of metabolic factors. Reactive oxygen species (ROS) in the human body, such as superoxide radicals ( ^‧ O ₂ ^- ) and hydrogen peroxide (H ₂ O ₂ ), can be converted into highly active hydrogen and oxygen free in the presence of metal ions. Base ( ^‧ OH), which causes the most direct damage to human tissue. Excessive free radicals accumulate in the skin, causing damage to the DNA in the epidermis, dermal connective tissue, lipid peroxidation, and denaturation of various collagen proteins, causing connective tissue to lose its original function, resulting in skin aging phenomena such as wrinkles and loss. Elastic and dry.

In view of the above, it is urgent to propose a plant material of a plurality of organisms for DNA repair, inhibition of melanin production, and oxidation resistance, in order to provide a cosmetic composition and/or a food additive.

Therefore, one aspect of the present invention provides a peony bark extract, wherein the peony bark extract is an acetophenone compound and has a plurality of biological activities including DNA repair, inhibition of melanin production and oxidation resistance, and Further applied to cosmetic compositions and/or food additives.

Next, another aspect of the present invention provides a method for producing a peony bark extract, which comprises obtaining a peony bark from a peony bark material by using at least one partitioning extraction step and at least one column separation step using a plurality of organic solutions. The extract, the peony bark extract is an acetophenone compound and has multiple biological activities including DNA repair, inhibition of melanin production, and oxidation resistance.

According to the above aspect of the present invention, there is proposed a peony bark extract which is 2,5-dihydroxy-4-methylacetophenone (2,5-dihydroxy- as shown in the formula (I). 4-methylacetophenone): Moreover, this peony bark extract has multiple biological activities including DNA repair, inhibition of melanin production and oxidation resistance.

According to another aspect of the present invention, a cosmetic composition is provided, characterized in that the cosmetic composition comprises at least an effective amount of the above-mentioned peony bark extract to provide a plurality of biological activities for DNA repair, inhibition of melanin production and oxidation resistance. .

According to still another aspect of the present invention, a food additive is provided, characterized in that the cosmetic composition comprises at least an effective amount of the above-mentioned peony bark extract to provide a plurality of biological activities for DNA repair, inhibition of melanin production and oxidation resistance. .

According to still another aspect of the present invention, a method of producing a peony bark extract is provided. In one embodiment, the peony bark material is first subjected to at least one crude extraction step using a first organic solution to obtain a crude extract. Next, the crude extract is subjected to a first partition extraction step using a second organic mixed solution to divide the first organic phase and the second organic phase, wherein the first organic phase comprises the first extract. Then, the first extract is subjected to a second partition extraction step using a third organic aqueous solution to divide the third organic phase and the aqueous phase, wherein the third organic phase comprises the second extract. Thereafter, the first column separation step is performed on the third organic phase to sequentially obtain the first eluate and the second dissolving solution according to the molecular weight. Subsequently, the second separation column is subjected to a second column separation step to separate the peony bark extract, which is a 2,5-dihydroxy-4-methylacetophenone as shown in the above formula (I).

According to an embodiment of the invention, the first organic solution may be, for example, an ethanol solution. In one example, after the at least one crude extraction step, at least a concentration step is performed to remove the first organic solution of the crude extract.

The second organic solution may be, for example, a n-hexane/methanol solution, and the third organic solution may be, for example, an ethyl acetate/water solution, and the first organic phase may be, for example, a methanol phase. In one example, after the first partitioning extraction step described above, at least the methanol from the first organic phase is removed to obtain a first extract.

According to an embodiment of the invention, the third organic solution may be, for example, Ethyl acetate/water solution, and the third organic phase described above may be, for example, an ethyl acetate phase.

According to an embodiment of the invention, the first column separation step is performed by using the first column and the first extract. In one example, the first column can be, for example, a Sephadex LH-20 gel chromatography column, and the first extract can be, for example, methanol.

According to an embodiment of the invention, after the first partitioning extraction step, at least the methanol of the first organic phase is removed to obtain the first extract.

According to an embodiment of the present invention, the second column separation step is performed by using the second column and sequentially using the second extract liquid, the third extract liquid and the fourth extract liquid to sequentially separate the first step. a third eluent, a fourth eluate and a fifth eluate, and the fourth eluate comprises the above-mentioned peony bark extract. In one example, the second column can be, for example, a mesh size of 70 mesh to 230 mesh Silica gel 60 column, and the second extract is n-hexane/ethyl acetate, third. The extract was ethyl acetate and the fourth extract was methanol.

According to an embodiment of the present invention, the above-mentioned peony bark extract or crude extract combines a plurality of biological activities of DNA repair, inhibition of melanin production and oxidation resistance.

The peony bark extract of the present invention and the method for producing the same are provided by at least one partitioning extraction step and at least one column separation step to obtain a peony bark extract, wherein the peony bark extract is of the formula (I) Acetophenone compounds. The acetophenone compound has both biological effects of DNA repair, inhibition of melanin production, and antioxidant activity, and can be 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 partitioning extraction step and at least one column separation step to obtain biological activities such as DNA repair, inhibition of melanin production, and oxidation resistance. Mudan peel extract.

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. After the peony bark used in the present invention is confirmed by Dr. Lin Hanqin, the acetophenone compound-like peony bark extract is obtained by the method disclosed in the present invention.

Please refer to FIG. 1 , which is a flow chart showing a method for manufacturing a peony bark extract according to an embodiment of the present invention. First, the method 100 of producing a peony bark extract can provide a peony bark material as shown in step 101. In an illustration, the peony bark material may be a dry powder, which may be broken at room temperature or slightly below room temperature, and the peony bark is broken by a pulverizer to form a dry powder of peony bark.

Next, as shown in step 103, the aforementioned peony skin material 101 is subjected to a crude extraction step using a first organic solution, such as an ethanol solution, to obtain a crude extract. In one example, the concentration of the aforementioned ethanol solution may be, for example, at least 95 volume percent [% (v/v)].

It should be further noted that any one of ordinary skill in the art to which the present invention pertains should be able to understand that the solid-liquid separation process may be further performed by using the gauze, filter paper, centrifugation or the like. After one treatment or several times of repetition, the residue of the peony skin material 101 is removed, and the resulting filtrate is combined.

After the solid-liquid separation treatment, the filtrate may be selectively subjected to a concentration step, and the ethanol solution of the filtrate may be removed by a conventional method such as a vacuum concentration method or a vacuum concentration method to obtain a crude extract.

Thereafter, at least one partitioning extraction step and at least one column separation step are utilized to obtain a peony bark extract. As used herein, "at least one partitioning extraction step" refers to the partition extraction of peony bark material using an organic solvent system of different polarity.

In an embodiment, as shown in step 105, the first extraction extraction step may be performed on the crude extract by using a second organic mixed solution, such as a n-hexane/methanol solution, to partition. a first organic phase and a second organic phase, wherein the first organic phase is a methanol phase and the first organic phase comprises a first extract. In one example, the methanol of the second organic mixed solution is a 95% (v/v) methanol solution, and the volume ratio of n-hexane to methanol of the second organic mixed solution may be, for example, 1:1.

After step 105, the methanol of the first organic phase described above may be selectively removed to obtain a first extract. As for the manner of removing the methanol, the same concentration step as the above-mentioned crude extract can be used or other conventional methods, and details are not described herein.

Then, as shown in step 107, the first extract is subjected to a second partition extraction step using a third organic aqueous solution, such as an ethyl acetate/water solution, to fractionate a third organic phase and an aqueous phase, wherein the third organic phase It is an ethyl acetate phase and the third organic phase contains a second extract. In an example, the volume ratio of ethyl acetate to water of the third organic aqueous solution may be, for example, 3:1.

Thereafter, as shown in step 109, a first column separation step is performed on the third organic phase. In an example, the first column separation step is to pump the third organic phase through the first column and with the first extract, wherein the first extract may be, for example, methanol, and the first column may be For the commercially available molecular sieve chromatography column, according to the difference in molecular weight, the first eluent (large molecular weight) and the second eluent (smaller molecular weight) can be sequentially obtained, wherein the second dissolving liquid contains the second extraction Things.

Subsequently, as shown in step 111, the second separation column is subjected to a second column separation step to separate the peony bark extract. In an example, the second column separation step is a second elution solution passing through a second column, such as a commercially available silicone column, and sequentially using a second extract having a different polarity, a third extract, and a third The fourth extract is for example, the second extract may be, for example, n-hexane/ethyl acetate (smaller polarity), and the third extract may be, for example, ethyl acetate (polar centered), the fourth extract. For example, it may be methanol (more polar), and the volume ratio of n-hexane to ethyl acetate of the second extract may be, for example, 1:3. The third eluent (smaller polarity), the fourth eluent (polar centered) and the fifth eluent (larger polarity) can be sequentially separated by the different polarity of the extract, wherein the fourth eluate contains An extract of peony bark, wherein the peony bark extract is an acetophenone compound, in an example, 2,5-dihydroxy-4-methylacetophenone as shown in formula (I):

In one embodiment, after the second column separation step, a recrystallization step is further selectively performed to obtain crystals of the peony bark extract from the fourth solution described above using methanol.

The 2,5-dihydroxy-4-methylacetophenone obtained above is known to inhibit platelet aggregation and inhibit the formation of thromboxane (Thromboxane A2, TXA2) and prostaglandin D2 (Prostaglandin D2) by Arachidonic acid. The product, regulates vasodilation to achieve sweating and promotes body heat dissipation. However, the present invention further confirmed by in vitro tests that the 2,5-dihydroxy-4-methylacetophenone obtained above has the biological activity of DNA repair, inhibition of melanin production and oxidation resistance, and thus can be applied to cosmetic compositions and / or food additives.

