TWI483742B - Method of treating cells in vitro with composition containing extract of peony root cortex and application thereof - Google Patents

Description

Method for treating cells in vitro by using composition containing peony bark extract and application thereof

The present invention relates to a peony bark extract and a method for producing the same, and more particularly to a plurality of biologically active peony bark extracts having both DNA repair and inhibition of melanin production and a method for producing the same.

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

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.

Currently, whitening ingredients on the market, such as vitamin C, hydroquinone (HQ), acelaic acid and arbutin, although the whitening effect is significant, but some whitening agents It achieves whitening effect by directly destroying melanocytes, and still has the disadvantage of cytotoxicity. Therefore, it is still necessary to find a whitening ingredient with cell safety.

In addition, the traditional Chinese herbal medicine for women's beauty care, mostly for qi and blood circulation, adjust the body of warming medicine, although there are a few single, the compound is directly used in skin whitening, acne, anti-inflammatory, etc., but for Chinese herbal extracts The mechanism analysis of active ingredients and their formulation in cosmetics are still not clear enough. The development of low-dose, high-performance whitening and anti-oxidant makeup maintenance products from traditional Chinese herbal medicines will help to enhance the cosmetics technology industry.

In view of the above, there is a need to provide a plant material for DNA repair and a plurality of organisms that inhibit melanin production, in order to provide a cosmetic composition and/or a food additive.

Accordingly, one aspect of the present invention provides a composition of a peony bark extract for preparing repair DNA and inhibiting melanin production, wherein the peony bark extract is a monoterpene glycoside compound such as peony saponin. D (mudanpioside D, or bD-glucopyranoside), which has both biological activity for repairing DNA and inhibiting melanin production, can be further applied to cosmetic compositions and/or food additives.

According to the above aspect of the present invention, a peony bark extract is prepared for preparing a repair DNA which inhibits melanin production, and the peony bark extract is a peony rind D as shown in the formula (I):

And the composition has an effective amount of peony bark extract to simultaneously repair DNA and inhibit melanin production.

According to an embodiment of the invention, the composition is a cosmetic composition and/or a food additive.

The peony bark extract of the present invention is used for preparing a composition for repairing DNA and inhibiting melanin production, and the peony saponin D can be used for cosmetic composition and/or food addition because of both DNA repair and bioactivity of inhibiting melanin production. Things.

As described above, the present invention provides a composition of the peony bark extract for preparing repair DNA and inhibiting melanin production, which comprises an effective amount of peponin D to simultaneously repair DNA and inhibit melanin production.

The paper referred to here, "Paeonia" means Ranunculaceae (Ranunculaceae) Paeonia (P aeonia) Peony (Paenoina Suffruticosa) root bark (root cortex). Mudanpi is a kind of Chinese herbal medicine. It is mainly used in traditional Chinese medicine to provide functions of cooling blood, clearing away heat, promoting blood circulation and dispersing phlegm. The peony bark used in the present invention may be a commercially available product, or a mudanpioside D (or bD-glucopyranoside) obtained by extracting peony bark by a conventional dispensing extraction and/or column separation step.

Referring to Fig. 1, there is shown a flow chart showing a method for producing a peony bark extract of the prior art, which is referred to by the inventor of the present invention in Phytochemistry, Vol. 41, No. 1, pp. 237-242 (1996). The article "Monoterpene Glycosides From Paeonia suffruticosa " and the master's thesis (titled "Study on the Chemical Constituents of Mudanpi", 1994) are hereby incorporated by reference.

Briefly, the method 100 of making the peony bark extract can provide the peony bar 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 during the above-mentioned crude extraction step, using gauze, filter paper, centrifugation or other means. After repeating several times, the residue of the peony skin material 101 was removed, and the obtained filtrate was 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, using at least one partitioning extraction step and at least one column separation Steps 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. The first organic phase and the second organic phase. 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 can be selectively removed. 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 organic phase is subjected to a second partitioning extraction step using a third organic aqueous solution, such as an ethyl acetate/water solution, to fractionate the third organic phase and the first aqueous phase, wherein the third The organic phase is the ethyl acetate phase. In one example, the volume ratio of ethyl acetate to water of the third organic aqueous solution may be, for example, 1:1.

Thereafter, as shown in step 109, a third partitioning extraction step is performed on the first aqueous phase using a fourth organic solution, such as n-butanol, to separate the fourth organic phase from the second aqueous phase.

Then, as shown in step 111, a first rubber column chromatography step is performed on the third organic phase. In an example, the first cartridge column chromatography step can be performed using a commercially available silicone chromatography column and eluted with a first extract, wherein the first extract can be, for example, chloroform and a mixture of chloroform and methanol. (wherein the volume ratio of chloroform to methanol can be, for example, 97:3) to obtain the first dissolution Liquid and residue.

Subsequently, as shown in step 113, the residue and the fourth organic phase are combined, and a second rubber column chromatography step is performed and the second extraction liquid is used for the extraction, wherein the second liquid extract may be, for example, chloroform and a mixture of methanol (wherein the volume ratio of chloroform to methanol can be, for example, 17:3), and the second cartridge column chromatography step can be the same as that used for the first cartridge column chromatography step to obtain a corundum skin a second solution of the extract. In one example, the above-mentioned peony bark extract is a monoterpene glycoside compound, ie, pepiside D as shown in formula (I):

In one embodiment, after the second xylene column chromatography step, the second eluate is selectively subjected to a reverse phase chromatography column separation/HPLC step to purify the peony saponin D.

The above-mentioned purified pepiside D has been confirmed by in vitro tests to have both biological activity for DNA repair and inhibition of melanin production, and thus can be applied to cosmetic compositions and/or food additives.

The biological activity of "DNA repair" as referred to herein means that the above-mentioned 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 above-obtained pepiside D is exemplified by the following examples. The cell viability, the comet assay, and the like after UV irradiation do have biological activity for repairing DNA.

The biological activity of "inhibiting melanin production" as referred to herein means that the peony skin extract obtained above can effectively inhibit the activity of tyrosinase, thereby reducing the production of dopaquinone and melanin, thereby achieving 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 above-obtained pepiside D is exemplified by inhibiting the activity of tyrosinase, inhibiting extracellular and intracellular melanin content, and down-regulate The expression of related proteins (eg, MC1R, MITF, tyrosinase, TRP-1, TRP-2, etc.) during melanogenesis to inhibit melanin production.

