Application of peach extract in preparation of composition for improving skin and / or anemia

Abstract

The invention relates to application of a peach extract to preparation of a composition for improving skin and / or anemia, wherein the peach extract is extracted from red pulp of peaches.

Description

Technical Field

[0001] This invention relates to peach extract, and more particularly to the use of peach extract in the preparation of compositions for improving skin and / or improving anemia. Background Technology

[0002] The peach (Prunus persica), also known as the blood peach or wine peach, belongs to the subgenus Prunus (P. subg. Amygdalus) of the genus Prunus in the family Rosaceae.

[0003] In addition to health, modern women also pursue delicate skin and a rosy complexion, hence the development of more diverse and natural ingredients to assist women in their pursuit of beauty. Summary of the Invention

[0004] In view of this, the present invention provides a use of peach extract for preparing compositions that improve skin and / or anemia.

[0005] In some embodiments, the use of a peach extract for preparing a composition for improving skin, wherein the peach extract is extracted from the red pulp of a peach.

[0006] In one embodiment, peach extract is used to combat blue light damage.

[0007] In one embodiment, peach extract was used to enhance mitochondrial activity.

[0008] In one embodiment, peach extract is used to promote the production of elastin in the skin.

[0009] In one embodiment, peach extract is used to reduce skin texture.

[0010] In one embodiment, peach extract is used to reduce skin wrinkles.

[0011] In one embodiment, peach extract is used to shrink skin pores.

[0012] In one embodiment, peach extract is used to enhance the antioxidant capacity of skin fibroblasts.

[0013] In other embodiments, the use of a peach extract for preparing a composition to improve anemia is described, wherein the peach extract is extracted from the red pulp of a peach.

[0014] In one embodiment, peach extract is used to increase heme levels.

[0015] In one embodiment, peach extract is used to increase the iron content in the blood.

[0016] In one embodiment, the peach extract includes at least hyperoside, isoquercitrin, pyroside, and rutin.

[0017] In one embodiment, hyperoside, isoquercitrin, styracin, and rutin are used to combat blue light damage.

[0018] In one embodiment, hyperoside, isoquercitrin, styracin, and rutin are used to enhance the antioxidant capacity of skin fibroblasts.

[0019] In summary, according to any embodiment of the present invention, peach extract can be used to prepare compositions that improve skin condition or anemia. In some embodiments, the aforementioned compositions also have one or more of the following functions: resisting blue light damage, enhancing the antioxidant capacity of skin fibroblasts, enhancing mitochondrial activity, promoting the production of skin elastin, reducing skin texture, reducing skin wrinkles, minimizing skin pores, increasing heme content, and increasing the iron content in the blood. Attached Figure Description

[0020] Figure 1 This is a graph showing the experimental results of the blue light protection effect of peach extract.

[0021] Figure 2 This is a graph showing the experimental results of the antioxidant activity of peach extract.

[0022] Figure 3 This is a graph showing the experimental results of the relative elastin content of peach extract.

[0023] Figure 4 This is a graph showing the results of an experiment on the relative mitochondrial activity of peach extract.

[0024] Figure 5 This is a diagram showing the experimental results of peach extract on the amount of wrinkles in human skin.

[0025] Figure 6 This is a diagram showing the experimental results of peach extract on human skin texture.

[0026] Figure 7 This is a graph showing the results of an experiment on the relative pore size of human skin using peach extract.

[0027] Figure 8 This is a graph showing the experimental results of the heme content in human blood obtained from peach extract.

[0028] Figure 9 This is a graph showing the experimental results of iron content in human blood from peach extract.

[0029] Figure 10 This is a graph showing the analytical results of bioactive substances in peach extract.

[0030] Figure 11 This is the fingerprint spectrum of peach extract.

[0031] Figure 12 This is a graph showing the experimental results of the anti-blue light effects of bioactive substances in peach extract.

[0032] Figure 13 This is a graph showing the experimental results of the antioxidant activity of bioactive substances in peach extract.

[0033] In the attached figures, the following labels are used:

[0034] TCI-AP-01: Hyperoside

[0035] TCI-AP-02: Isoquercitrin

[0036] TCI-AP-07: Fireworks Glycoside

[0037] TCI-AP-08: Rutin Detailed Implementation

[0038] The concentration symbol "wt%" used in this article usually refers to weight percentage concentration, while the concentration symbol "vol%" usually refers to volume percentage concentration.

[0039] In some embodiments, peach extract is a product obtained by solvent extraction of peaches or by extraction followed by at least one post-processing procedure such as filtration, concentration, and sterilization, wherein the peach is the mature fruit of the peach plant (scientific name: Prunus persica). The extraction solvent may be water, alcohol, or a combination thereof.

[0040] In some embodiments, the method for preparing peach extract includes extracting peaches with water as the extraction solvent to obtain a primary extract, filtering the primary extract to remove solid particles to obtain a filtrate (hereinafter referred to as the first filtrate), concentrating the first filtrate to obtain a concentrated product (which may be a solid and / or a liquid), and sterilizing the concentrated product to obtain a sterile product.

[0041] In some embodiments, a peach refers to a peach with red flesh having a Pantone color scale of 504C to 506C. In some embodiments, a peach is a fresh peach or a frozen peach. In some embodiments, a peach can refer to a whole peach (i.e., the fruit including the peel, flesh, and pit), the flesh of a whole peach with the pit removed and the peel on, or the flesh of a whole peach without the peel and pit removed. For example, a peach can be the flesh of a frozen peach imported from France with the peel on (i.e., without the pit).

[0042] In some embodiments, the peach may be peach pieces or puree that have undergone at least one physical processing procedure to change its appearance, such as cutting, crushing, grinding, or extruding. For example, the peach pieces are frozen peaches with the skin on, which have been crushed into pieces, and the diameter of the pieces may be less than or equal to 12 mm. The peach pieces can be coarsely crushed using a grinder and then sieved through a filter with a mesh size of approximately 12 mm.