The biological activity of "DNA repair" as used herein means that the aforementioned peony peel extract can effectively promote DNA repair in cells, reduce damage caused by UV irradiation, and thereby improve cell survival rate. In one example of the present invention, the biological activity of the aforementioned DNA repair can be evaluated by cell viability after UV irradiation, comet assay, and the like.

The biological activity referred to herein as "inhibiting melanin production" means that the aforementioned peony bark extract can effectively inhibit the activity of tyrosinase and further reduce The production of dopaquinone and melanin to achieve skin whitening effect. 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. Therefore, in one example of the present invention, the aforementioned biological activity for inhibiting melanin production can be inhibited by the activity inhibition rate of tyrosinase, extracellular melanin content, intracellular melanin content, and activity of DOPA oxidase. Rate evaluation.

The "antioxidant" biological activity referred to herein means that the aforementioned peony bark extract can be inhibited. In one example of the present invention, the aforementioned antioxidative biological activity can be formed by hydroxyl radicals formed by photolysis of hydrogen peroxide (H ₂ O ₂ ) by UV irradiation, thereby simulating the damage of DNA by free radicals. Evaluate.

It is worth mentioning that the peony bark extract of the present invention is subjected to at least one partitioning extraction step in the order of solvent polarity from high to low, and then extracted by the different column chromatography and extraction to obtain the formula (I). The peony bark extract. Since the method of the present invention can find a component which is better and more effective for DNA repair, inhibition of melanin production and oxidation resistance in the peony bark, it can be applied to a cosmetic composition and/or a food additive.

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 uses several embodiments to illustrate the application of the present invention, but it is not The present invention is intended to be limited to those skilled in the art, and various modifications and changes can be made without departing from the spirit and scope of the invention.

Example 1: Preparation of Cortex Mouth Extract

This example produces a peony bark extract using the method 100 of Figure 1. First, as shown in step 101, a crude extraction step is carried out by using a pulverizer to break up about 10.0 kg of peony bark material at room temperature or slightly below room temperature.

Next, as shown in step 103, a crude extraction step is performed, after the pulverized peony bark material is immersed in a 95 volume percent ethanol solution, and subjected to a solid-liquid separation step, which is treated by gauze, filter paper, centrifugation or the like. After one or several iterations, the residue of the peony bar material 101 was removed and the resulting filtrate was combined. After the solid-liquid separation treatment, the ethanol solution of the above filtrate was removed by a reduced pressure concentration method to obtain a crude extract of about 1.48 kg of peony bark.

Thereafter, as shown in step 105, the crude extract is subjected to a first partition extraction step using a volume ratio of 1:1 n-hexane/95% (v/v) methanol solution to fractionate 95% (v/v) methanol. Phase (about 1.10 g) and n-hexane phase (about 330.0 g), wherein the 95% (v/v) methanol phase comprises the first extract.

After step 105, the aforementioned 95% (v/v) methanol phase methanol may be selectively removed using the same concentration step as described above to obtain the crude extract or other conventional means to obtain the first extract. As for the above-mentioned concentration step or other conventional methods, as described above, it is no longer ambiguous here.

Then, as shown in step 107, the first extract is subjected to a second partition extraction step using an ethyl acetate/water solution having a volume ratio of 3:1 to fractionate the ethyl acetate phase (about 38.0 g) and the aqueous phase ( About 1100.0g), of which acetic acid The ethyl ester phase contains a second extract.

Thereafter, as shown in step 109, the first column separation step is performed on the ethyl acetate phase to pass the ethyl acetate phase through a commercially available molecular sieve column, such as a glucose gel chromatography column (Sephadex LH-20). </ br> </ br> </ br> Smaller) wherein the second eluent contains the second extract described above.

Subsequently, as shown in step 111, the second separation column is subjected to a second column separation step to pass the second solution to a column of silica gel 60 (silica gel 60) having a particle size of 70, for example, to a mesh size of (Jingming Chemical Co., Ltd., ACME01-000000-40NL, India), and using 1:3 by volume of n-hexane/ethyl acetate (smaller polarity), ethyl acetate (polar centered) and methanol (polarity) Large) For the extraction, each 250 ml (mL) of the solution is collected into a bottle according to the order of collection. The obtained eluent is subjected to a thin layer chromatography of tannin extract (the developing solution is n-hexane/ethyl acetate (1:4)), and the detector is an ultraviolet light or an iodine reagent detecting component, and the same component of the elution solution is combined. The three parts can be separated in sequence, which are the third eluent, the fourth dissolving solution and the fifth dissolving solution. The fourth eluent contains a peony bark extract and is recrystallized from methanol to obtain a compound 2,5-dihydroxy-4-methylacetophenone having a structure represented by the formula (I):

The physicochemical properties of the 2,5-dihydroxy-4-methylacetophenone obtained above were as follows: molecular weight (MW) was 166; melting point was 141 ° C to 142 ° C; UV (MeOH) λ _max (log ε): 211 (4.07), 232 (4.16), 265 (3.97), 359 (3.64) nm; IR (KBr) v _max : 3330, 1630 cm ^-1 ; ¹ H-NMR (δ mult. ( J in Hz ), CDCl ₃ ): H-9 (2.25, s), H-8 (2.53, s), H-3 (6.75, s), H-6 (7.08, s); ¹³ C-NMR (δ, CDCl ₃ ): C-9 (16.7), C-8 (26.6), C-6 (115.2), C-1 (118.0), C-3 (121.1), C-4 (140.1), C-2 (145.3) ), C-5 (156.6), C-7 (203.5); EI-MS m/z (rel.int.%): 166 ([M] ⁺ , 68), 151 (100).

The above 2,5-dihydroxy-4-methylacetophenone was dissolved in dimethyl sulfoxide (DMSO), and was placed at a concentration of 100 mg/mL, and left at room temperature for use.

Example 2: Evaluation of cytotoxicity of peony bark extract

In this example, the first test of the peony bark extract 2,5-dihydroxy-4-methylacetophenone of Example 1 was cytotoxic to skin cells, melanocytes or hepatocytes. Skin cells suitable for testing can be, for example, human skin keratinocytes (HaCaT; provided by Dr. Xu Hanming, National Chengchi University School of Medicine) and human skin fibroblast strain (Hs68; BCRC number: 60038), which can be used for testing melanocytes. Mouse melanin Tumor cell line (B16; ATCC number: CRL-6322), the hepatocytes for testing can be, for example, normal rat liver cells (BNL CL.2; ATCC TIB-73), wherein ATCC is a typical American species center (American Abbreviation for Type Culture Collection; ATCC, BCRC is the abbreviation of Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan.

The above cell lines are cultured in a cell culture medium using aseptic techniques. The cell culture medium of the HaCaT cell strain is Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island, NY) and an additional 10 volume percent of fetal bovine serum (FBS; Hazelton Product, Denver, PA, USA) and 1 mM sodium pyruvate. The cell culture medium of B16 cell line, BNL CL.2 cell line and Hs68 cell line was DMEM (Gibco, Grand Island, NY) and 10% (v/v) FBS (fetal bovine serum; Hazelton Product, Denver, PA, USA).

The cells of the HaCaT cell line, the B16 cell line, the BNL CL.2 cell line, and the Hs68 cell line were cultured in a 5% CO ₂ , 95% air, and a cell culture incubator at 37 ° C depending on the cell condition. The culture medium was changed approximately every 2 to 3 days. When the cell has a confluence of about 80% to about 100% in the culture dish, the culture solution is removed and washed with double (1×) phosphate buffered saline (PBS), and then utilized. Trypsin (1×) allowed the cells to form a single cell suspension, which was then subcultured or tested.

At this time, the examples were evaluated for cell viability using the MTT method. In other words, the cells of the HaCaT cell line, the B16 cell line, the BNL CL.2 cell line, and the Hs68 cell line were planted in a well of a 96-well plate at a cell density of 1.0×10 ⁵ /mL, and then at 37 ° C. Incubate for at least 24 hours while maintaining a humidity of 5% carbon dioxide. Thereafter, different concentrations (0 to 100 μM) of the peony bark extract of Example 1, 2,5-dihydroxy-4-methylacetophenone or vitamin C (ascorbic acid, AA; ascorbic acid; Sigma Chemical Co, St.) Louis, USA), respectively, was added to the above cells, wherein 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 result is shown in Fig. 2. 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 FIGS. 2A to 2F, which show HaCaT cell strain, B16 cell strain, BNL CL.2 cell strain and Hs68 cell strain according to several embodiments of the present invention, and the peony bark extract or vitamin of Example 1. A graph of cell viability after 72 hours of C action. Figures 2A to 2F show the cell survival rate (%) of the untreated control group as 100%, and others The cell survival rate of the treatment group was compared with that of the untreated control group, and the value obtained by the conversion, wherein the figure number ● represents the cell survival curve of the HaCaT cell line, and the figure number ▲ represents the cell survival rate curve of the B16 cell line. , Figure No. ◆ represents the cell viability curve of BNL CL.2 cell line, Figure No. ■ represents the cell viability curve of Hs68 cell line, and figure No. ☆ represents the addition of Peony peel extract of Example 1 2,5- The cell viability curve of dihydroxy-4-methylacetophenone, and the figure ▽ represents the cell viability curve of vitamin C addition. Each value is derived from at least 3 samples or more.