It is worth mentioning that the present invention proves that puerarin D has both the function of repairing DNA and inhibiting melanin production by in vitro and in vivo tests, and thus can be applied to cosmetic compositions and/or food additives. . For example, when the pepiside D obtained by the present invention is 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 any combination thereof. cosmetic. Further, when the pepiside D obtained by the present invention is applied to a food additive, the form thereof may include, but is not limited to, a nutraceutical or a health food.

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

Example 1: Preparation of Cortex Mouth Extract

This example produces a peony bark extract using the method 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 45.0 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 time, or after extracting the residue with a 70 volume percent ethanol solution and repeating several times, the residue of the peony skin material 101 is removed, and the filtrate obtained as described above is 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 6.8 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 5.09 kg) and n-hexane phase (about 1.61 kg), of which 95% (v/v) methanol phase contains 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 1:1 to fractionate the ethyl acetate phase (about 0.178 kg) and the first water. phase.

Thereafter, as shown in step 109, the first aqueous phase is treated with n-butanol. A third partitioning extraction step is performed to divide the fourth organic phase from the second aqueous phase.

Then, as shown in step 111, the first ethyl acetate phase is subjected to a first xylene column chromatography step to obtain a first eluent and a residue. In the above example, the first cartridge column chromatography step may utilize a commercially available silicone column, such as Kiesilgel 60 (70-230 mesh or 230-400 mesh; Merk, Darmstadt, Germany), and utilizes chloroform and A mixture of chloroform and methanol (in which the volume ratio of chloroform to methanol was 97:3) was extracted as the first extract.

Subsequently, as shown in step 113, the residue and the fourth organic phase are combined, and the second rubber column chromatography step is carried out and washed with chloroform and methanol in a volume ratio of 19:1, 9:1 or 17:3, respectively. In the above, the leaching solution containing the peony bark extract is obtained by pulverizing with 17:3 by volume of chloroform and methanol as the second extract. The obtained second eluent can be selectively subjected to a reverse phase chromatography column separation/HPLC step, and the pepiside D as shown in the formula (I) can be purified, and has a structure represented by the formula (I):

The physicochemical properties of the above obtained pepiside D were as follows: molecular weight (MW) was 510; melting point was 122 ° C to 127 ° C; UV (EtOH) λ _max nm (log ε): 258 (4.20); IR (KBr) v _max cm ^-1 : 3393,1702,1603,1510; ¹ H-NMR (δ mult. ( J in Hz), pyridine-d ₅ ): OCH ₃ (3.67, s), H-3 (2.31 (d, 12.2) and 2.49 (d, 12.2)), H-5 (3.09 (d, 6.8),), H-6 (2.32 (d, 10.5) and 2.91 (dd, 10.5, 6.8)), H- 8 (5.12 (d, 12.1) and 5.23 (d, 12.1)), H-9 (5.94, s), H-10 (1.67, s); ¹³ C-NMR (δ, pyridine-d ₅ ): OCH3 ( 55.2), C-1 (88.7), C-2 (85.8), C-3 (44.6), C-4 (105.8), C-5 (43.7), C-6 (23.3), C-7 (71.6) ), C-8 (60.9), C-9 (101.6), C-10 (19.6); FAB-MS m/z (rel.int.%): 509 ([MH ⁺ ](20), 151 (100 ), 137 (15), 121 (13).

The above-mentioned peony saponin D 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 saponin D

In this example, first, it was tested whether the peony saponin D of Example 1 is 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. For example, a mouse melanoma cell line (B16; ATCC number: CRL-6322), and the hepatocytes for testing may be, for example, a normal rat liver cell (BNL CL.2; ATCC TIB-73), wherein the ATCC is typical of the United States. Abbreviation for the American Type Culture Collection (ATCC), BCRC is the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan. Abbreviation for).

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 each 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 saponin D or vitamin C (ascorbic acid, AA; ascorbic acid; Sigma Chemical Co, St. Louis, USA) of Example 1 were separately added to the above cells, wherein the vitamins The C line 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 saponin D or vitamin of Example 1. A graph of cell viability after 72 hours of C action. Fig. 2A to Fig. 2F show the cell survival rate (%) of the untreated control group as 100%, and the cell survival rate of the other treatment groups was converted to a value obtained by comparison with the untreated control group, wherein Figure No. ● represents the cell viability curve of HaCaT cell line, the figure number ▲ represents the cell viability curve of B16 cell line, and the figure number ◆ represents the cell viability curve of BNL CL.2 cell line, and the figure number is representative. The cell survival rate curve of the Hs68 cell line, the figure number ☆ represents the cell viability curve of the addition of the peony saponin D of Example 1, and the figure number ▽ represents the cell survival rate curve of the addition of vitamin C. Each value is at least greater than or equal It was obtained from 3 sample numbers.

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, as is apparent from the results of Fig. 2E, the higher the concentration of the peony bark extract of Example 1 added to the HaCaT cell line, the higher the HaCaT cell survival rate. In contrast, as can be seen from the results of the 2D graph, the cell viability of the high concentration (100 μM) of vitamin C in the BNL CL.2 cell line is only about 50%, and it is speculated 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 efficacy of pecitin D in inhibiting mushroom tyrosinase activity in vitro

Tyrosine is catalyzed by tyrosinase to form dominophenylalanine (DOPA). When DOPA is catalyzed by tyrosinase, it forms a series of DOPAquinone. The catalytic reaction causes melanogenesis, and the tyrosinase activity inhibition test of the extracellular preliminary screening is performed by the tyrosinase obtained in the mushroom.