[0043] In some embodiments, the initial extract is obtained by extracting peaches and water at a ratio of 1:3 to 8. In some embodiments, the initial extract is obtained by extracting peaches and water at a ratio of 1:5.

[0044] In some embodiments, extraction refers to heating a mixture of peaches and water to a specific temperature and maintaining it for a specific period of time. In some embodiments, extraction refers to heating water to a specific temperature, adding peaches, and then maintaining the mixture at the aforementioned specific temperature for a specific period of time. In one embodiment, the specific temperature is between 100°C and 75°C. In one embodiment, the specific temperature is 85±5°C. In one embodiment, the specific time is between 40 and 80 minutes. In one embodiment, the specific time is 60 minutes. For example, water is heated to 85±5°C, then chopped pieces of frozen peach flesh with peel imported from France are added, and the mixture is kept at 85±5°C for 60 minutes to obtain a peach extract.

[0045] In one embodiment, the extraction further includes an acceptance procedure, which involves measuring the Brix value of the initial extract. The acceptance procedure is completed when the measured Brix value of the initial extract is greater than or equal to 1.2 Brix°. In another embodiment, the acceptance procedure further includes extending the Brix value of the initial extract for a specific time when the measured Brix value is less than 1.2 Brix°, measuring the Brix value of the initial extract again after the specific time extension, and repeating the extension of time and Brix value measurement until the measured Brix value of the initial extract is greater than or equal to 1.2 Brix°, at which point the acceptance procedure is completed.

[0046] In some embodiments, when the concentrated product is a liquid, the concentrated product may be filtered once or multiple times to obtain a filtrate (hereinafter referred to as the second filtrate), and then the second filtrate may be sterilized to obtain a sterile product. It should be understood that the term "peach extract" as used herein may refer to any of the initial extract, filtrate, concentrated product, and sterile product obtained during the preparation process.

[0047] For example, pitted, peel-on red flesh from imported French frozen peaches is pulverized into small pieces. The pulverized peaches are then mixed with water at a weight ratio of 1:5 (peach pulverization to water) and extracted at 85±5℃ for 0.5–2 hours to obtain a primary extract. The primary extract is then filtered through a 400-mesh filter to obtain a filtrate, which is then extracted at 60±5℃ and a pressure of 1±0.2 kg / cm². 2 The filtrate was concentrated under reduced pressure to obtain a concentrated product. The concentrated product was then filtered and sterilized to obtain peach extract.

[0048] In some embodiments, the sugar content of the initial extract is greater than or equal to 1.2 Brix. In some embodiments, the sugar content of the concentrated product is approximately 9 ± 0.5 Brix. In some embodiments, the pH value of the peach extract is less than or equal to 3.7. In some embodiments, the acidity of the peach extract is 1.3 ± 0.5. In some embodiments, the peach extract includes malic acid to adjust the acidity to 1.3 ± 0.5.

[0049] In some embodiments, peach extract can be used to improve skin condition, and this peach extract is extracted from the red flesh of a peach (with or without skin).

[0050] In one embodiment, peach extract can be used to combat blue light damage. In one embodiment, peach extract at concentrations of 0.125 mg / mL or 0.0625 mg / mL can significantly reduce blue light damage to skin fibroblasts.

[0051] In one embodiment, peach extract can be used to enhance the antioxidant capacity of skin fibroblasts. In one embodiment, compared to untreated skin fibroblasts, peach extract significantly enhances the antioxidant capacity of skin fibroblasts at concentrations of 0.125 mg / mL or 0.0625 mg / mL.

[0052] In one embodiment, peach extract can be used to enhance mitochondrial activity. In one embodiment, compared to untreated peach extract, peach extract significantly enhanced mitochondrial activity in skin fibroblasts at concentrations of 0.125 mg / mL or 0.0625 mg / mL.

[0053] In one embodiment, peach extract can be used to promote the production of elastin in the skin. In one embodiment, compared with untreated peach extract, peach extract can significantly promote the production of elastin in skin fibroblasts at concentrations of 0.125 mg / mL or 0.0625 mg / mL.

[0054] In one embodiment, peach extract can be used to reduce skin texture. In one embodiment, peach extract can be used to reduce skin wrinkles. In one embodiment, peach extract can be used to minimize skin pores. In one embodiment, peach extract can be used to increase heme levels. In one embodiment, peach extract can be used to increase blood iron levels. In one embodiment, the effective dosage of peach extract is 0.9 g / day.

[0055] In some embodiments, any of the aforementioned compositions may be a pharmaceutical product. In other words, this pharmaceutical product contains an effective amount of peach extract.

[0056] In some embodiments, the aforementioned pharmaceutical products may be manufactured using techniques known to those skilled in the art into dosage forms suitable for enteral, parenterally, oral, or topically administration.

[0057] In some embodiments, enteric or oral dosage forms may be, but are not limited to, tablets, troche, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, or the like. In some embodiments, non-enteral or localized dosage forms may be, but are not limited to, injections, sterile powders, external preparations, or the like. In some embodiments, injections may be administered via subcutaneous injection, intraepidermal injection, intradermal injection, or intralesional injection.

[0058] In some embodiments, the aforementioned pharmaceutical product may comprise a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing technologies. In some embodiments, a pharmaceutically acceptable carrier may be one or more of the following: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. The type and quantity of carrier selected fall within the scope of expertise and routine practice of those skilled in the art. In some embodiments, the solvent that can be used as a pharmaceutically acceptable carrier may be water, normal saline, phosphate-buffered saline (PBS), or an aqueous solution containing alcohol.