From the results of Fig. 2A to Fig. 2F, it was found that a high concentration (100 μM) of peony bark extract was added to HaCaT cell line, B16 cell line, BNL CL.2 cell line and Hs68 cell line to affect the degree of cell growth. Less, the cell survival rate is still maintained above about 70%, so the peony bark extract of Example 1 is not cytotoxic to the above cell lines at the added concentration of up to 100 μM. Next, from the results of the 2C and 2E, it is understood that the higher the concentration of the peony bark extract of the first embodiment added to the HaCaT cell line and the B16 cell line, the higher the cell survival rate of the two cells. In contrast, as can be seen from the results of the 2D graph, the cell survival rate of the high concentration (100 μM) of vitamin C in the BNL CL.2 cell line is only about 55%, and it is presumed that the vitamin C is added at a higher concentration, affecting the cells. The pH of the culture medium of the strain further inhibits the growth of the cells.

Example 3: Assessing the ability of the peony bark extract to protect skin cells

This example further evaluated the protective ability of skin cells after UVB irradiation using the peony bark extract of Example 1.

1. Establish a suitable dose of UVB irradiation

First, HaCaT cell line and Hs68 cell line were seeded in a 96-well plate at a cell concentration of 1.5 × 10 ⁵ /mL for 24 hours, and the supernatant was removed, rinsed with PBS, and drained. Next, after irradiating the above cells with UVB of different irradiation energies (0 to 1000 J/m ² ), the serum-free fresh medium was immediately added to the cells of each well of the 96-well plate for 4 hours. Then, the cell survival rate was evaluated by the same MTT method as in Example 2, and the results are shown in Fig. 3.

Please refer to FIG. 3, which is a graph showing cell survival rate of HaCaT cell line and Hs68 cell line after UVB irradiation with different energies according to an embodiment of the present invention, wherein the horizontal axis is the irradiation energy of UVB (J/m ^{2 ).} ), the vertical axis is the cell survival rate (%).

From the results of Fig. 3, on the whole, HaCaT cells are more resistant to ultraviolet rays. Next, after the HaCaT cell line and the Hs68 cell line were irradiated with UVB of an irradiation energy of 10, 20 J/m ² , the cell survival rate of the above two kinds of cells was reduced to about 80%. However, after the UVB effect of the irradiation energy of 50 to 1000 J/m ² , the cell survival rate decreased by 70% as the irradiation energy increased. In particular, after the irradiation energy of 150 J/m ² and the UVB of 1000 J/m ² , the difference in cell viability of the above two kinds of cells was more remarkable.

Therefore, the following selection of UVB irradiation energy (for survival ≧70%) for HaCaT cell line and Hs68 cell line is, for example, 10 J/m ² , 20 J/m ² or 50 J/m ² in the presence of Or in the absence of the peony bark extract, the ability of the peony bark extract to protect the skin cells after UVB irradiation is evaluated based on the ability to protect the DNA and promote DNA replication.

2. Assessment (I) - cell viability

First, the HaCaT cell line and the Hs68 cell line were seeded in a 96-well plate at a cell concentration of 1.5 × 10 ⁵ /mL for 24 hours, and then the cell supernatant was removed, and the serum-free fresh medium was replaced and placed in the cell. Culture in an incubator. After 4 hours of culture, the supernatant was removed, rinsed with PBS, and drained. Next, after UVB irradiation with different irradiation energies (10, 20, 50 J/m ² ) or no irradiation (ie, control group) of the above cells, immediately add serum-free fresh medium to each well of the 96-well plate to continue. Cultured for 4 hours, wherein the serum-free medium was not added (ie, UVB control group, only UVB was irradiated) or 10 μg/mL or 20 μg/mL of the first example of the peony bark extract (ie, experimental group, irradiation) Add peony bark extract after UVB). Then, cell survival rate was evaluated by the same MTT method as in Example 2, and changes in cell type were observed in an optical microscope imaging system, and the results of cell viability were as shown in Figs. 4A and 4B.

Please refer to FIG. 4A and FIG. 4B, which are graphs showing the cell viability of the peony bark extract of the HaCaT cell line and the Hs68 cell line after UVB irradiation according to Example 1 of several embodiments of the present invention, wherein On the horizontal axis, different doses of peony bark extract were used for different UVB irradiation energies (J/m ² ), and the vertical axis was cell viability (%).

From the results of Figs. 4A and 4B, it was found that with the same UVB irradiation energy, as the concentration of the effect of the peony bark extract of Example 1 was increased, the cell survival rate of the HaCaT cell line and the Hs68 cell line was significantly increased as a whole.

Secondly, in the UVB control group (UVB only), the cell survival rate of HaCaT cell line increased slightly with the increase of UVB irradiation energy, but Hs68 The cell line did not exhibit a dose response as the UVB irradiation energy increased.

Furthermore, in the experimental group (50 μM or 100 μM of the peony bark extract of Example 1 after UVB irradiation), the HaCaT cells after UVB irradiation were significantly reduced as the concentration of the peony bark extract of Example 1 was increased. The phototoxic reaction caused by it. Taking UVB irradiation energy 10 J/m ² as an example, compared with the experimental group irradiated only by UVB (UVB irradiation energy is 10 J/m ² , the peony bark extract of Example 1 is 0 μM), with the examples The concentration of the effect of the peony bark extract is increased, which significantly increases the cell survival rate, as shown in Fig. 4A. However, under the same UVB irradiation energy of 10 J/m ² , the test results of the peony bark extract of Example 1 in Hs68 cells did not exhibit this trend, as shown in Fig. 4B. Therefore, the following HaCaT cells were selected to evaluate the DNA protection of the peony bark extract of Example 1 and the ability to promote DNA replication.

3. Evaluation (II) - Comet test

The comet assay analysis method was performed under a fluorescent microscope, and the degree of DNA damage in a single cell was directly detected by single cell gel electrophoresis (SCGE), thereby evaluating the peony bark extract of Example 1.

First, the HaCaT cell strain was seeded at a density of 1.5 × 10 ⁵ /mL in a 3 cm ² culture dish. After 24 hours of culture, it was divided into control group (no UVB and no reaction with peony bark extract), UVB control group (only UVB was irradiated but not reacted with peony bark extract) and experimental group (before and after UVB irradiation with 50 μM) Group I) or 100 μM (experimental group II) of the peony bark extract was reacted for 4 hours]. Among them, the UVB control group and the experimental group were irradiated with UVB under the same conditions as the evaluation (I) of the above Example 3, and the experimental group was reacted with the peony bark extract before UVB irradiation to evaluate its protective ability against DNA. The reaction with the peony bark extract after UVB irradiation was used to evaluate its ability to repair DNA. Then, each of the above groups of cells was subjected to the following DNA damage assessment by single cell colloidal electrophoresis.

After the above reaction time was reached, the supernatant was separately collected into a 15 mL centrifuge tube, and the cells were removed by trypsin (1×). After removing the supernatant by centrifugation, 1 mL of PBS was added to redisperse the cells. 100 μL of the cells were counted for cell density and viability using a fully automated cell counter (eg, the brand name Countess® automatic cell counter, Invitrogen CountessTM ⁾ , and 500 μl of cell fluid and 1 μL of ethidium bromide (ethidium) were taken. Bromide; EtBr) was mixed and allowed to stand at 4 ° C for 5 minutes. Next, apply gelatinized slides (FEA, Microscope slides ground edges, thickness 1-1.2 mm) to 300 μl of 0.75% normal melting agarose (NMA) and cover the coverslip at 4 °C. After standing for 5 minutes, a mixture of 100 μl of a 1:1 volume of cell fluid and 0.5% low melting agarose (LMA) was added to 0.75% NMA, covered with a coverslip and placed at 4 ° C. 5 minutes. Then, add 100 μl of 0.5% LMA, cover with a cover glass and leave at 4 ° C for 5 minutes.

Then, the slide was completely immersed in a lysing buffer (2.5 M NaCl, 100 mM EDTA, 100 Mm Tris-HCl, 10% DMSO, 1% Triton X100) and allowed to stand at 4 ° C for 1 hour. The cell membrane is broken.

Then, the aforementioned slide is immersed in an enzyme buffer solution (enzyme reaction) Buffer; containing 40 mM Hepes, 0.1 M KCl, 0.5 mM EDTA, and 0.2 mg/ml BSA, adjusted to pH 8 with KOH) was placed at 4 ° C for 15 minutes to remove most of the protein such as cell membrane and nuclear membrane.

Thereafter, the slides were further immersed in an alkaline buffer solution (alkaline buffer; containing 0.3 M NaOH and 1 mM EDTA in deionized water, pH 13.5) at 4 ° C for 15 minutes to expand the double-stranded DNA to form a single strand to increase the analysis. Sensitivity.

Subsequently, the slide was placed in a DNA electrophoresis tank, and electrophoresis was carried out for 30 minutes at 4 ° C in a dark environment, and the DNA of the cells in the slide was observed under a fluorescence microscope (400 x). If the DNA of the cell is fragmented or the like, the broken DNA will move out of the cell to form a tailing phenomenon. At this time, the DNA has a head and a tail, and a comet image can be observed.

The slides of the same conditions were taken from the above slides, and the damage ratio of 500 cells was randomly counted, and 25 cells with comet-type cells were randomly sampled for fluorescence image storage analysis. Then, using commercially available analytical software (such as CASP-Comet Assay Software Project, CASPlab), according to head length, tail length, head intensity (or head amount of DNA), Parameters such as tail intensity (or tail DNA amount), respectively calculate the percentage of head DNA {or head brightness percentage; head DNA intensity (%); that is, [head brightness / (head brightness + tail brightness) ×100〕}, the percentage of tail DNA {or the percentage of tail brightness; tail DNA intensity (%); that is, [tail brightness / (head brightness + tail brightness) × 100]} and tail moment (tail moment) ×% of tail DNA)). If the above-mentioned percentage of tail length, tail brightness percentage, and tail momentum are more High indicates that the damage of DNA is more serious, and the result is shown in Fig. 5.