In this test, the peony saponin D of Example 1 was formulated to a concentration of 0, 5 μM, 25 μM, 50 μM, and 100 μM. In the control group, vitamin C powder was taken and freshly prepared to a concentration of 0, 5 μM, 25 μM, 50 μM, and 100 μM, and stored in the dark. Next, take 2 μL of Mucortin D (test group) or Vitamin C (positive control group) in 18 μL of DMSO, add to 96-well plate, or add 25 μL of 100 Units of mushroom tyramine. The acidase solution was reacted at room temperature for 10 minutes to determine the background value of the mushroom tyrosinase at a wavelength of 475 nm. Subsequently, 155 μL of a 2.5 mM L-DOPA solution was added in the dark to mix at room temperature. Then, using a spectrophotometer, e.g. Synergy ^TM 2 multifunction machine microplate readings (Multi-Mode Microplate Reader; BioTek , USA), measuring the absorbance of each well of mushroom tyrosinase, and then by the formula (II) The inhibition rate of tyrosinase activity was calculated, and the inhibitory activities of the compounds at different concentrations on the mushroom tyrosinase were calculated. The results are shown in Fig. 3:

In formula (II), Ab stands for solvent, tyrosine, PBS, tyrosinase, but does not contain pepiside D or vitamin C; At represents peony glucoside D and tyrosine, PBS, tyramine Acidase; and A0 represents peony saponin D or vitamin C, tyrosine, PBS, but does not contain tyrosinase.

Please refer to Fig. 3, which is a histogram showing inhibition of mushroom tyrosinase activity by the use of the peony glucoside D of the first embodiment, wherein the horizontal axis is tyrosinase or vitamin C, and the vertical axis is mushroom tyrosinase. The inhibition activity rate (%). As is apparent from the results of Fig. 3, the pebbardin D of Example 1 inhibited tyrosinase activity and reached an inhibition rate of about 60% to 70% of tyrosinase. Compared with the degree of inhibition of tyrosinase activity by the same concentration of vitamin C, the degree of inhibition of tyrosinase activity by peony saponin D of Example 1 is about 80% of that of vitamin C. right.

2. Analysis of extracellular melanin content

This assessment measures the amount of extracellular melanin. Briefly, B16 cells per well in 96-well plates Species 1.0 × 10 ⁵ cells, and then maintained under a carbon dioxide concentration of 5% humidity of at least 24 hours culture at 37 ℃. Thereafter, 5 μM to 100 μM of puerarin D or vitamin C (AA) was mixed in a cell culture medium DMEM containing 100 nm of α-MSH and containing no phenol red. After the B16 cells were treated for 72 hours at 37 ° C, 100 μL of the cell culture solution per well was added to a 96-well plate, and the cell culture solution of each well was measured by a spectrophotometer, such as the above-mentioned multifunctional microplate reader. The absorbance at 405 nm was compared with the control group (final concentration of DMSO 0.1%) and the positive control group (vitamin C). Then, the absorbance of the peony saponin D or the vitamin C itself is calculated, and converted into the ability of melanin D or vitamin C to inhibit melanin production after acting on the cells.

Please refer to FIG. 4A, which is a histogram for evaluating the relative ratio of pepiside D to inhibiting extracellular melanin content in Example 1, wherein the horizontal axis is saponin D and vitamin C, and the vertical axis is inhibition. The relative ratio (%) of extracellular melanin content. The long strips filled with the upper right oblique line indicate treatment with lycopene D at a concentration of 5 μM and vitamin C. The long strips filled with the upper left oblique line indicate treatment with peony saponin D at a concentration of 25 μM and vitamin C. Filling the strips of the cross-line means treating with 50 μM of puerarin D and vitamin C. Filling the black strips means treating with 100 μM of puerarin D and vitamin C.

From the results of Figure 4A, it can be seen that compared with the control group, pepiside D can effectively reduce the formation of melanin outside B16 cells, and with vitamin C. The effect is quite. Therefore, the above results confirmed that the peony saponin D of Example 1 can effectively reduce the formation of extracellular melanin.

3. Analysis of intracellular melanin content

This evaluation is based on the determination of the extracellular melanin content described above to further evaluate the intracellular melanin content determination. After the cells were washed with PBS and then drained, the cells were lysed by adding 100 μL of 1N NaOH, and then the cell lysate was measured at 405 nm using a spectrophotometer, such as the above-mentioned multifunctional microplate reader. The absorbance value was compared with the control group (final concentration of DMSO 0.1%) and the positive control group (vitamin C). Then, the absorbance of the peony saponin D or the vitamin C itself is calculated and converted into melanin content of the peony saponin D or vitamin C.

Please refer to FIG. 4B, which is a histogram for evaluating the relative ratio of the peony saponin D to inhibiting the melanin content in the cells, wherein the horizontal axis is melanin D and vitamin C, and the vertical axis is inhibition. The relative ratio (%) of melanin content in cells. The long strips filled with the upper right oblique line indicate treatment with lycopene D at a concentration of 5 μM and vitamin C. The long strips filled with the upper left oblique line indicate treatment with peony saponin D at a concentration of 25 μM and vitamin C. Filling the strips of the cross-line means treating with 50 μM of puerarin D and vitamin C. Filling the black strips means treating with 100 μM of puerarin D and vitamin C.

As can be seen from the results of Fig. 4B, pepiside D was effective in reducing the formation of melanin in B16 cells compared with the control group, and was equivalent to the effect of vitamin C. Therefore, the above results confirmed that the peony saponin D of Example 1 can effectively reduce the formation of melanin in cells.

4. Assess the performance of related genes and proteins during melanin production (1) Western blotting analysis (I) (1.1) Extracting RNA

B16 cells were seeded in a 6 well plate at a density of 1 × 10 ⁵ /ml, cultured for 24 hours, and 100 μM of pecitin D and vitamin C were separately mixed in a fresh medium containing 100 nM α-MSH. After 0, 24, 48, and 72 hours, total RNA was extracted and quantitatively analyzed.

The RNA concentration obtained above can be known by a conventional method using a spectrophotometer to measure the absorbance at OD _{260 nm} and OD _{280 nm} . Those of ordinary skill in the art to which the present invention pertains should be well known and will not be further described herein.

After the total RNA obtained is quantified, the real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) is used to detect whether the peony peel extract of the first embodiment regulates the NER system. Related gene expression.

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 55 ° C for 50 seconds to 60 seconds at 55 ° C The primer is bonded to 60 ° C for 50 seconds to 60 seconds, and the DNA is extended at 72 ° C for 50 seconds to 2 minutes, thereby repeating the reaction for 40 cycles, and then repeating the reaction for 40 cycles. It is carried out at 72 ° C for 5 minutes to 7 minutes. After the cycle is completed, the PCR product is subjected to agar electrophoresis analysis, and the fluorescence intensity of the related gene of the obtained NER system is analyzed by using a commercially available software such as an amplification plot or other functional replacement software, and the result is as shown in FIG. 5A. Show.