[0059] In some embodiments, any of the foregoing compositions may be an edible composition. In other words, the edible composition contains a specific amount of peach extract. In some embodiments, the foregoing edible compositions may be a food product or a food additive. In some embodiments, food products may be, but are not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements.

[0060] In some embodiments, any of the foregoing compositions may be a cosmetic or skincare product. In other words, the cosmetic or skincare product contains a specific amount of peach extract.

[0061] In some embodiments, the aforementioned cosmetics or skincare products may be any of the following forms: toner, gel, gel mask, mud mask, lotion, cream, lipstick, foundation, pressed powder, loose powder, cleansing oil, cleansing milk, facial cleanser, body wash, shampoo, conditioner, sunscreen, hand cream, nail polish, perfume, serum, and face mask. In some embodiments, the aforementioned cosmetics or skincare products may, as needed, include topical acceptable ingredients. In some embodiments, topical acceptable ingredients may be, for example, emulsifiers, penetration enhancers, softeners, solvents, excipients, antioxidants, or combinations thereof.

[0062] The following experiments all use the student t-test to determine whether there is a statistically significant difference between two sample groups, i.e., to calculate their p-value.

[0063] Unless otherwise specified, all experiments below were conducted at room temperature, which is 25±5℃.

[0064] Example 1: Preparation of peach extract

[0065] peach:

[0066] We selected frozen peaches imported from France whose red flesh was between 504C and 506C. After pitting the selected peaches, we crushed them with a coarse 12mm pore size to obtain the crushed peach flesh with the skin on.

[0067] Production process:

[0068] First, heat five times the weight of the peaches in water (RO water). When the water temperature reaches 85°C, add the peaches and maintain the water temperature at 85±5°C for extraction to obtain a preliminary extract. The Brix content of the preliminary extract is then measured. If the Brix content of the preliminary extract is greater than 1.2 Brix, proceed to the next step. The extraction time is 60 minutes.

[0069] The initial extract was then filtered through a 400-mesh filter to obtain the filtrate, which was then subjected to further treatment at a temperature of 60±5℃ and a pressure of 1±0.2 kg / cm². 2 The filtrate was then concentrated under reduced pressure to obtain a concentrated product. The sugar content of this concentrated product was 9 Brix.

[0070] Next, the concentrated product was filtered sequentially using a 400-mesh filter and a 5µm filter to obtain peach extract.

[0071] Example 2: Experiment on resistance to blue light damage

[0072] Materials and Instruments:

[0073] 1. Cell line: Human skin fibroblasts CCD-966sk, obtained from the Bioresource Conservation and Research Center (BCRC); Cat.60153, hereinafter referred to as human skin fibroblasts.

[0074] 2. Cell culture medium: Earle's balanced salt type MEM (Minimum essential medium) culture medium containing 0.1M non-essential amino acids, 1.5g / L sodium bicarbonate, 0.1M pyruvate, and 10% fetal bovine serum (purchased from Gibco) was added.

[0075] 3. Phosphate-buffered saline (PBS): purchased from Gibco, product number 10437-028.

[0076] 4. DCFH-DA solution: The fluorescent dye DCFH-DA (purchased from Sigma / SI-D6883-50MG) was prepared with dimethyl sulfoxide (DMSO) to a concentration of 5 μg / ml, which can stain reactive oxidizing agents (ROS).

[0077] 5. Trypsin-EDTA: 10X Trypsin-EDTA (purchased from Gibco) diluted 10 times with PBS.

[0078] 6. Flow cytometry, BD Accuri C6 Plus.

[0079] Experimental procedure:

[0080] 1. Add 2 mL of cell culture medium to each well of a six-well culture dish, and seed 2 × 10⁶ cells per well. 5 Human skin fibroblasts.

[0081] 2. Place the culture dish in a CO2 incubator and incubate at 37°C for 24 hours to allow the cells to adhere.

[0082] 3. After cell attachment, human skin fibroblasts were divided into the following three groups and subjected to corresponding processing procedures.

[0083] Blank group: Each well was replaced with 2 mL of simple cell culture medium (i.e., without peach extract of any of the examples) and cultured at 5% CO2 and 37°C for 1 hour. Then, 2 μL of DCFH-DA solution was added to each well and incubated for 15 minutes. After that, the wells were moved to the dark and incubated for another 15 minutes (at room temperature).

[0084] Control group: Each well was replaced with 2 mL of simple cell culture medium (i.e., without peach extract of any of the examples) and cultured at 5% CO2 and 37°C for 1 hour. Then, 2 μL of DCFH-DA solution was added to each well and incubated for 15 minutes. After that, the well was transferred to a blue light chamber and irradiated with blue light (wavelength of 500 nm) for 15 minutes (at room temperature).

[0085] Experimental Group A: Each well was replaced with cell culture medium containing 0.0625 mg / mL peach extract (prepared in Example 1) and cultured at 5% CO2 and 37°C for 1 hour. Then, 2 μL of DCFH-DA solution was added to each well and incubated for 15 minutes. After that, the well was transferred to a blue light box and irradiated with blue light (wavelength 500 nm) for 15 minutes (at room temperature).

[0086] Experimental Group B: Each well was replaced with cell culture medium containing 0.125 mg / mL peach extract (prepared in Example 1) and cultured at 5% CO2 and 37°C for 1 hour. Then, 2 μL of DCFH-DA solution was added to each well and incubated for 15 minutes. After that, the well was transferred to a blue light box and irradiated with blue light (wavelength 500 nm) for 15 minutes (at room temperature).

[0087] 4. Then, rinse the cells in each of the above groups twice with PBS.

[0088] 5. Next, add 200 μL of trypsin to each well and react for 5 minutes in the dark. After the reaction, add cell culture medium to each well to stop the reaction.

[0089] 6. Collect the cells from each group into their respective centrifuge tubes containing cell culture medium, and centrifuge the tubes containing cells and cell culture medium at 400×g for 10 minutes.