Referring to Fig. 5, there is shown a photograph of a comet assay of a Haca T cell strain after UVB irradiation according to Example 1 of several embodiments of the present invention, wherein the reference line length of Fig. 5 is 50 μm. In Fig. 5, the first picture from the left side of the first line represents the control group. The second to fourth photographs from the left side of the first row represent the results of UVB irradiation in the UVB control group. The results of the second and third rows of photographs represent the results of the experimental group after UVB irradiation. The square in the upper right corner of each photo magnifies the DNA pattern of the cells randomly sampled.

The results from Figure 5 show that as the UVB irradiation energy increases, the tailing phenomenon caused by damage to the cell DNA is more pronounced, as shown in the UVB control group in Figure 5 (ie, the second to fourth photos from the left side of the first line) ) shown. Secondly, with the increase of the addition amount of the peony bark extract of the first embodiment, the comet tailing phenomenon caused by DNA damage can be effectively reduced, as shown in the experimental group of FIG. 5 (ie, the photos of the second row to the third row). It is proved that the peony bark extract of the first embodiment has the ability to protect and repair skin cells.

4. Assessment (III) - Cell Cycle

This example further evaluates the effect of the peony bark extract of Example 1 on the cell cycle. In summary, cell cycle analysis mainly quantitatively analyzes DNA content and ploidy state, and uses a fluorescent dye that can penetrate cell membranes (such as red propidium iodide (PI) dye solution) to dye the molecule. After embedding the DNA molecule, the red fluorescence intensity at a wavelength of 488 nm was observed by flow cytometry. When the cells are damaged or undergo apoptosis, the DNA is fragmented and released outside the cell. At this time, the DNA content of the cells embedded in the PI is decreased, and the relative fluorescence intensity is also weakened, thereby evaluating the cells. The degree of apoptosis.

First, HaCaT cell line and Hs68 cell line were seeded at a density of 1.5 × 10 ⁵ /mL in a 3 cm ² culture dish. After 24 hours of culture, it was divided into control group (no UVB and no reaction with peony bark extract), UVB control group (only UV was irradiated but not reacted with peony bark extract) and experimental group (both before and after UVB irradiation) Reaction, in which the experimental group was reacted with 50 μM (experimental group I) or 100 μM (experimental group II) of the peony bark extract for 4 hours before UVB irradiation, after UVB for 2 hours or 4 hours, and then The same concentration of the peony bark extract was reacted for 2 hours or 4 hours.

After the above reaction time was reached, the supernatant was separately collected into a 15 mL centrifuge tube, and the cells were removed by trypsin (1×). After removing the supernatant by centrifugation, 300 μl of PBS was added to redisperse the cells. . Next, 700 μl of 99.9% alcohol-fixed cells were slowly shaken, and a cell fixed solution having a final alcohol concentration of 70% was placed and fixed at 4 ° C for 24 hours. Then, centrifuge at 1200 rpm for 5 minutes, remove the alcohol fixative, add 445 μL of PBS to evenly disperse the cells, add 5 μL of RNase (10 mg/mL) and 50 μL of 10% Triton X-100. The solution was reacted at 37 ° C for 30 minutes to decompose RNA. After centrifugation for 5 minutes, remove the supernatant, add 500 μl of PBS to disperse the cells, add 5 μL of propidium iodide (PI) dye solution for DNA staining, and place at 4 ° C in the dark. minute. Subsequently, the cell membrane filter of 0.45 μm and collected in a liquid flow sorter (fluorescence-activated cell sorter; FACS ) dedicated tubes, using a commercially available flow cytometry (Flow cytometer), e.g. FACScan ^TM System (FACScan ^TM system; Becton Dickson), red wavelength fluorescence generated by laser excitation (for example, parameter set to FL3 LIN), analysis of 10,000 cells, analysis of cell cycle distribution with Winmdi computer software, and cell cycle Sub- G ₁ , G ₀ /G ₁ , S, and G ₂ /M were subjected to numerical quantitative analysis, and the results are shown in Fig. 6.

Referring to Figure 6, there is shown a cell cycle distribution of a peony bark extract of HacaT cells irradiated with or without UVB according to Example 1 of several embodiments of the present invention, wherein the horizontal axis of Figure 6 represents Time, while the vertical axis is the cell logarithmic value. In Fig. 6, the first photograph from the left of the first row and the left of the second row shows the cell cycle distribution of the control group. The second cycle from the left of the first row to the left and the second from the left of the second row show the cell cycle distribution of the UVB control group. The cell cycle distribution of the experimental group is shown in the third to fourth photos from the left side of the first line and the third to fourth pictures from the left side of the second line.

The results shown by FIG. 6, compared to the control group, the control group were UVB 2 hours after the UVB irradiation, the cells of the G ₀ / G ₁ phase decreased, but increased Sub-G ₁ and S phase. There was also a consistent result 4 hours after UVB irradiation. In the UVB control group, the G ₀ /G ₁ phase of the cells continued to decrease 4 hours after UVB irradiation, but the Sub-G ₁ and G ₂ /M phases also increased. . However, when the peony bark extract of Example 1 was added to the experimental group, compared with the UVB control group, the cells of the experimental group significantly increased the DNA synthesis phase of SG ₂ /M with the reaction time, but Sub-G phase ₁ There is a tendency to decrease, demonstrating that the peony bark extract of Example 1 does affect cell cycle distribution and enhances the DNA synthesis phase of SG ₂ /M.

5. Evaluation (IV)-BrdU immunofluorescence staining

When cells are subjected to DNA synthesis, an analog of thymidine, such as 5-bromo-deoxy-nucleoside (5-bromo-deoxy-), is added. Uridine; BrdU), which can cause misincorporation and is embedded in DNA, thereby calibrating DNA and detecting the activity of DNA synthesis in living cells. In the process of replicating DNA during cell proliferation, a newly synthesized DNA sequence can be incorporated to replace thymine (T), and the fluorescence content of BrdU-FITC can be used to detect whether the compound has a cell proliferation effect.

First, HaCaT cell line and Hs68 cell line were seeded at a density of 1.5 × 10 ⁵ /mL, respectively, in each well of a 96-well plate. After 24 hours of culture, it was divided into control group (no UVB and no reaction with peony bark extract) and multiple experimental groups (including UVB with irradiation of ⁰ , 10 J/m ² , 20 J/m ² , 50 J/m ^{2 )} . Thereafter, it was reacted with 0, 5 μM, 25 μM, 50 μM, 100 μM of peony bark extract for 4 hours). The UVB control group and the experimental group described above were irradiated with UVB under the same conditions as the evaluation (I) of the above Example 3.

After each of the above groups reached the reaction time, the cells were washed with PBS. Next, 50 μL of 100% ice methanol was added to each well of the cells, and the cells were fixed by reacting at 4 ° C for 10 minutes. Then, the supernatant was removed and air-dried for 1 hour, and then 1% (w/v) of bovine serum albumin (BSA) was added to fill the cell gap to reduce the background value produced by non-specific binding. After shaking uniformly for 1 hour at room temperature, the supernatant was removed, and 40 μL of the primary antibody was added to each well of the cells, and allowed to react at 4 ° C for 24 hours. The primary antibody was a monoclonal antibody against mouse BrdU (mouse mAb; Santa Cruz Biotechnology; dilution concentration of immunofluorescence staining was 1:100).

Thereafter, the cells were washed twice with PBS, and a FITC secondary antibody that binds to fluorescence was added thereto, and the reaction was allowed to stand at room temperature for 3 hours in the dark. Then, the cells were washed twice with PBS, 100 μL of PBS was added to each well, and the amount of fluorescence of FITC (Excitation: 485 nm, Emission: 528 nm) was detected by an enzyme immunoassay analyzer (BioTek, Synergy ^TM 2, USA); 10 μL of a 1:250 Hoechst 33342 dye solution (10 μg/mL) was added to each well, and after uniform mixing, nuclear staining was observed under a microscope. Subsequently, the supernatant was removed, and 100 μL of PBS was added to each well, and the antigen in Hoechst 33342 (Excitation: 360 nm, Emission: 460 nm) cells was detected by an enzyme immunoassay analyzer (BioTek, Synergy ^TM 2, USA). The fluorescence expression produced by antibody binding was calculated and the fluorescence ratio was calculated. The results are shown in Figures 7A and 7B.

Please refer to FIGS. 7A and 7B, wherein FIG. 7A shows the BrdU firefly of the peony bark extract of HacaT cell line and Hs68 cell line irradiated with or without UVB according to Example 1 of several embodiments of the present invention. Light intensity histogram, and Fig. 7B shows photographs of cells stained with immunofluorescence of HaCaT cell line according to Example 1 of several embodiments of the present invention (Fig. 7B, magnification: 200 times) .

In Figure 7A, the upper panel shows the BrdU fluorescence intensity histogram of the Hs68 cell line, and the lower panel shows the BrdU fluorescence intensity histogram of the HaCaT cell line, where the horizontal axis represents the control group and the experimental group with different conditions, while the vertical axis Represents different BrdU fluorescence intensities (%).