Please refer to FIG. 5A, which is a photograph showing the agar colloid of the related gene expression of the B16 cell line in the melanin production using the peony saponin D of Example 1. The value above each band represents the gene related to melanin production. The gene expression amount (density ratio; obtained from fluorescence intensity) relative to β-actin.

From the results of Figure 5A, the B16 cell line significantly inhibited the genes involved in melanin production after 24 hours of reaction with pepiside D (eg MC1R, MITF, tyrosinase, TRP- compared to the control group). 1. The amount of TRP-2, etc., shows that the peony bark extract of Example 1 can inhibit the expression of related genes when melanin is produced, and contributes to the inhibition of melanin production.

(2) Western blotting analysis (I) (2.1) Extracting proteins

The experimental group B16 cells of the above 10 cm ² culture dish were reacted with 100 nM α-MSH and different concentrations of pepiside D or vitamin C for 72 hours, and then the supernatant was separately collected into a 15 mL centrifuge tube and reused. Trypsin (1×) was used to remove the cells, and the supernatant was removed by centrifugation, followed by protein extraction, and the protein expression was analyzed by Western blotting.

The protein concentration obtained above can be determined by a conventional method using different concentrations of BSA, and the absorbance of the extracted protein is brought into a linear regression formula to be converted. 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, the standard protein MW marker is sequentially added to After the proteins of the above groups, the electrophoresis was carried out 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, trade name PolyScreen PVDF Hybridization Transfer Membrane, PK-NEF1002, PerkinElmer, USA), is immersed 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 added with FITC secondary antibody (1:1000) at 4 ° C for 2-3 hours. After that, the PVDF transfer was washed with 0.1% PBST at 4 °C. The film was reacted 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 luminescence image analyzer described above can detect the intensity of Luminol (Cyclic phthalhydrazides) released from the ground state when excited to an excited state and then return to the ground state according to the stored image, and quantitatively analyze.

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

Please refer to FIG. 5B, which is a Western blot analysis diagram showing the expression of related proteins of the B16 cell line in the melanin production using the peony saponin D of Example 1. The value above each band represents the time when melanin is produced. The protein expression of the related gene relative to β-actin (density ratio; obtained from fluorescence intensity).

As can be seen from Fig. 5B, compared with the control group, the B16 cell line can significantly inhibit the formation of melanin after reacting with pepiside D for 24 hours. The amount of protein (for example, MC1R, MITF, tyrosinase, TRP-1, TRP-2, etc.) shows that the peony bark extract of Example 1 can inhibit the expression of related proteins in melanin production, which is helpful to achieve Inhibits melanin production.

The above results show that the peony saponin D of Example 1 can inhibit the synthesis of mRNA and protein of related genes when melanin is produced, thereby inhibiting melanin production.

(3) Evaluation of related protein expression during melanin production (II)

The experimental group B16 cells of the above 10 cm ² culture dish were reacted with 100 nM α-MSH and different concentrations of pepiside D or vitamin C for 72 hours, and then the supernatant was separately collected into a 15 mL centrifuge tube and reused. Trypsin (1×) was used to remove the cells. After removing the supernatant by centrifugation, 500 μL of 4% paraformaldehyde was added to react at 4 ° C for 1 hour to fix the cells and analyze the expression of related proteins.

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 a primary antibody such as the fourth panel 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. 5C.

Please refer to FIG. 5C, which is a three-dimensional graph showing the expression of related proteins of the B16 cell line in the melanin production by using the peony saponin D of the first embodiment, wherein the x-axis represents the fluorescence intensity and the y-axis represents the different groups. Z-axis generation The number of cells in the table. The y-axis is from the first to the sixth, respectively, from the first to the sixth, wherein the first track represents the expression of melanin-related protein in the negative control group (B16 cell line without any treatment), and the second channel represents α-MSH. The expression level of the melanin-producing protein in the control group cells was treated, and the third lane indicates the expression level of the melanin-producing protein in the experimental group cells (treated by α-MSH and 5 μM of peponin D), and the fourth lane indicates the experimental group. The expression of melanin-producing proteins in cells (treated with α-MSH and 25 μM of puerarin D), and the fifth lane indicates melanin-producing proteins of experimental cells (treated with α-MSH and 50 μM of peponin D) The amount of expression, lane 6 indicates the amount of melanin production-related protein in the experimental group cells (treated with α-MSH and 100 μM of puerarin D).

As can be seen from Fig. 5C, the performance of MC1R, MITF, tyrosinase, TRP-1, and TRP-2 was decreased after 72 hours of treatment with 100 μM of peony glucoside D in the experimental group compared with the control group. Example 1, pepiside D can inhibit the expression of related proteins in the production of melanin in cells, and help to inhibit the production of melanin in cells.

Example 4: Evaluation of the ability of pepiside D to protect skin cells

This example uses the peony saponin D of Example 1 to further evaluate the protective ability of skin cells after UVB irradiation with an irradiation energy of 10 J/m ² , 20 J/m ² or 50 J/m ² .

1. 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. The culture was carried out for 4 hours, wherein the serum-free medium was not added (ie, the UVB control group was irradiated only with UVB) or 5 μM, 25 μM, 50 μM, and 100 μM of the peony saponin D of Example 1 (ie, the experimental group was added after irradiation with UVB). Mudan peel extract). Then, the cell survival rate was evaluated by the MTT method of Example 2, and the change in cell type was observed in an optical microscope imaging system, and the results of cell viability were as shown in Figs. 6A and 6B.

Please refer to FIGS. 6A and 6B for cell viability of the HaCaT cell line (Fig. 6A) and Hs68 cell line (Fig. 6B) after treatment with UVB using the peony saponin D of Example 1. The graph, in which the horizontal axis is the irradiation energy of different UVB (J/m ² ), the different doses of the peony bark extract are used, and the vertical axis is the cell survival rate (%).

From the results of Figs. 6A and 6B, 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), as the UVB irradiation energy increased, the Hs68 cell line showed a gradual decline with the increase of UVB irradiation energy. However, the cell survival rate of HaCaT cell line decreased, but no such trend.