[0090] 7. After centrifugation, remove the supernatant from each centrifuge tube, and then wash the cell pellet in each centrifuge tube with PBS.

[0091] 8. Next, centrifuge each centrifuge tube again at 400×g for 10 minutes.

[0092] 9. After centrifugation again, remove the supernatant from each centrifuge tube and add 1 ml of PBS to suspend the cell pellet in each centrifuge tube to obtain the cell solution to be tested.

[0093] 10. The fluorescence signal of DCFH-DA in the cell culture of each tube was detected using a flow cytometer (BD Accuri C6 Plus Flow Cytometer 660517) to quantify the intracellular ROS content. The excitation wavelength used for fluorescence signal detection was 450-490 nm, and the emission wavelength was 510-550 nm. Since DCFH-DA is first hydrolyzed into DCFH (dichlorodihydrofluorescein) after entering the cell, and then oxidized by ROS into DCF (dichlorofluorescein) which emits green fluorescence, the fluorescence intensity of cells treated with DCFH-DA reflects the intracellular ROS content, thereby indicating the proportion of cells with high ROS expression to the original cell count.

[0094] Experimental results:

[0095] Therefore, the human skin fibroblasts in the control group were not exposed to blue light, meaning they grew under normal physiological metabolic conditions. Furthermore, considering the ROS content of the control group as 100% relative ROS production, the ROS content of the other groups was converted to relative ROS production as a percentage (%) using interpolation, as shown in Table 1. Figure 1 As shown.

[0096] Table 1

[0097] Relative ROS generation Blank group 100％ control group 72211.1％ Experimental Group A 305.6％ Experimental Group B 250.0％

[0098] exist Figure 1 In the above, compared with the control group, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001; compared with the control group, "#" represents a p-value less than 0.05, "##" represents a p-value less than 0.01, and "###" represents a p-value less than 0.001. The more "*" or "#" symbols present, the more statistically significant the difference.

[0099] Refer to Table 1 and Figure 1 In the control group, human skin fibroblasts produced a relative ROS level as high as 72211.1% after 15 minutes of blue light irradiation. This indicates that blue light significantly promotes ROS production in human skin fibroblasts, potentially causing substantial damage to human skin.

[0100] Continue with reference to Table 1 and Figure 1 In experimental group A, human skin fibroblasts treated with peach extract at a concentration of 0.0625 mg / mL showed a relative ROS production of 305.6% even under blue light irradiation. Compared to the control group, the ROS production in experimental group A was significantly reduced.

[0101] Continue with reference to Table 1 and Figure 1 In experimental group B, human skin fibroblasts treated with peach extract at a concentration of 0.125 mg / mL showed a 250% increase in relative ROS production. Compared to the control group, the ROS production in experimental group B was significantly reduced.

[0102] Therefore, the experimental results show that peach extract can significantly prevent the production of reactive antioxidants caused by blue light. This means that peach extract can prevent damage to skin fibroblasts caused by blue light exposure. Peach extract can enhance the skin's resistance to blue light, preventing damage caused by blue light exposure.

[0103] Example 3: Antioxidant Experiment

[0104] Reactive oxygen species (ROS) are one of the key determinants of oxidative stress in the body, which damages cells, impairs their function, and promotes apoptosis. This experiment measures ROS levels to determine the ability to combat oxidative stress. Many factors contribute to oxidative stress, such as chronic diseases, stress and anxiety, and being overweight. External factors should also not be ignored, such as pollution, smoking, alcohol consumption, medications, sunlight, strenuous physical activity, or a sedentary lifestyle.

[0105] Materials and Instruments:

[0106] 1. Cell line: Human skin fibroblasts CCD-966sk, obtained from the Bioresource Conservation and Research Center (BCRC); Cat.60153, hereinafter referred to as human skin fibroblasts.

[0107] 2. Cell culture medium: basal medium containing 10 vol% FBS (purchased from Gibco). The basal medium was prepared by adding components to Eagle's minimum essential medium (MEM, purchased from Gibco, product number 15188-319) to include 1 mM sodium pyruvate (purchased from Gibco), 1.5 g / L sodium bicarbonate (purchased from Sigma, Cat. S5761-500G), and 0.1 mM non-essential amino acid solution (purchased from Gibco).

[0108] 3. Phosphate-buffered saline (PBS) solution: purchased from Gibco, product number 10437-028.

[0109] 4. DCFH-DA solution: Dissolve 2,7-dichloro-dihydro-fluorescein diacetate (DCFH-DA; product number SI-D6883, purchased from Sigma) in dimethyl sulfoxide (DMSO, purchased from Sigma, product number SI-D6883-50MG) to prepare a 5 μg / ml DCFH-DA solution.

[0110] 5. Flow cytometry, BD Accuri C6 Plus.

[0111] 6. Hydrogen peroxide (H2O2): Purchased from Sigma-Aldrich, product model 95299-1L.

[0112] 7. Trypsin-EDTA: 10X Trypsin-EDTA (purchased from Gibco) diluted 10 times with PBS.

[0113] Experimental procedure:

[0114] 1. Human skin fibroblasts were cultured at a density of 1 × 10⁶ cells per well. 5 The cells were inoculated in a 6-well culture dish containing 2 mL of cell culture medium per well using a specific method.

[0115] 2. Place the culture dish in 5% CO2 at 37°C and incubate for 24 hours.

[0116] 3. After culturing, the cells were divided into four groups, and each group was treated differently.

[0117] Blank group: Each well was replaced with 2 mL of simple cell culture medium (i.e., without the peach extract of this invention).

[0118] Control group: Each well was replaced with 2 mL of simple cell culture medium.

[0119] Experimental Group A: Each well was replaced with 2 mL of cell culture medium containing 0.0625 mg / mL of peach extract from Example 1.