In Figure 7B, the first line of the picture is the HaCaT cell image observed by the phase contrast optical microscope, the second line is the HaCaT cell nuclear image observed by Hoechst 33342 fluorescent staining, and the third line is the BrdU fluorescence. DNA images of HaCaT cell lines observed by staining. The first photo from the first row to the third row from the top represents the control group. The middle photo from the first row to the third row up represents the UVB control group. The photos on the right side of the first row to the third row represent the experimental group. The larger square in the lower right or lower right corner of each photo magnifies the cell pattern randomly sampled within the smaller square.

From the results of Fig. 7A, the BrdU fluorescence of the HaCaT cell line and the HaCaT cell line which did not react with the peony bark extract was significantly decreased as the UVB irradiation dose was increased as compared with the control group. However, HaCaT cells significantly increased the fluorescence content of BrdU after reacting with the peony bark extract. Especially in the experimental group with UVB irradiation dose of 10 J/m ² , the peony bark extract significantly enhanced cell proliferation. However, no regular dose response was observed in the Hs68 cell line.

Secondly, the results of Figure 7B show that the HaCaT cell line of the experimental group (pictures from the first row to the third row in the upper row) is compared with the UVB control group (the first to third intermediate photos) The 100 μM peony bark extract reacted significantly to increase the fluorescence content of BrdU. It was proved that the peony bark extract of Example 1 can promote DNA replication, allowing BrdU-FITC to be inserted into the new synthetic sequence of DNA replication, greatly enhancing BrdU. Fluorescent performance.

In summary, the above evaluation (II)-comet assay, evaluation (III)-cell cycle, evaluation (IV) BrdU immunofluorescence staining and other experiments confirmed that the peony bark extract of Example 1 does have DNA protection and promotion. The ability to replicate DNA.

Example 4: Assessing the ability of peony bark extract to repair skin cells

This example further evaluated the repair ability of skin cells after UVB irradiation using the peony bark extract of Example 1.

1. Evaluation of related gene and protein expression in nucleotide excision repair system

The nucleotide excision repair (NER) system primarily repairs DNA damage caused by ultraviolet radiation. When cells perform NER repair DNA, it is known that the DNA damage is identified by XPC combined with HR23E protein. TFIIH, XPB and XPD helicases loosened the DNA at the lesion, and the complex formed by XPA and RPA binds to the DNA damage to promote the endonuclease XPF/ERCC1 splicing 5' end and XPG cleavage 3' end. Subsequently, phosphorylation of RPA attached to the sheared single-stranded DNA by CDK2 mainly promotes the binding of the PCNA-binding DNA polymerase δ to the resected DNA damage fragments, thereby repairing the DNA damage.

First, the HaCaT cell line and the Hs68 cell line were inoculated at a density of 1.5 × 10 ⁵ /mL, respectively, in a 6-well plate or a 10 cm ² culture dish. After 24 hours of culture, it was divided into control group (no UVB and no reaction with peony bark extract), UVB control group (only 10 J/m ² UVB but not with peony bark extract) and experimental group (irradiation 10) The UVB of J/m ² reacts with the peony bark extract before and after). The UVB control group and the experimental group were irradiated with UVB of 10 J/m ² or 50 J/m ² under the same conditions as the evaluation (I) of the above Example 3. The experimental group was reacted with 50 μM or 100 μM of peony bark extract for 4 hours before irradiation with UVB (the control group was cultured in serum-free fresh medium for 4 hours), irradiated with UVB, and reacted with the same concentration of peony bark extract. After 2 hours, 4 hours, 6 hours or 8 hours (the control group was cultured for 2 hours, 4 hours, and 6 hours in serum-free fresh medium), the related gene and protein expression of the nucleotide excision repair system were measured.

(1) Assessment of related gene expression in the NER system

After the above reaction time was inoculated in each of the 6-well plates, the supernatant was separately collected into a 15 mL centrifuge tube, and the cells were removed by trypsin (1×), and the supernatant was removed by centrifugation. The method is known to extract total RNA. After the total RNA obtained is quantified, a real-time reverse-transcription polymerase chain reaction (real-time reverse-transcription polymerase chain reaction; Real-time RT-PCR) was used to test whether the peony bark extract of Example 1 regulates the related gene expression of the NER system.

The above real-time RT-PCR can be carried out by referring to a conventional method or the following manner. First, a reverse transcription reaction solution was prepared which adjusted the concentration of the total RNA obtained to 2 mg/mL with 0.1% DEPC-H ₂ O, and the volume was made up to 12.5 μL, and 1 μL of oligo-thymidylate was added. [oligo (dT)] The primer was uniformly mixed and reacted at 70 ° C for 2 minutes. Then, the above reaction mixture was quickly placed on ice, and then 4 μL of 5 times (5×) Moloney murine leukemia virus (MMLV) reverse transcription reaction buffer solution (MMLV-RT buffer solution) was added. 1 μL of 10 mM dNTPs, 0.5 μL of RNase inhibitor and 1 μL of MMLV reverse transcriptase (MMLV RTase), reacted at 42 ° C for 1 hour and reacted at 94 ° C for 5 minutes to synthesize cDNA. Thereafter, the volume of cDNA was made up to 100 μL by adding iced deionized water (ddH ₂ O), and the cDNA was prepared and stored at -80 °C.

Next, take the above 10 μL of cDNA (about 10 ng to 50 ng), mix with 12.5 μL of SYBR Green mixture, 2.5 μl of 10 μM gene primer pair (including upstream primer and upstream primer) and ddH ₂ O. A total volume of 25 μL of the reaction solution. Specific examples of the upstream primer and the upstream primer for each of the aforementioned genes are shown in Table 1, for example:

After the above reaction solution was centrifuged at a low rotation speed, real-time quantitative PCR was carried out under the following conditions: separation of the double-stranded template DNA (dDNA denaturation) at 52 ° C for 15 seconds at 52 ° C to 60 ° C The primer was annealed for 45 seconds, and the DNA was extended at 72 ° C for 1 minute to carry out an extension of the DNA, and the reaction was repeated for 40 cycles, and then carried out at 72 ° C for 5 minutes. After the cycle is completed, the fluorescence intensity of the gene related to the NER system obtained above is analyzed using a commercially available software such as an amplification plot or other functional replacement software, and the results are shown in Fig. 8A.

Please refer to FIG. 8A, which shows the related gene expression of the nucleotide excision repair system (NER) of the Haca T cell line with or without UVB irradiation according to Example 1 of several embodiments of the present invention. , wherein the horizontal axis is the time (hours) of the reaction between the HaCaT cell line and the peony bark extract, and the vertical axis is the gene expression of the NER system related gene relative to β-actin (density ratio; converted from fluorescence intensity) ).

From the results of Fig. 8A, the HaCaT cell line was reacted with 50 μM of peony bark extract for 4 hours before irradiation with UVB, and reacted with 50 μM of peony bark extract after irradiation with UVB. Hours can significantly increase the amount of XPC gene expression. As for other genes, for example The expression levels of XPA gene, RPA gene, ERCC1 gene, XPF gene, XPG gene and PCNA gene also increased with the increase of reaction time, indicating that the peony peel extract of Example 1 can increase the gene expression related to the NER system. Helps to repair DNA damage caused by UV irradiation of HaCaT cells.

(2) Evaluation of related protein performance of NER system (I)

(2.1) Extracting proteins

After the reaction time was reached in each of the above 10 cm ² culture dishes (the experimental group was irradiated with 10 J/m ² of UVB and then reacted with the same concentration of the peony bark extract for 8 hours), the supernatant was separately collected to 15 mL for centrifugation. The tube was removed by trypsin (1×), and the supernatant was removed by centrifugation, followed by protein extraction, and the protein expression was analyzed by Western blotting.

First, a standard curve can be established using different concentrations of BSA using a conventional method, and the absorbance of the extracted protein can be brought into a linear regression formula to convert the protein concentration of each group. Those of ordinary skill in the art to which the present invention pertains should be familiar with establishing standard curves and converting protein concentrations, which are not described herein.

(2.2) Protein electrophoresis analysis

Next, the proteins of the above groups can be evaluated using a sodium dodecyl sulfate-polyacrylamide colloidal electrophoresis assay (SDS-PAGE electrophoresis assay). First, 10% of the running gel was prepared according to the second table and injected into the gluer. Distilled water was added to the upper layer of the colloid to flatten the colloidal liquid surface. After solidification, the upper layer of ddH ₂ O was removed and the remaining moisture was absorbed. .

Then, 7.5% of a stacking gel was prepared according to the third table, and after being injected into the lower layer of the underlayer, the tooth mold was inserted, and the tooth mold was removed after solidification.

Subsequently, the proteins of the above groups were converted, mixed with 5× Loading dye buffer in a volume ratio of 5:1, treated at 100 ° C for 5 minutes, and then moved to 4 ° C for cooling.

The film prepared above was placed in a commercially available electrophoresis tank (for example, trade name miniVE, Complete, GE-80-6418-77, GE Healthcare, .U.S.A.), and pre-run for 15 minutes to 20 minutes at a voltage of 90 volts. Then, a standard protein MW marker and a protein of each of the above groups were sequentially added, followed by electrophoresis analysis at a voltage of 130 volts.

Then, the above-mentioned SDS-PAGE electrophoresis colloid was taken out and subjected to Western blotting assay. First, a commercially available transfer film such as polyvinylidene difluoride membrans (PVDF membrane; for example, the trade name PolyScreen PVDF) Hybridization Transfer Membrane, PK-NEF1002, PerkinElmer, U.S.A.) was soaked in methanol for 5 minutes to change the polarity of the PVDF membrane. Next, put the sponge pad, filter paper and electrophoresis gel in the Gel holder cassette, cover the PVDF transfer film, remove the bubbles, and then put the filter paper, sponge pad and card in sequence. The crucible is clamped and placed in a commercially available western transfer tank (for example, trade name Mighty small transphor, TE 22, Amersham biosciences, USA), and subjected to 60 volts at a voltage of 200 volts at 4 ° C. Minutes, the protein was transferred to a PVDF transfer film. Thereafter, the PVDF transfer film with protein was completely washed with 0.1% PBST (Tween-20/PBS), and immunostaining of specific protein expression was performed.