Furthermore, in the experimental group (50 μM or 100 μM of the peony saponin D of Example 1 after UVB irradiation), the HaCaT cells after UVB irradiation were significantly reduced as the concentration of peponin D in 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 saponin D of Example 1 is 0 μM), with the examples The concentration of peony saponin D increased, which significantly increased cell viability, as shown in Figure 6A. 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. 6B. Therefore, the following HaCaT cells were selected to evaluate the DNA protection of peony saponin D of Example 1 and the ability to promote DNA replication.

2. 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 or no reaction with peponin D), UVB control group (UVB only but not with peponin D) and experimental group (50 μM before and after UVB irradiation) Group I) or 100 μM (experimental group II) of pepiside D 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 peponin D before UVB irradiation to evaluate its protective ability against DNA. The reaction with peony glucoside D 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 slide was immersed in an enzyme reaction buffer (40 mM Hepes, 0.1 M KCl, 0.5 mM EDTA, and 0.2 mg/ml BSA, adjusted to pH 8 with KOH) at 4 ° C for 15 minutes to remove Most of the proteins such as cell membranes and nuclear membranes.

Thereafter, the slides were further immersed in an alkaline buffer solution (alkaline buffer; containing 0.3 M NaOH and 1 mMm 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 sensitivity of the analysis.

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) × Peripheral DNA percentage)]. The higher the value of the tail length percentage, the tail brightness percentage, and the tail momentum obtained above, the more serious the DNA damage, and the results are shown in Figs. 7A and 7B.

Please refer to Fig. 7A, which is a photograph of a comet assay of HaCaT cell line treated with the peony saponin D of Example 1 and irradiated with UVB, wherein the reference line length of Fig. 7A is 50 μm. In Fig. 7A, the first picture from the left side of the first line represents the control group. From the left side of the first line The 2 to 4 photographs 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 left or upper right corner of each photo magnifies the DNA pattern of the cells randomly sampled.

The results from Figure 7A show that as the UVB irradiation energy increases, the tailing phenomenon caused by damage to the cellular DNA is more pronounced, as in the UVB control group of Figure 7A (ie, the second to fourth photos from the left side of the first line). ) shown. Secondly, with the increase of the addition amount of pepiside D in the first embodiment, the comet tailing phenomenon caused by DNA damage can be effectively reduced, as shown in the experimental group (Fig. 7 to the third row) of Fig. 7A. It is proved that the peony saponin D of the first embodiment has the ability to protect and repair skin cells. After the DNA damaged cells of Fig. 7A were counted, the results are shown in Fig. 7B.

Please refer to FIG. 7B, which is a histogram showing the number of damaged cells of the HaCaT cell line treated with the peony saponin D of Example 1 and irradiated with UVB, wherein the horizontal axis is the irradiation dose of UVB, and the vertical axis. The number of cells damaged by DNA per 1000 cells, and the control group represents HaCaT cells that were not irradiated with UVB.

As is apparent from the results of Fig. 7B, as the amount of the addition of pepiside D in the first embodiment was increased, the number of cells damaged by DNA was effectively reduced, and it was confirmed that the peony saponin D of Example 1 has the ability to protect cellular DNA.

3. Assessment (III) - Cell Cycle

This example further evaluates the effect of the peony saponin D of Example 1 on the cell cycle. In summary, the cell cycle analysis mainly quantitatively analyzes the DNA content and the ploidy state, and uses a fluorescent dye that can penetrate the cell membrane (for example, a red propidium iodide (PI) dye solution) to dye the dye. After the molecules were embedded in 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 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 peponin D), UVB control group (only UV was irradiated but not reacted with pepiside D), and experimental group (both erythroside before and after UVB irradiation) D reaction), in which the experimental group was reacted with 50 μM (experimental group I) or 100 μM (experimental group II) of pepiside D for 4 hours before UVB irradiation, after UVB irradiation for 2 hours or 4 hours, and then The same concentration of pepiside D 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 numerically analyzed, and the results are shown in Fig. 8.

Please refer to Fig. 8, which shows the cell cycle distribution of the HaCaT cell line treated with or without UVB using the peony saponin D of Example 1, wherein the horizontal axis of Figure 8 represents time and the vertical axis represents cells. Logarithmic value. In Fig. 8, the first photograph from the left side of the first line and the left side of the second line shows the cell cycle distribution map 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.

From the results of Fig. 8, it was revealed that the cells of the UVB control group had a decrease in the G ₀ /G ₁ phase of the cells 2 hours after UVB irradiation, but the Sub-G ₁ and S phases increased compared with the control group. 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 saponin D 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 saponin D of Example 1 does affect cell cycle distribution and enhances the DNA synthesis phase of SG ₂ /M.

4. Evaluation (IV)-BrdU immunofluorescence staining

When cells are subjected to DNA synthesis, an analog of thymidine, such as 5-bromo-deoxy-uridine (BrdU), may be added to cause misincorporation and insertion into the DNA. Thereby, DNA can be calibrated and the activity of DNA synthesis in living cells can be detected. 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 peponin D) and multiple experimental groups (including UVB irradiated with 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 puerarin D 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 fluorescent-conjugated FITC secondary antibody was added thereto, and the reaction was allowed to stand at room temperature for 3 hours in the dark. Then, it was 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 fluorescent expression produced by antibody binding was calculated and the fluorescence ratio was calculated. The results are shown in Figures 9A to 9C.

Please refer to Fig. 9A and Fig. 9B, respectively, showing BrdU of HacaT cell line (Fig. 9A) and Hs68 cell line (Fig. 9B) using the peony saponin D of Example 1 for or without UVB irradiation. Fluorescence intensity histogram, where the horizontal axis represents the control group and the experimental group with different conditions, and the vertical axis represents the different BrdU fluorescence intensity (%).

Fig. 9C is a photograph showing the cells of the HaCaT cell line stained with immunofluorescence using the peony saponin D of Example 1 (magnification: 200 times), wherein the 9C chart is counted from the left side (straight) The photographs are HaCaT cell images observed by phase contrast optical microscopy, the second row (straight) photographs are HaCaT nuclear images observed by Hoechst 33342 fluorescent staining, and the third row (straight) photographs are HaCaT observed by BrdU fluorescent staining. DNA image of the cell line. From the upper left side, the first column (horizontal) from the first to the third photo represents the control group, the second column (horizontal) from the first to the third photo represents the UVB control group, and the third column (horizontal) is the first The third piece of Zhang Zhi represents the experimental group. The larger square in each photo is enlarged to show random sampling in the middle smaller square. Cell type.