[0120] Experimental Group B: Each well was replaced with 2 mL of cell culture medium containing 0.125 mg / mL of peach extract from Example 1.

[0121] 4. After the above four groups were treated, the culture trays were placed in a 5% CO2 environment at 37°C for 1 hour.

[0122] 5. Add 2 μL of DCFH-DA solution to each well and allow it to react for 15 minutes.

[0123] 6. Add H2O2 and react at 37°C for 1 hour. That is, add 1 mM hydrogen peroxide to each group to simulate oxidative damage.

[0124] 7. Then, rinse each well twice with 1 mL of PBS.

[0125] 8. Add 200 μL of trypsin to each well and react in the dark for 5 minutes. After the reaction, add cell culture medium to each well to stop the reaction.

[0126] 9. Collect the cells and cell culture medium from each group into their respective centrifuge tubes, and centrifuge the centrifuge tubes containing cells and cell culture medium at 400xg for 10 minutes.

[0127] 10. After centrifugation, remove the supernatant from each centrifuge tube and then reconstitute the cell pellet in each centrifuge tube with PBS.

[0128] 11. Next, centrifuge each centrifuge tube again at 400xg for 10 minutes.

[0129] 12. After centrifugation again, remove the supernatant from each centrifuge tube and suspend the cell pellet in 1 mL of PBS in the dark to obtain the cell solution to be tested.

[0130] 13. Flow cytometry was used to detect the fluorescence signal of DCFH-DA in the cell culture of each tube to quantify the intracellular ROS content. The excitation wavelength used for fluorescence detection was 450-490 nm, and the emission wavelength was 510-550 nm. Since the experiment was performed in triplicate, the average value of the measurements from each group's triplicate experiments was used as the ROS content.

[0131] Experimental results:

[0132] Therefore, the human skin fibroblasts in the control group were not damaged by hydrogen peroxide, meaning they were under normal physiological metabolic conditions; thus, it was assumed that their relative ROS production was 100%. Furthermore, considering the ROS content of the control group as 100% relative ROS production, the ROS content of the other groups was converted to relative ROS production as a percentage using interpolation, as shown in Table 2. Figure 2 As shown.

[0133] Table 2

[0134] Relative ROS generation Blank group 100.0％ control group 20225.0％ Experimental Group A 7300.0％ Experimental Group B 7100.0％

[0135] exist Figure 2In the above, compared with the control group, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001; compared with the control group, "#" represents a p-value less than 0.05, "##" represents a p-value less than 0.01, and "###" represents a p-value less than 0.001. The more "*" or "#" symbols present, the more statistically significant the difference.

[0136] like Figure 2 As shown in the comparison of the blank group and the control group, it can be seen that after hydrogen peroxide treatment, the relative amount of ROS generated (high fluorescence performance) increased significantly (up to 20225%, which is statistically significant, p value less than 0.001); this shows that hydrogen peroxide treatment does promote the production of intracellular ROS, thereby causing damage to skin fibroblasts.

[0137] Continue to refer to Table 2 and Figure 2 In experimental group A, human skin fibroblasts treated with peach extract at a concentration of 0.0625 mg / mL showed a relative ROS production of 7300.0% even after hydrogen peroxide treatment. Compared to the control group, the ROS production in experimental group A was significantly reduced.

[0138] Continue with reference to Table 1 and Figure 1 In experimental group B, human skin fibroblasts treated with peach extract at a concentration of 0.125 mg / mL showed a relative ROS production of 7100.0%. Compared to the control group, the ROS production in experimental group B was significantly reduced.

[0139] The above experimental results show that the peach extract of this embodiment can effectively reduce the production or accumulation of ROS in cells. In other words, the peach extract of this embodiment can act as a ROS scavenger. That is, the peach extract of this embodiment can reduce oxidative damage to cells caused by ROS by lowering the content of ROS in cells. Based on this, the peach extract has antioxidant function, preventing skin from being damaged by the environment, maintaining the normal function and structure of skin cells, reducing cell apoptosis caused by oxidative stress, and resulting in healthier skin.

[0140] Example 4: Experiment promoting elastin production

[0141] Materials and Instruments:

[0142] 1. Cell culture medium: Basal medium containing 10 vol% fetal bovine serum (FBS; brand: Gibco). The basal medium was prepared by adding 1 mM sodium pyruvate to Eagle's minimum essential medium (MEM, purchased from Gibco, product number 15188-319).

[0143] 2. Cell line: Human dermal fibroblasts CCD-966Sk (obtained from BCRC No. 60153), hereinafter referred to as human dermal fibroblasts.

[0144] 3. Human Elastin ELISA Kit (model ab239433, brand: Abcam).

[0145] 4. Enzyme Immunoassay Analyzer (Brand: BioTek).

[0146] Experimental procedure:

[0147] 1. Human skin fibroblasts were cultured at a density of 1 × 10⁶ cells per well. 4 Each cell was inoculated into a 24-well culture dish containing 0.5 mL of cell culture medium per well.

[0148] 2. Place the culture dish in a 5% CO2 environment at 37°C and incubate for 24 hours.

[0149] 3. After culturing, the cells were divided into experimental and control groups and the following treatment procedures were performed accordingly.

[0150] Blank group: Each well was replaced with simple cell culture medium (i.e., cell culture medium without peach extract).

[0151] Experimental Group A: Each well was replaced with cell culture medium containing peach extract prepared in Example 1 at a concentration of 0.0625 mg / mL.

[0152] Experimental Group B: Each well was replaced with cell culture medium containing peach extract prepared in Example 1 at a concentration of 0.125 mg / mL.

[0153] 4. After processing, place the culture tray in a 5% CO2 environment at 37°C for 1 hour.