The above-mentioned PVDF transfer film was immersed in 0.1% PBST (Tween-20/PBS) containing 5% skim milk powder and shaken gently for 60 minutes for non-specific antigen blocking. . Next, after washing the PVDF transfer film with 0.1% PBST, the primary antibody (1:1000) was prepared using 0.1% PBST, and the PVDF transfer film was completely immersed in the primary antibody and reacted at 4 ° C until overnight. Then, the PVDF transfer film was washed with 0.1% PBST, and a secondary antibody (1:1000) was added thereto to act at 4 ° C for 2-3 hours. Thereafter, the PVDF transfer film was washed with 0.1% PBST at 4 ° C for 2 hours, and a coloring agent was added to react with the PVDF transfer film for 3 minutes. Then, a color developer mixing reaction was added, and images were stored using a cold light image analyzer (Avegene Life Science, LIAS ChemLite Series, ChemLite 200FA/400FA). The aforementioned luminescence image analyzer can detect Luminol (Cyclic phthalhydrazides) from the ground state according to the stored image. The intensity of luminescence is released when excited into an excited state and then returned to the ground state, and quantitative analysis is performed.

Please refer to FIG. 8B, which shows the protein expression of the nucleotide excision repair system (NER) of the Haca T cell line with or without UVB irradiation according to Example 1 of several embodiments of the present invention. The Western blotting analysis chart, in which the first track represents the performance of the NER-related protein in the control group, the second track represents the performance of the NER-related protein in the UVB control group, and the third track represents the performance of the NER-related protein in the experimental group, wherein The examples were reacted with 50 μM of peony bark extract for 4 hours before UVB irradiation, and reacted with the same concentration of peony bark extract for 8 hours after UVB irradiation at 10 J/m ² .

As can be seen from the results of Fig. 8B, compared with the UVB control group, the HaCaT cell line of the experimental group was reacted with 50 μM of the peony bark extract for 4 hours before irradiation with UVB, and 50 μM of peony bark after irradiation with UVB. After 8 hours of reaction, the expression of XPC, XPA, RPA, ERCC1, XPF, XPG and PCNA can be increased.

(3) Assessment of related protein expression in the NER system (II)

After the reaction time was reached in each of the above groups (the experimental group was irradiated with 50 J/m ² of UVB and then reacted with the same concentration of peony bark extract for 4 hours), the supernatant was separately collected into a 15 mL centrifuge tube, and trypsin was used again. Trypsin, 1×) Remove the cells, remove the supernatant by centrifugation, add 500 μL of 4% paraformaldehyde at 4 ° C for 1 hour to fix the cells and analyze the protein expression. Next, 5 μL of 10% Triton X-100 was added and the mixture was allowed to act at 4 ° C for 5 minutes, and then the supernatant was removed by centrifugation, and the primary antibody was added and reacted at 4 ° C for 24 hours. Then, FITC secondary antibody (1:500) was added to avoid light and react at room temperature for 3 hours. Subsequently, the cell liquid was transferred to a FACS-dedicated test tube, and 10,000 cells were analyzed by a green wavelength fluorescence (FL1 LOG) generated by laser light excitation using a flow cytometer, and analyzed by the Winmdi computer software after the action of the compound. The amount of fluorescent expression of various proteins was quantified, and the results are shown in Fig. 8C.

Specific examples of the above applicable primary antibodies can be as shown in Table 4:

Please refer to FIG. 8C, which shows a nucleotide excision repair system for the HaCaT cell line with or without UVB irradiation using the peony bark extract of Example 1 of the flow cytometer according to several embodiments of the present invention. A three-dimensional histogram of the related protein representation of NER), where the x-axis represents the fluorescence intensity, the y-axis represents the different groups, and the z-axis represents the number of cells. The y-axis is from the first to the fifth, respectively, from the first to the fifth, wherein the first lane represents the expression of the NER-related protein in the negative control group (HaCaT cell line not irradiated with UVB), and the second lane represents the NER-related protein in the control group. The amount of performance, the third track represents the performance of the NER-related protein in the UVB control group, and the fourth track represents the performance of the NER-related protein in the experimental group I (reacted with 50 μM of the peony bark extract), and the fifth track represents the experimental group II. (Reacts with 100 μM of Cortex Extract) NER Phase The amount of protein expression.

As can be seen from Fig. 8C, the performance of XPC, XPA, RPA, ERCC1, XPF, XPG and PCNA was decreased in the UVB control group after UVB (50 J/m ² ) for 4 hours compared with the control group. However, the protein performance of the experimental group increased significantly with the increase of the reaction concentration of the peony bark extract. It was confirmed that the peony bark extract of Example 1 can repair the DNA of HaCaT cells after ultraviolet irradiation by increasing the expression of NER-related protein. Damaged place.

The above results show that the peony bark extract of Example 1 can enhance the DNA and protein synthesis of NER-related genes, thereby promoting cell repair.

Example 5: Assessing the ability of peony bark extract to repair living skin cells

This example was prepared by using the peony bark extract of Example 1 to prepare a gel for application to living skin, and further evaluating its ability to repair skin cells after UVB irradiation.

First, a peony bark extract gel was prepared, and the peony bark extract of Example 1 was set to a concentration of 100 μM, and 10 μL of each was added to 1 mL of physiological saline to be dispersed, and 2 g of hydroxyl was weighed. Ethyl cellulose (HEC) gel was added and completely dissolved, sealed with a parafilm seal to protect the light, and the colloid was thickened and agglomerated over 24 hours to prepare a peony bark extract gel (including 2% HEC and 20 μg/mL of peony bark extract) were used.

At the same time, placebo was prepared by dissolving 2 g of HEC gel in 1 mL of physiological saline (pH adjusted to 6.47), sealing with a sealing wax film, and thickening and aggregating the colloid over 24 hours to make a comfort. Agent (including 2% HEC) use.

In addition, 30 C57BL/6JNarl strains of 5-week-old male rats (purchased from the National Animal Research Center of the National Science Association) were housed in a cage with 5 air conditioners in an air-conditioned animal house, in which the feeding temperature was maintained. 21 ± 2 ° C, humidity maintained at 50% to 60% and maintained a 12-hour switching light dark cycle. The feed and water supply are not restricted during the feeding period, and the remaining feeding conditions are carried out in accordance with the relevant experimental animal management guidelines announced by the National Institutes of Health. The mice were subjected to an experiment (6 weeks old) after one week of adaptation to the experimental environment, and all the experimental procedures were performed during daylighting. Before the experiment, the mice were depilated for 24 hours, and each mouse was weighed and administered with abdominal anesthesia (1 g/10 μl), and 30 mice were treated according to the following groups.

Five mice were randomly selected from the control group, and the L, a, and b values of each mouse were recorded by skin colorimeter, without any UVB irradiation and drug treatment, and at 0, 2, 4, 6, and 8 hours. Point, the L, a, b values were measured at different time points of the color difference meter, and the punching of the skin tissue was simultaneously performed. The skin layer of the removed skin tissue was stained with red medicinal water and immersed in 4% paraformaldehyde and stored at 4 °C.

In the placebo-controlled group, 5 mice were randomly selected, and the L, a, and b values of each mouse were recorded by a skin color difference meter, each of which applied 0.05 g of placebo without any UVB irradiation, and at 0, 2, At 4, 6 and 8 hours, the L, a, and b values were measured with a color difference meter, and a punch of the skin tissue was simultaneously performed. The skin layer of the removed skin tissue was stained with red medicinal water and immersed in 4% paraformaldehyde and stored at 4 °C.

Five mice were randomly selected from the placebo-treated UVB control group. The L, a, and b values of each mouse were recorded by skin colorimeter. After depilation, each mouse was given a 0.05 g consolation at each irradiation site. The agent was allowed to act for 4 hours, and after UVB (2000 J/m ² ) irradiation, the L, a, and b values of each mouse were recorded using a skin color difference meter. Then, 0.05 g of placebo was applied to each of the mice at the irradiation site, and the L, a, and b values were recorded and recorded for 0, 2, 4, 6, and 8 hours, respectively. Subsequently, a drilled section of the skin tissue is performed at the irradiation site. The skin layer of the removed skin tissue was stained with red medicinal water and immersed in 4% paraformaldehyde and stored at 4 °C.

In the experimental group, 15 mice were randomly selected, and the L, a, and b values of each mouse were recorded by skin color difference meter. Five experimental mice in each group of the smearing compound were randomly distributed. After depilation, each mouse was irradiated. Each of the 0.05 g of the peony bark extract gel was applied and reacted for 4 hours. Then, UVB (2000 J/m ² ) irradiation was performed, and L, a, and b values of each mouse at 0 hours, 2 hours, 4 hours, 6 hours, and 8 hours were recorded and measured by a skin color difference meter, and each small A 0.05 g layer of peony bark extract was applied again to the irradiated area of the mouse. Thereafter, L, a, and b values recorded at 0 hours, 2 hours, 4 hours, 6 hours, and 8 hours were measured with a skin color difference meter. Subsequently, a drilled section of the skin tissue is performed at the irradiation site. The epidermis layer of the removed skin tissue was stained with red medicinal water and immersed in a 10% neutral formalin solution and stored at 4 °C.