From the results of Fig. 9A and Fig. 9B, the BrdU fluorescence of the HaCaT cell line and the HaCaT cell line which did not react with pepiside D 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 pepiside D. Especially in the experimental group with UVB irradiation dose of 10 J/m ² , pepiside D significantly enhanced cell proliferation. However, no regular dose response was observed in the Hs68 cell line.

Secondly, the results from the 9C chart show that compared to the UVB control group (the second column (horizontal) from the top left (the first to the third photo)), the experimental group (from the left side of the third column (horizontal The 1st to 3rd HaCaT cell lines significantly increased the fluorescence content of BrdU by reacting with 100 μM of peony glucoside D, demonstrating that the peony glucoside D of Example 1 can promote DNA replication and BrdU- FITC is able to embed the new synthetic sequence of DNA replication, greatly improving the fluorescent performance of BrdU.

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

Example 5: Evaluation of the ability of peponin D to repair skin cells

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

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

Nucleotide excision repair (NER) system mainly repairs due to ultraviolet radiation DNA damage. When cells perform NER repair DNA, it is known that the DNA damage is identified by XPC combined with HR23E protein, and the DNA of the lesion is loosened by TFIIH, XPB and XPD helicase, and the complex formed by XPA and RPA binds to DNA damage. At the 5' end of the cleavage of the DNA endonuclease XPF/ERCC1 and the 3' end of the XPC. 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 peponin D), UVB control group (only 10 J/m ² UVB but not with peponin D) and experimental group (irradiation 10) J/m ² is reacted with peony saponin D before and after UVB). 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 pepiside D 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 peponin D. 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, the real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) is used to detect whether the peony saponin D of the first embodiment regulates the NER system. Related gene expression.

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 to which the above respective genes are applied are shown, for example, in Table 5.

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. 10A.

Please refer to FIG. 10A, which shows the related gene expression of the nucleotide excision repair system (NER) of the HaCaT cell line with or without UVB irradiation using the peony saponin D of Example 1, wherein the horizontal axis is HaCaT cells. The time (hours) in which the strain reacts with peponin D, and the vertical axis is the phase of the NER system. Gene expression relative to β-actin (density ratio; obtained from fluorescence intensity).

From the results of Fig. 10A, the HaCaT cell line was reacted with 50 μM of pepiside D for 4 hours before irradiation with UVB, and reacted with 50 μM of peony saponin D after irradiation with UVB. Hours can significantly increase the amount of XPC gene expression. As for other genes, such as XPA gene, RPA gene, ERCC1 gene, XPF gene, XPG gene and PCNA gene, the expression amount also increased with the increase of reaction time, indicating that the peony saponin D of Example 1 can increase the NER system. The amount of gene expression helps to repair DNA damage caused by HaCaT cells after UV irradiation.

(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 pepiside D 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, a 10% running gel was prepared according to the second table of Example 2, and then 7.5% of the stacking gel was prepared according to the third table of Example 2, and it is also not mentioned here.

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, trade name PolyScreen PVDF Hybridization Transfer Membrane, PK-NEF1002, PerkinElmer, USA), is immersed 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. Minute Clock, transfer the protein onto the 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 luminescence image analyzer described above can detect the intensity of Luminol (Cyclic phthalhydrazides) released from the ground state when excited to an excited state and then return to the ground state according to the stored image, and quantitatively analyze.

Please refer to FIG. 10B, which shows a Western blot analysis of the protein expression of the nucleotide excision repair system (NER) of the HaCaT cell line with or without UVB irradiation using the peony saponin D of Example 1. Wherein, the first track represents the expression amount of the NER-related protein in the control group, the second track represents the expression amount of the NER-related protein in the UVB control group, and the third track represents the expression amount of the NER-related protein in the experimental group, wherein the embodiment is before the UVB irradiation It was reacted with 50 μM of pepiside D for 4 hours, and then reacted with the same concentration of peponin D for 8 hours after UVB irradiation at 10 J/m ² .

From the results of Fig. 10B, it was found that the HaCaT cell line of the experimental group was reacted with 50 μM of puerarin D for 4 hours before irradiation with UVB, and with 50 μM of peony bark after UVB irradiation, compared with the UVB control group. After 8 hours of reaction with glycoside D, the expression levels of XPC, XPA, RPA, ERCC1, XPF, XPG and PCNA were 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 pepiside D 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. Subsequently, 5 μL of 10% Triton X-100 was added thereto for 5 minutes at 4° C., and the supernatant was removed by centrifugation, and the primary antibody shown in the fourth table of Example 2 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. 10C.

Please refer to FIG. 10C, which shows a nucleotide excision repair system for HaCaT cell line with or without UVB irradiation using the flow cytometry Example 1 according to several embodiments of the present invention. NER) A three-dimensional graph of the expression of related proteins, 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 performance of the third lane indicates the expression of NER-related protein in the UVB control group, and the fourth lane indicates the performance of the NER-related protein in the experimental group I (reacted with 50 μM of peponin D), and the fifth lane indicates the experimental group II. (Reaction with 100 μM of puerarin D) The amount of NER-related protein.

As can be seen from Fig. 10C, 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 pepiside D. It was confirmed that the peony saponin D 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 saponin D of the first embodiment can enhance the DNA and protein synthesis of the NER-related gene, thereby promoting cell repair.

Example 6: Evaluation of the ability of pepiside D to repair living skin cells

This example was prepared by using the peony saponin D of Example 1 to be applied to a living skin to further evaluate its ability to repair skin cells after UVB irradiation.

First, a pebbina D gel was prepared by disposing the peony saponin D of Example 1 at a concentration of 100 μM, and each 10 μL was added to 1 mL of physiological saline to be dispersed, and 2 g of hydroxyl was weighed. Ethyl cellulose The (hydroxyethyl cellulose; HEC) gel was added and completely dissolved, and sealed with a parafilm seal to protect the light. The colloid was thickened and agglomerated over 24 hours to prepare a peony saponin D gel (containing 2% HEC). With 20 μg/mL of peponin D) spare.

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. The agent (containing 2% HEC) is ready for 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.