[0154] 5. Next, after processing the human skin fibroblasts in each group according to the standard procedure of the elastin detection kit, the amount of elastin produced in each group of human skin fibroblasts was detected by an enzyme immunoassay analyzer.

[0155] Experimental results:

[0156] Therefore, the elastin content measured in the blank group is considered as 100% (i.e., the relative elastin production under normal physiological metabolic conditions is 100%), and the elastin content measured in the experimental group is converted into elastin production expressed as a percentage, such as... Figure 3 As shown. In Figure 3 In the p-values, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001. The more "*" symbols there are, the more statistically significant the difference.

[0157] Please see Figure 3 With the elastin production in the control group being 100%, the elastin production in experimental group A was 126.7%. The elastin production in the experimental group was significantly increased by 26.7%, indicating that human skin fibroblasts treated with peach extract for 24 hours can produce more elastin.

[0158] See also Figure 3 With the elastin production in the control group being 100%, the elastin production in experimental group B was 180.1%. The elastin production in the experimental group was significantly increased by 80.1%, indicating that human skin fibroblasts treated with peach extract for 24 hours can produce more elastin.

[0159] Therefore, the peach extract in this embodiment has the function of promoting the synthesis of elastin in cells. Based on this, the peach extract has the function of promoting the synthesis of elastin in cells. When cells are treated with peach extract, it can promote the production of elastin in skin cells, thereby improving skin elasticity.

[0160] Example 5: Mitochondrial activity experiment

[0161] Mitochondria are key organelles for cellular energy production, regulating cellular metabolism and survival through oxidative phosphorylation (or the electron transport chain). Mitochondrial dysfunction leads to abnormal ROS proliferation, potentially causing mutations in the mitochondrial genome (mtDNA), apoptosis, and cell damage. This experiment used JC-1 stain to observe the mitochondrial membrane potential of human skin cells. When the mitochondrial membrane potential increases, JC-1 aggregates can accumulate on the inner mitochondrial membrane, forming polymers. In other words, a high level of JC-1 aggregation can be considered as increased mitochondrial activity within skin cells, indicating healthier skin cells.

[0162] Materials and Instruments:

[0163] 1. Cell line: Human skin fibroblasts CCD-966sk (BCRC No. 60153).

[0164] 2. Cell culture medium: basal medium containing 10 vol% FBS (purchased from Gibco). The basal medium was prepared by adding components to Eagle's minimum essential medium (MEM, purchased from Gibco, product number 15188-319) to include 1 mM sodium pyruvate (purchased from Gibco), 1.5 g / L sodium bicarbonate (purchased from Sigma), and 0.1 mM non-essential amino acid solution (purchased from Gibco).

[0165] 3. Phosphate-buffered saline (PBS): purchased from Gibco.

[0166] 4. Mitochondrial membrane potential detection kit (BDTM MitoScreen (JC-1) kit, model 551302). This kit includes JC-1 dye (lyophilized) and 10X analysis buffer. Before use, dilute the 10X analysis buffer 10-fold with PBS to prepare the 1X analysis buffer. Add 130 μL of DMSO to the JC-1 dye (lyophilized) to prepare the JC-1 stock solution. Then, further dilute the JC-1 stock solution with the 1X analysis buffer to prepare the JC-1 working solution (i.e., JC-1 mitochondrial-specific dye). The dilution ratio is 1:100 between the JC-1 stock solution and the 1X analysis buffer.

[0167] 5. Trypsin: 10X Trypsin-EDTA (purchased from Gibco) diluted 10 times with 1X PBS.

[0168] 6. Flow cytometer: purchased from BD Pharmingen, model BDTM Accuri C6Plus.

[0169] 7. Peach extract: Peach extract prepared by the preparation method described in Example 1 of this case.

[0170] Experimental steps:

[0171] 1. Human skin fibroblasts were cultured at a density of 1 × 10⁶ cells per well. 5 The cells were inoculated in a 6-well culture dish containing 2 mL of cell culture medium per well using a specific method.

[0172] 2. Replace the culture medium in each well of the culture dish with 2 mL of experimental culture medium. Specifically, experimental group A used cell culture medium containing 0.0625 mg / mL peach extract, experimental group B used cell culture medium containing 0.125 mg / mL peach extract, and the control group used simple cell culture medium (i.e., without peach extract).

[0173] 3. Place the culture dish in a 5% CO2 environment at 37°C and incubate for 24 hours.

[0174] 4. Remove the experimental culture medium from the culture dish and rinse twice with 1 mL of PBS.

[0175] 5. Add 200 μL of trypsin to each well and react in the dark for 5 minutes. After the reaction, add cell culture medium to stop the reaction. Collect the cells and cell culture medium from each well into their respective centrifuge tubes, and centrifuge the tubes containing cells and cell culture medium at 400 x g for 10 minutes.

[0176] 6. After centrifugation, remove the supernatant and reconstitute the cell pellet with 1 mL of PBS or transfer it to another centrifuge tube to obtain a centrifuge tube containing cell suspension.

[0177] 7. Centrifuge the centrifuge tube containing the cell suspension at 400xg for 5 minutes.

[0178] 8. After centrifugation, remove the supernatant from each centrifuge tube and add 100 μL of JC-1 working reagent to each centrifuge tube.

[0179] 9. Vortex the cell pellets in each centrifuge tube with JC-1 working reagent until homogeneous, and incubate for 15 minutes in the dark.

[0180] After 10.15 minutes, centrifuge each centrifuge tube at 400xg for 5 minutes.

[0181] 11. After centrifugation, remove the supernatant from each centrifuge tube, reconstitute the cell pellet in each centrifuge tube with 1 mL of PBS, and centrifuge at 400 x g for 5 minutes.

[0182] 12. After centrifugation, remove the supernatant from each centrifuge tube, reconstitute the cell pellet in each centrifuge tube with 1 mL of PBS, and centrifuge at 400 x g for 5 minutes.