The aforementioned skin tissue soaked in 4% paraformaldehyde and 10% neutral formalin solution is subsequently paraffin-embedded to gradually replace the water in the tissue with liquid paraffin. Thereafter, the embedded cassette was agglutinated at 4 ° C for about 10 minutes, and then placed in a freezer to increase the paraffin hardness to perform paraffin tissue sectioning. The obtained paraffin tissue sections were further stained with hematoxylin-eosin (H&E) to observe the morphology of the cell tissues. Whether the structure changes or not, the result is as shown in Fig. 9A. The paraffin embedding, tissue sectioning, H&E staining and the like described above are well known to those of ordinary skill in the art to which the present invention pertains, and are not described herein.

In addition, the above tissue sections can be further subjected to immunohistochemical staining (IHC) analysis, which is in situ stained with an antibody calibrated with fluorescein isothiocyanate (FITC) to specifically detect tissue sections. NER related proteins and localized. Briefly, the paraffin tissue sections were dewaxed and hydrated, soaked in 3% H ₂ O ₂ at 4 ° C for 20 minutes, washed with PBS, immersed in citric acid buffer solution, and microwaved at 4 W. After heating for 3 minutes, the cold citric acid buffer solution was replaced and cooled at 4 ° C for 15 minutes, and then heated in a microwave oven at 4 W for 2 minutes. The tissue sections were washed with 1×PBS for 5 minutes, and then reacted by adding a blocking buffer for 30 minutes to fill the interstitial space. Then, one-stage antibody (1:100) as shown in Table 4 was dropped on the tissue section, and after treatment at room temperature for 2 hours, the tissue was washed twice with PBS, then immersed in PBST for 10 minutes, and finally with water. Wash for 30 minutes. Thereafter, secondary antibody (1:500) was dropped on the tissue section, and the reaction was allowed to stand at room temperature for 2 hours in the dark, and then the cells were incubated twice with PBS, and then immersed in PBST for 30 minutes. Then, the FITC fluorescence staining was observed under a microscope, and Hoekst 33342 staining solution (10 μg/ml) at a ratio of 1:250 was applied to the tissue for 10 minutes, and the tissue cells and staining were observed under a microscope. As shown in the "nuclei" of Figures 9B to 9I. Then, the cells were washed twice with PBS, and the coverslips were covered with a cover gel, and the edges of the coverslips were fixed on the slides with transparent nail polish, and fluorescence imaging analysis was performed under a microscope. The results are shown in Fig. 9B. Fluorescent staining of the NER-related protein to Figure 9I is shown.

Referring to Figs. 9A to 9I, there are shown results of histochemical staining of a gel prepared by using the peony bark extract of Example 1 on a living skin according to several embodiments of the present invention. From the results of H&E stain in Fig. 9A, the epidermal layer of the UVB control group applied only with placebo was slightly thickened, but the tissue of the experimental group coated with the peony bark extract was relatively complete. Secondly, the BrdU results from Fig. 9B show that the amount of BrdU expression increases as the reaction time of the peony bark extract gel increases. Furthermore, the results from Fig. 9C to Fig. 9I show that the expression of NER-related proteins such as XPC, XPA, RPA, ERCC1, XPF, XPG and PCNA by NER-related proteins after compound (1) and (2) The reaction time of the peony bark extract gel increased, and it was confirmed that the peony bark extract of the first embodiment can repair the damage of the DNA of the living skin cells after ultraviolet irradiation by increasing the expression of the NER-related protein.

Example 6: Assessing the ability of peony bark extract to protect DNA from oxidation

1. Establish H ₂ O ₂ Suitable dose with UVB treatment

Skin cells that are exposed to ultraviolet radiation or physiological metabolism can cause oxidative stress and cause DNA damage. This embodiment uses different concentrations of H ₂ O ₂ and/or UVB irradiation of different energies to attack the plastid DNA via photodegradation of hydroxyl radicals, thereby simulating the damage of DNA by free radicals. The ability of the peony bark extract of Example 1 to protect DNA against oxidation.

First, pUC119 plastid DNA (0.5 μg/μL; Takara, Japan) and DNA molecular weight standard marker (DNA MW standard marker; N-Hind III cleavage, hereinafter referred to as λHind III label; 0.5 μg / μL; Takara, Japan), after dilution with 8 volumes of PBS, respectively, was treated according to the following groups.

2 μL of pUC119 plastid DNA (0.056 μg/μL) diluted with PBS or 2 μL of λHind III labeled (0.056 μg/μL) diluted with PBS were added to the control group, respectively, and 8 μL of PBS was added. Nucleic acid electrophoresis analysis was performed using an agar gel.

In the control group, 2 μL of the above pUC119 plastid DNA (0.056 μg/μL) diluted with PBS was added, and 6.5 μL of ddH ₂ O, 1 μL of restriction enzyme reaction solution (H buffer) and 0.5 μL of restriction enzyme EcoRI were added. (15 unit/μL; Takara, Japan), after 1 hour of action at 37 ° C, nucleic acid electrophoresis analysis was performed using an agar gel.

The H ₂ O ₂ and UVB treatment groups were treated with pUC119 plastid DNA (0.056 μg/μL) or λHind III (0.056 μg/μL) diluted with PBS, and 2 μL of each was added to 3 μL of PBS and 5 μL. H ₂ O ₂ (concentrations of 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, respectively). Next, the pUC119 DNA Plasmid was irradiated with UVB of 10 J/m ² , 20 J/m ² , 50 J/m ² , 100 J/m ² , and 200 J/m ² , and the λ Hind III label was irradiated with 1000 J/m ² , After UVB of 2000 J/m ² , 3000 J/m ² , 3500 J/m ² , and 4000 J/m ² , the two were subjected to nucleic acid electrophoresis analysis using an agar gel.

The above nucleic acid electrophoresis was carried out by electrophoresis using a 0.8% agar gel for 30 minutes. Then, using the image capture analysis system to take the electrophoretic film and perform image analysis, the DNA ribbon concentration of the control group is 100%, and the supercoil (SC) type DNA and open loop (open circular) of each group are respectively calculated. OC) type DNA and percentage (%) of linear (LN) DNA.

In general, pUC119 plastid DNA is mainly supercoil (SC) structure (not shown), and after treatment with EcoRI restriction endonuclease, pUC119 plastid DNA can be cut into linear (LN) structure ( The figure is not shown). When the pUC119 plastid DNA is exposed to 5 mM H ₂ O ₂ and the UVB irradiation energy reaches 200 J/m ² , the plastid DNA can also be completely sheared into a linear (LN) structure, such as Figure 10A H ₂ O _{2 .} and UVB UVB treated group of 200 J / m ² of the ink ribbon shown in FIG. The higher the concentration of H ₂ O ₂ , the lower the UVB irradiation energy required to completely shear the pUC119 plastid DNA into a linear (LN) structure. When the pUC119 plastid DNA is exposed to 40 mM H ₂ O ₂ , the UVB irradiation energy required to completely cleave pUC119 plastid DNA into a linear (LN) structure is required for exposure to 20 mM H ₂ O ₂ The UVB irradiation energy is very similar (not shown).

Similar results were obtained for DNA linear fragments, exemplified by the λHind III label, which includes DNA linear fragments of various molecular weights, and the damage of the DNA fragments is more severe as the energy of UVB irradiation increases. When the λHind III label is exposed to 5 mM H ₂ O ₂ and the UVB irradiation energy reaches 3500 J/m ² , the λHind III DNA can be cut into smaller fragments (not shown). As the H ₂ O ₂ concentration is higher, the UVB irradiation energy required to shear the λHind III DNA into smaller fragments is lower (not shown).

Based on the above results, the following selection was performed to expose pUC119 plastid DNA and λHind III DNA to 5 mM and 20 mM H ₂ O ₂ , respectively, and to use UVB at an irradiation dose of 200 J/m ² and 3500 J/m ² , respectively. Irradiation, thereby evaluating whether the peony bark extract of Example 1 can protect DNA from oxidation and avoid DNA damage.

2. Evaluation of peony bark extract to protect DNA from oxidation and avoid DNA damage Injury ability

In this example, 50 μM or 100 μM of the peony bark extract of Example 1 was added to pUC119 plastid DNA or λHind III DNA, and then induced by UVB irradiation and H ₂ O _{2 to} induce pUC119 plastid DNA or λHind III DNA. were exposed to H ₂ O ₂ 5 mM and 20 mM, the irradiation dose and then were 200 J / m ² and 3500 J / m UVB ² of irradiated, and then verify that extracts of Paeonia protected by the above evaluation method of anti-DNA Oxidize and avoid DNA damage. The control group, the control group, the H ₂ O ₂ and UVB treatment groups evaluated here, the H ₂ O ₂ and UVB treatment methods, and the nucleic acid electrophoresis analysis are the same as those in the first embodiment of the sixth embodiment, and no rumors are made here.

For the experimental group I, take 2 μL of the above-mentioned pUC119 plastid DNA diluted with PBS (0.056 μg/μL), and add 3 μL of the peony bark extract of Example 1 (concentration: 50 μM or 100 μM), and then separately and 5 After mixing μL of H ₂ O ₂ (5 mM or 20 mM), UVB (200 J/m ² ) was irradiated, and nucleic acid electrophoresis analysis was performed using an agar gel.