5 mice were randomly selected from the placebo control group and measured with a skin colorimeter. The L, a, and b values of each mouse were recorded, and 0.05 g of each placebo was applied without any UVB irradiation, and L, a was measured by a color difference meter at 0, 2, 4, 6, and 8 hours. , b value, and simultaneously punching the skin tissue. 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 quercetin D 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 paeonin D gel 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 whether or not the morphological structure of the cell tissues was changed, and the results are shown in Fig. 11A. 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, the 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 washed 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 11B to 11I. Then, the cells were washed twice with PBS, and the coverslips were covered with a sealing gel. The edges of the coverslips were fixed on the glass slides with transparent nail polish, and fluorescence imaging analysis was performed under a microscope. The result is shown in Fig. 11B. The fluorescent staining of the NER-related protein to Figure 11I is shown.

Please refer to FIGS. 11A to 11I, which show histochemical staining (Fig. 11A) and nuclear staining of a gel prepared by using the peony saponin D of Example 1 according to several embodiments of the present invention. (Fig. 11B to Fig. 11I) and NER-related protein fluorescence staining (Fig. 11B to Fig. 11I) results. From the results of the H&E stain of Fig. 11A, 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 D-gel was relatively complete. Secondly, the results of nuclear staining (BrdU) from Fig. 11B showed that the amount of BrdU expression increased as the reaction time of the peony bark extract gel increased. Furthermore, the results from Fig. 11C to Fig. 11I show that the expression of NER-related proteins such as XPC, XPA, RPA, ERCC1, XPF, XPG and PCNA after NER-related proteins are treated by compounds (1) and (2) The reaction time of the paeonin D gel increased and it was confirmed that the peony saponin D of Example 1 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 7: 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 pepiside D 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) The two were diluted with 8 volumes of PBS and 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 and 20 mM, respectively). Next, the pUC119 DNA Plasmid was irradiated with 200 J/m ² of UVB, and the λ Hind III label was irradiated with 3500 J/m ² and 4000 J/m ² of UVB, and 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). After treatment with the above 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 is 200 J/m ² , the plastid DNA can also be completely cut into a linear (LN) structure (not shown). 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. Evaluate the ability of peony bark extract to protect DNA against oxidation and avoid DNA damage

In this example, 50 μM or 100 μM of peony D 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} separate pUC119 plastid DNA or λHind III DNA. Exposure to 5 mM and 20 mM H ₂ O ₂ , and irradiation with UVB at an irradiation dose of 200 J/m ² and 3500 J/m ² , respectively, and then using the above evaluation method to confirm whether peponin D can protect DNA against oxidation 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 were the same as described above, and no rumors are made here.

For the experimental group I, take 2 μL of the above pUC119 plastid DNA diluted with PBS (0.056 μg/μL), and add 3 μL of the peony saponin D of Example 1 (concentration 50 μM or 100 μM), and then 5 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 the experimental group II, 2 μL of the above-mentioned λHind III labeled with PBS (0.056 μg/μL) was added, and 3 μL of the peony saponin D of Example 1 (concentration of 50 μM or 100 μM) was added, and then each and 5 μL. After mixing with 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 12A to 12B.

Please refer to Fig. 12A to Fig. 12B, which are diagrams showing the electrophoresis of nucleic acid against the oxidation of DNA by using the peony saponin D of the first embodiment, wherein the 12A map shows that the pUC119 plastid DNA is subjected to different concentrations of H ₂ O _{2 .} And/or nucleic acid electrophoresis patterns after UVB treatment of different energies, and Fig. 12B shows nucleic acid electrophoresis patterns of λHind III labeling treated with different concentrations of H ₂ O ₂ and/or UVB of different energies.

The results of the nucleic acid electrophoresis patterns from Fig. 12A to Fig. 12B show that the plastid DNA of pUC119 is mainly a supercoiled (SC) structure (as shown in the control group of Fig. 12A), 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 12A). When pUC119 plastid DNA was first added to the peony saponin D of Example 1, and then exposed to 5 mM or 20 mM H ₂ O ₂ and UVB irradiation energy of 200 J/m ² , 50 μM or 100 μM peony saponin D is effective in protecting and avoiding that most of the plastid DNA is not sheared into a linear (LN) structure, as shown by the band of experimental group I in Figure 12A.

Similar results can be obtained with Figure 12B. The results of the electrophoresis pattern of the nucleic acid shown in Fig. 12B 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 12B. When the λHind III label was added to the peony saponin D 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 peponin D 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 12B.

Based on the results of the above examples, the peony saponin D of the first embodiment 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 the present invention is exemplified by specific process conditions, specific cell strains, specific analytical methods or specific instruments, and illustrates the application of the peony bark extract of the present invention and a method for producing the same, but any of the technical fields of the present invention. It is to be understood by those skilled in the art that the present invention is not limited thereto, and the peony peel extract of the present invention can be carried out using other process conditions, other cell strains, other analytical methods or instruments without departing from the spirit and scope of the present invention. Further, when the pepiside D obtained by the present invention is applied to a cosmetic composition, the form thereof may include, but is not limited to, a liquid, an emulsion, an ointment, a powder, a whitening agent, a spotting agent, a freckle agent, or a cosmetic of any combination thereof. Further, when the pepiside D 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.

It can be seen from the above embodiments of the present invention that the peony bark extract of the present invention and the method for producing the same have the advantages that the peony glucoside D of the monoterpene glycoside compound 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 quercetin D of the monoterpene glycoside compound of the present invention has various biological activities, the obtained pepiside D can be applied to a cosmetic composition and/or a food additive.

Although the invention has been disclosed above in several embodiments, it is not intended to The invention is defined by the general knowledge of the present invention, and various modifications and refinements can be made without departing from the spirit and scope of the invention. The scope is defined.

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 partition extraction step on the first extract with a third organic aqueous solution to obtain a third organic phase

109‧‧‧Step of using the fourth organic solution to carry out the third partition extraction step of the aqueous phase to obtain the fourth organic phase

111‧‧‧Step of performing a first xylene column chromatography step on the third organic phase to obtain the first eluent and residue

113‧‧‧ Combining the residue with the fourth organic phase, performing a second xylene column chromatography step to obtain a second eluate containing the peony bark extract

The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; .

2A to 2F show the HaCaT cell line, the B16 cell line, the BNL CL.2 cell line, and the Hs68 cell line according to several embodiments of the present invention, and the peony saponin D or vitamin C of Example 1 for 72 hours. Post-cell survival rate graph.