[0183] 13. After centrifugation, remove the supernatant from each centrifuge tube and resuspend the cells in 500 μL of PBS to obtain the cell solution to be tested.

[0184] 14. The membrane potential of mitochondria in the test solution of cells in each well was measured by flow cytometry to perform mitochondrial activity analysis.

[0185] Experimental results:

[0186] Therefore, the experimental results of the blank group are considered as 100% (i.e., the relative JC-1 aggregation amount is 100%), and the experimental results of the experimental group are converted into the corresponding relative JC-1 aggregation amount expressed as a percentage, such as... Figure 4 As shown, a high level of JC-1 aggregation indicates more viable cells, while a low level of JC-1 aggregation indicates that the cells are closer to an apoptotic state.

[0187] See Figure 4 The relative JC-1 aggregation in experimental group A was approximately 105.0%. In other words, compared to the control group, the mitochondrial activity of human skin fibroblasts in experimental group A was increased by 5%. This indicates that peach extract can enhance the mitochondrial activity of skin cells.

[0188] See also Figure 4 The relative JC-1 aggregation in experimental group B was approximately 117.8%. In other words, compared to the control group, the mitochondrial activity of human skin fibroblasts in experimental group B was significantly increased by 17.8%. This indicates that peach extract can enhance mitochondrial activity in skin cells.

[0189] Therefore, peach extract has the function of promoting mitochondrial activity in cells. When cells are treated with peach extract, it can promote the prevention of apoptosis in mitochondria in skin cells, enabling mitochondria to provide more energy to cells. It can also prevent gene mutations or apoptosis caused by abnormal ROS proliferation and reduce the production of free radicals in cells.

[0190] Example 6: Human Experimentation

[0191] Subjects: 7 subjects (adults who reported poor complexion or loose skin, aged between 20 and 55 years old).

[0192] Test items and instruments:

[0193] 1. Skin Wrinkles: The VISIA advanced digital skin analyzer sold by Canfield (USA) was used for measurement. A high-resolution camera lens photographed the same subject's facial skin, especially the area around the eyes, before and after drinking the product. Standard white light was used to detect changes in fine lines, thus identifying the location of the wrinkles and obtaining a numerical value representing the smoothness of the skin. After measurement, the wrinkle quantity of the control group was used as a baseline (i.e., the relative expression rate of wrinkles in the control group was 100%), and the relative expression rate (%) of skin wrinkles in the experimental group was calculated.

[0194] 2. Skin Texture: The VISIA advanced digital skin texture analyzer sold by Canfield in the United States was used to analyze the facial skin of the subjects. The principle is to capture high-resolution skin images using visible light, and then use built-in software to analyze the roughness of the skin based on its depressions and protrusions to obtain a skin texture value. The higher the skin texture measurement value, the rougher the skin. After measurement, the skin texture performance of the control group was used as a baseline (i.e., the relative performance rate of the skin texture of the control group was 100%), and the relative performance rate (%) of the skin texture of the experimental group was calculated.

[0195] 3. Pores (Trichopore): The VISIA advanced digital skin analyzer sold by Canfield in the United States was used to analyze the facial skin of the subjects. The principle is to measure the amount of pores on the subject's face using a high-resolution camera lens. Standard white light is used to create shadows in the recessed areas of the pores, making the pores appear darker than the surrounding skin, thus allowing the detection of the number and area of ​​pores. Software then analyzes the number and area of ​​pores to obtain a numerical value for pore quality; a higher value indicates a larger number and area of ​​pores. After measurement, the pore quality of the control group was used as a baseline (i.e., the relative pore quality rate of the control group's skin was 100%), and the relative pore quality rate (%) of the experimental group's skin was calculated.

[0196] 4. Blood heme and iron levels: Tested by Liren Medical Laboratory. Higher heme and iron levels increase oxygen metabolism, improve anemia, and thus provide more oxygen to skin cells, promoting skin repair. This results in a more rosy and radiant complexion, indicating a healthier appearance.

[0197] Experimental procedure:

[0198] Subjects were instructed to consume a 50mL bottle of peach sample daily, containing 0.9g of the peach extract prepared in Example 1, for four consecutive weeks. Blood samples were collected from each subject before the first administration (the control group) and after four weeks (the experimental group), and facial skin was analyzed using the aforementioned digital skin analyzer.

[0199] Each bottle of peach sample contains 0.9g of peach extract prepared in Example 1, 0.05g of citric acid, 0.0075g of steviol glycosides, 0.05g of liquid peach flavoring A, 0.05g of liquid peach flavoring B, and 48.9425g of water.

[0200] Experimental results:

[0201] Depend on Figure 5As can be seen, while the relative expression rate of skin wrinkles in the control group was 100%, the relative expression rate of skin wrinkles in the experimental group was 86.2%. This means that after the subjects consumed peach extract continuously for four weeks, the number of wrinkles on their facial skin decreased compared to before use. In other words, peach extract can reduce wrinkles, increase skin smoothness, improve skin aging, and thus make the skin more delicate and radiant.

[0202] Depend on Figure 6 As can be seen, while the relative expression rate of skin texture in the control group was 100%, the relative expression rate of skin texture in the experimental group was 90.5%. This means that after the subjects consumed peach extract continuously for four weeks, the skin texture on their faces was reduced compared to before use. In other words, peach extract can reduce skin texture, thereby improving rough skin, increasing skin smoothness, and ultimately making the skin more delicate and radiant.

[0203] Depend on Figure 7 As can be seen, while the relative pore size manifestation rate was 100% in the control group, it was 90.6% in the experimental group. This means that after the subjects consumed peach extract continuously for four weeks, the pores on their facial skin were reduced compared to before use. In other words, peach extract can shrink pores on the skin, improving rough skin, tightening pores, and ultimately making the skin smoother and more radiant.