In addition, in the experimental group II, 2 μL of the above-mentioned λHind III labeled (0.056 μg/μL) diluted with PBS was added, and 3 μL of the peony bark extract of Example 1 (concentration of 50 μM or 100 μM) was added, and then each and 5 After mixing μL of H ₂ O ₂ (5 mM or 20 mM), UVB (200 J/m ² ) was irradiated, and nucleic acid electrophoresis analysis was performed using an agar gel. The above results are shown in Figures 10A to 10B.

Please refer to FIG. 10A to FIG. 10B, which are diagrams showing the electrophoresis of nucleic acid against the oxidation of DNA by using the peony bark extract of the first embodiment according to several embodiments of the present invention, wherein the 10A map shows the pUC119 plastid DNA. The nucleic acid electrophoresis pattern after treatment with different concentrations of H ₂ O ₂ and/or UVB of different energies, and the 10B graph shows that the λHind III labeling nucleic acid treated with different concentrations of H ₂ O ₂ and/or different energy UVB Electropherogram.

The results of the nucleic acid electrophoresis patterns from 10A to 10B show that the pUC119 plastid DNA is mainly a supercoiled (SC) structure (as shown in the control group of Fig. 10A), and after treatment with EcoRI restriction endonuclease, The pUC119 plastid DNA was cut into a linear (LN) structure (as shown in the control group of Figure 10A). When pUC119 plastid DNA was first added to the peony bark extract of Example 1, and then exposed to 5 mM or 20 mM H ₂ O ₂ and UVB irradiation energy up to 200 J/m ² , 50 μM or 100 μM peony bark extract The material is effectively protected and prevents most of the plastid DNA from being sheared into a linear (LN) structure, as shown by the band of experimental group I in Figure 10A.

Similar results can be obtained with Figure 10B. The results of the electropherogram of the nucleic acid of Figure 10B show that when the λHind III label is exposed to 5 mM or 20 mM H ₂ O ₂ and the UVB irradiation energy reaches 3500 J/m ² , the λHind III DNA can be cut into smaller pieces. Small fragments are shown in the ribbon of Experiment Group II in Figure 10B. When the λHind III label was added to the peony bark extract of Example 1 and then exposed to 5 mM or 20 mM H ₂ O ₂ and the UVB irradiation energy reached 3500 J/m ² , 50 μM or 100 μM peony bark extract It is effective to protect and avoid most of the λHind III label from being cut into smaller fragments, as shown by the ribbon of Experiment Group II in Figure 10B.

Based on the results of the above examples, the peony bark extract of Example 1 not only inhibits melanin production, promotes DNA replication, and regulates the NER repair mechanism, but also effectively prevents and protects DNA from damage caused by H ₂ O ₂ and UVB irradiation.

It should be noted that although the present invention is exemplified by specific process conditions, specific cell strains, specific analytical methods or specific instruments, the peony of the present invention is illustrated. The application of the skin extract and the method of producing the same, but it is to be understood by those skilled in the art that the present invention is not limited thereto, and the peony bark extract of the present invention may be used without departing from the spirit and scope of the present invention. Use other process conditions, other cell lines, other analytical methods or instruments. 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 and the method for producing the same have the advantages that the peony ketone extract can be effectively obtained by using at least one partitioning extraction step and at least one column separation step. To provide DNA repair, inhibition of melanin production, and antioxidant biological activity. Since the peony skin extract using the acetophenone compound of the present invention has various biological activities, 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.

100‧‧‧ method

101‧‧‧Procedures for providing peony skin material

103‧‧‧Steps of crude extraction of the peony bark material using the first organic solution to obtain the crude extract

105‧‧‧Step of performing a first partition extraction step on the crude extract using the second organic solution to obtain the first extract

107‧‧‧Second partition extraction of the first extract using a third aqueous organic solution Step to obtain the second extract step

109‧‧‧Steps of performing a first column separation step on the second extract to obtain a first solution and a second solution

111‧‧‧Steps of separating the second eluate into a second column separation step to separate the cortex 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.

2A to 2F are diagrams showing the HaCaT cell strain, the B16 cell strain, the BNL CL.2 cell strain, and the Hs68 cell strain according to several embodiments of the present invention, and the cortex peel extract or vitamin C of Example 1 for 72 hours. Post-cell survival rate graph.

Fig. 3 is a graph showing cell survival rate of HaCaT cell line and Hs68 cell line after UVB irradiation with different energies according to an embodiment of the present invention.

Fig. 4A and Fig. 4B are graphs showing the cell viability of the peony bark extract of the HacaT cell line and the Hs68 cell line after UVB irradiation according to Example 1 of several examples of the present invention.

Fig. 5 is a photograph showing a comet assay of a Haca T cell strain after UVB irradiation according to Example 1 of several examples of the present invention.

Fig. 6 is a graph showing the cell cycle distribution of the peony bark extract of HahaT cells irradiated with or without UVB according to Example 1 of several embodiments of the present invention.

Fig. 7A is a BrdU fluorescence intensity histogram showing the HacaT cell line and the Hs68 cell line irradiated with or without UVB according to Example 1 of several examples of the present invention.

Fig. 7B is a photograph showing the cells of the peony bark extract subjected to immunofluorescence staining of the HaCaT cell line according to Example 1 of several embodiments of the present invention (Fig. 7B, magnification: 200 times).

Figure 8A shows an embodiment 1 according to several embodiments of the present invention. The peony bark extract is expressed for the gene related to the nucleotide excision repair system (NER) of the HaCaT cell line irradiated with or without UVB.

Figure 8B is a diagram showing the Western blotting of the protein expression of the nucleotide excision repair system (NER) of the HacaT cell line with or without UVB irradiation according to Example 1 of several embodiments of the present invention. Method analysis chart.

Figure 8C shows the correlation of the nucleoside excision repair system (NER) of the HacaT cell line with or without UVB irradiation using the flow cytometry sample of Example 1 in accordance with several embodiments of the present invention. A three-dimensional histogram of protein expression.

Fig. 9A to Fig. 9I show the results of histochemical staining of a gel prepared by using the peony bark extract of Example 1 on a living skin according to several embodiments of the present invention.

10A to 10B are diagrams showing the electrophoresis of nucleic acids for protecting DNA against oxidation of the peony bark extract of Example 1 according to several embodiments of the present invention.

<110> Jianan University of Pharmacy <120> Cortex peel extract and its manufacturing method and application <130> None <160> 16 <210> 1 <211> 17 <212> DNA <213> Artificial sequence <220> primer <223> PCR forward primer( XPC F) <400> 1 <210> 2 <211> 26 <212> DNA <213> Artificial sequence <220> primer <223> PCR reverse primer( XPC R) <400> 2 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> primer <223> PCR forward primer( XPA F) <400> 3 <210> 4 <211> 22 <212> DNA <213> Artificial sequence <220> primer <223> PCR reverse primer( XPA R) <400> 4 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> primer <223> PCR forward primer( RPA F) <400> 5 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> primer <223> PCR reverse primer( RPA R) <400> 6 <210> 7 <211> 19 <212> DNA <213> Artificial sequence <220> primer <223> PCR forward primer( ERCC1 F) <400> 7 <210> 8 <211> 21 <212> DNA <213> Artificial sequence <220> primer <223> PCR reverse primer( ERCC1 R) <400> 8 <210> 9 <211> 18 <212> DNA <213> Artificial sequence <220> primer <223> PCR forward primer( XPF(ERCC4) F) <400> 9 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> primer <223> PCR reverse primer( XPF(ERCC4) R) <400> 10 <210> 11 <211> 17 <212> DNA <213> Artificial sequence <220> primer <223> PCR forward primer( XPG(ERCC5) F) <400> 11 <210> 12 <211> 25 <212> DNA <213> Artificial sequence <220> primer <223> PCR reverse primer( XPG(ERCC5) R) <400> 12 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> primer <223> PCR forward primer( PCNA F) <400> 13 <210> 14 <211> 26 <212> DNA <213> Artificial sequence <220> primer <223> PCR reverse primer( PCNA R) <400> 14 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <220> primer <223> PCR forward primer( β - actin F) <400> 15 <210> 16 <211> 17 <212> DNA <213> Artificial sequence <220> primer <223> PCR reverse primer( β - actin R) <400> 16

100‧‧‧ method

101‧‧‧Procedures for providing peony skin material

103‧‧‧Steps of crude extraction of the peony bark material using the first organic solution to obtain the crude extract

105‧‧‧Step of performing a first partition extraction step on the crude extract using the second organic solution to obtain the first extract

107‧‧‧Step of performing a second partitioning extraction step on the first extract with a third organic aqueous solution to obtain a second extract

109‧‧‧Steps of performing a first column separation step on the second extract to obtain a first solution and a second solution

111‧‧‧Steps of separating the second eluate into a second column separation step to separate the cortex peel extract

Claims (5)

A method for treating animal cells in vitro, characterized in that an animal cell is treated in vitro using a composition comprising an effective dose of 50 μM to 100 μM of 2,5-dihydroxy-4-methylacetophenone, the 2,5 -Dihydroxy-4-methylacetophenone such as 2,5-dihydroxy-4-methylacetophenone of formula (I): And the 2,5-dihydroxy-4-methylacetophenone has the biological activity of DNA repair to repair the DNA of the animal cell in vitro. A method for treating animal cells in vitro according to the invention of claim 1, wherein the composition is a cosmetic composition. A method of treating an animal cell in vitro according to the first aspect of the invention, wherein the composition is a food composition. A method for treating animal cells in vitro according to the invention of claim 1, wherein one of the animal cells is derived from human or mouse. Treatment of animals in vitro according to the scope of claim 1 A method of cells, wherein the animal cell is a skin cell, a melanocyte or a hepatocyte.