Figure 3 is a histogram showing inhibition of mushroom tyrosinase activity by the use of peony saponin D of Example 1.

Figures 4A and 4B are graphs showing the relative ratios of the peony saponin D for inhibiting extracellular melanin content (Fig. 4A) to intracellular melanin content (Fig. 4B), respectively.

Fig. 5A is a photograph showing the agar colloid of the gene expression of the B16 cell line in the case of melanin production using the peony saponin D of Example 1.

Fig. 5B is a Western blot analysis showing the expression of related proteins of the B16 cell line in the case of melanin production using the peony saponin D of Example 1.

Figure 5C shows the use of the peony saponin D of Example 1 for B16 A three-dimensional graph of the expression of related proteins in cell lines during melanin production.

Fig. 6A and Fig. 6B are graphs showing the cell viability of the HaCaT cell line (Fig. 6A) and the Hs68 cell line (Fig. 6B) after the UVB irradiation using the peony saponin D of Example 1.

Fig. 7A is a photograph showing a comet assay of a HaCaT cell strain treated with the peony saponin D of Example 1 and irradiated with UVB.

Fig. 7B is a histogram showing the number of cells damaged by the HaCaT cell line treated with the peony saponin D of Example 1 and irradiated with UVB. Fig. 8 shows the use of the peony saponin D of Example 1. Or cell cycle profile of HaCaT cell lines that were not irradiated with UVB.

Fig. 9A and Fig. 9B show the BrdU fluorescence intensity histogram of the HaCaT cell line (Fig. 9A) and the Hs68 cell line (Fig. 9B) with or without UVB irradiation, respectively, using the peony saponin D of Example 1. Figure.

Fig. 9C shows a photograph of a cell which was immunofluorescently stained for HaCaT cell strain using the peony saponin D of Example 1 (magnification: 200 times).

Figure 10A is a histogram showing the expression of related genes of the nucleotide excision repair system (NER) of the HaCaT cell line with or without UVB irradiation using the peony saponin D of Example 1.

Figure 10B is a Western blot analysis showing the protein expression of the nucleotide excision repair system (NER) of the HaCaT cell line with or without UVB irradiation using the peony saponin D of Example 1.

Figure 10C shows the correlation of the nucleoside excision repair system (NER) of the HaCaT cell line with or without UVB irradiation using the flow cytometry Example 1 according to several embodiments of the present invention. A three-dimensional graph of protein expression.

11A to 11I are respectively showing histochemical staining (Fig. 11A), nuclear staining, and NER-related protein of a gel prepared by using the peony saponin D of Example 1 according to several embodiments of the present invention. Fluorescent staining (Fig. 11B to Fig. 11I) results.

Figures 12A and 12B show respectively different concentrations of H ₂ O ₂ and/or different energies of pUC119 plastid DNA (Fig. 12A) and λHind III label (Fig. 12B) according to several embodiments of the present invention. The nucleic acid electrophoresis pattern after UVB treatment.

<110> Jianan University of Pharmacy and Technology

<120> Mudan peel extract, its manufacturing method and application

<130> None

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<210> 1

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<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR forward primer ( MC1R F)

<400> 1

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<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR reverse primer ( MC1R R)

<400> 2

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<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR forward primer ( MITF F)

<400> 3

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<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR reverse primer ( MITF R)

<400> 4

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<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR forward primer(Tyrosinase F)

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<212> DNA

<213> Artificial sequence

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<223> PCR reverse primer (Tyrosinase R)

<400> 6

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<213> Artificial sequence

<220> primer

<223> PCR reverse primer ( TRP-1 R)

<400> 8

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR reverse primer ( TRP-2 F)

<400> 9

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR reverse primer ( TRP-2 R)

<400> 10

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR forward primer( β-actin F)

<400> 11

<210> 12

<211> 17

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR reverse primer( β-actin R)

<400> 12

<210> 13

<211> 17

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR forward primer ( XPC F)

<400> 13

<210> 14

<211> 26

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR reverse primer ( XPC R)

<400> 14

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR forward primer ( XPA F)

<400> 15

<210> 16

<211> 22

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR reverse primer ( XPA R)

<400> 16

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR forward primer ( RPA F)

<400> 17

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR reverse primer ( RPA R)

<400> 18

<210> 19

<211> 19

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR forward primer ( ERCC1 F)

<400> 19

<210> 20

<211> 21

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR reverse primer( ERCC1 R)

<400> 20

<210> 21

<211> 18

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR forward primer( XPF(ERCC4) F)

<400> 21

<210> 22

<211> 23

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR reverse primer (XPF(ERCC4) R)

<400> 22

<210> 23

<211> 17

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR forward primer( XPG(ERCC5) F)

<400> 23

<210> 24

<211> 25

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR reverse primer ( XPG(ERCC5) R)

<400> 24

<210> 25

<211> 24

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR forward primer ( PCNA F)

<400> 25

<210> 26

<211> 26

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR reverse primer ( PCNA R)

<400> 26

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR forward primer( β-actin F)

<400> 27

<210> 28

<211> 17

<212> DNA

<213> Artificial sequence

<220> primer

<223> PCR reverse primer( β-actin R)

<400> 28

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 partition extraction step on the first extract with a third organic aqueous solution to obtain a third organic phase

109‧‧‧Step of using the fourth organic solution to carry out the third partition extraction step of the aqueous phase to obtain the fourth organic phase

111‧‧‧Step of performing a first xylene column chromatography step on the third organic phase to obtain the first eluent and residue

113‧‧‧ Combining the residue with the fourth organic phase, performing a second xylene column chromatography step to obtain a second eluate containing the peony bark extract

Claims (3)

A method for treating cells in vitro, characterized in that a skin cell or a melanocyte is treated in vitro by using an effective amount of a composition of mudanpioside D as shown in formula (I): Moreover, the peony saponin D has the biological activity of repairing DNA and inhibiting melanin production, thereby repairing DNA of the skin cells in vitro and inhibiting melanin production of the melanocytes. The method for treating cells in vitro according to the first aspect of the invention, wherein the composition is a cosmetic composition and/or a food additive. The method for treating cells in vitro according to claim 1, wherein the skin cells and one of the melanocytes are derived from human or mouse.