[0204] Reference Figure 8 While the heme content in the control group was 13 g / dL, the heme content in the experimental group was 13.3 g / dL. This indicates that, compared to before consumption, the subjects' blood heme content significantly increased by 2.3% after four weeks of continuous consumption of peach extract. In other words, peach extract can increase the heme content in human blood.

[0205] Reference Figure 9 While the iron content in the control group was 101 g / dL, the iron content in the experimental group was 118.7 g / dL. This indicates that, compared to before consumption, the subjects' blood iron levels significantly increased by 17.4% after four weeks of continuous consumption of peach extract. In other words, peach extract can increase the iron content in the blood, thereby increasing oxygen metabolism, improving anemia, and thus providing oxygen to skin cells to promote skin repair. This also resulted in a more rosy and radiant complexion, indicating a better overall appearance.

[0206] Example 7: Analysis of bioactive components in peach extract

[0207] Natural plant extracts typically contain multiple components and are not pure substances. Different bioactive substances have varying solubilities in different solvents. This experiment utilizes immiscible solvents to transfer a specific component from peach extract to another solvent.

[0208] Instruments and equipment:

[0209] (1) Nuclear Magnetic Resonance Spectrometer (NMR). 1D and 2D spectra were obtained using an Ascend 400MHz instrument, Bruker Co., Germany. Chemical shift is expressed as δ in ppm.

[0210] (2) Mass spectrometer (MS) tandem mass spectrometry-two-dimensional ion trap tandem Fourier transform mass spectrometry and ESI-MS / MS: measured using Bruker amaZon SL system, unit is m / z.

[0211] (3) Medium pressure liquid chromatography (MPLC): Rf+, Teledyne ISCO, Lincoln NE, High Performance Liquid Chromatography (HPLC): Agilent 1200 series HPLC system; Agilent Vacuum Degassing Unit 1322A; Agilent Quadruple Pump G1311A for solvent delivery; Agilent G1314B Multiple Wavelength Detector (MWD); Agilent 1260 Infinity DAD VL G1315D for detection wavelengths of 210nm, 280nm, 320nm, and 365nm (Agilent Germany).

[0212] (4) Analytical Column: 5μm C18(2) (250 x 10 mm, Phenomenex, USA).

[0213] (5) Column Chromatography Packing Materials: Sephadex LH-20 (Pharmacia, Piscataway, NJ, USA), Diaion HP-20 macroporous resin (Mitsubishi Chemical Co., Japan), Merck Kieselgel 60 normal-phase silica gel (40-63um, Art. 9385), Merck reverse-phase silica gel. RP-18 (40-63um, Art.0250).

[0214] (6) Thin-layer chromatography was performed using TLC aluminum sheets (Silica gel 60F254, 0.25mm, Merck, Germany) and TLC aluminum sheets (RP-18F254-S, 0.25mm, Merck, Germany).

[0215] (7) Solvents: n-hexane, ethyl acetate, acetone, methanol, ethanol, acetonitrile (purchased from Merck Taiwan), chloroform-d1 (deuteration degree 99.5%), methanol-d4 (deuteration degree 99.5%), deuterium oxide (deuteration degree >99.8%), dimethyl sulfoxide-d6 (deuteration degree >99.9%) (Merck Taiwan).

[0216] Test process:

[0217] refer to Figure 10 First, 5 liters (L) of peach extract obtained in Example 1 were separated by liquid phase partitioning with an equal proportion of n-butanol and water to obtain n-butanol extract and aqueous extract, respectively. Next, the n-butanol extract was concentrated and dried under reduced pressure to obtain 34.2 g of n-butanol extract (BUF). The aqueous extract was concentrated and dried under reduced pressure to obtain 267.1 g of aqueous extract (WF).

[0218] Subsequently, a bioassay-guided fractionation method was used, and the n-butanol extract was subjected to Sephadex LH-20 column chromatography with methanol as the extraction solvent. Thin-layer chromatography was then used to combine extracts with similar results, resulting in three fractions: the first fraction (BUF1), the second fraction (BUF2), and the third fraction (BUF3).

[0219] Continued reference Figure 10 The second separation section (BUF2) was further separated using a reverse-medium pressure liquid chromatography (RP-MPLC) system to obtain multiple extracts. Here, the extract was linearly purified from water to methanol for 60 minutes at a flow rate of 10 mL / min. Subsequent thin-layer chromatography (TLC) analysis was performed using aluminum plates coated with silica gel 60F254 (0.25 mm) to merge extracts with similar results, resulting in multiple sub-separation sections, including BUF2-2, BUF2-5, BUF2-8, and BUF2-9.

[0220] The BUF2-2 separation section was purified by reverse-phase high-efficiency liquid chromatography (methanol / water = 2 / 3, v / v) to obtain the bioactive substance TCI-AP-01. Its chemical structure was analyzed by 1H-NMR and electrospray ionization mass spectrometry (ESIMS), confirming it as hyperoside. Its IUPAC name is 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one, and its chemical structural formula is as follows:

[0221]

[0222] The BUF2-5 separation unit was purified by reverse-phase high-efficiency liquid chromatography (methanol / water = 1 / 3, v / v) to obtain the bioactive substance TCI-AP-02. Its chemical structure was analyzed by 1H-NMR and electrospray ionization mass spectrometry (ESIMS), confirming it as isoquercitrin. Its IUPAC name is 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one, and its chemical structural formula is as follows:

[0223]

[0224] The BUF2-8 separation section was purified by reverse-phase high-efficiency liquid chromatography (methanol / water = 2 / 5, v / v) to obtain the bioactive substance TCI-AP-07. After analysis of its chemical structure by 1H-NMR and electrospray ionization mass spectrometry (ESIMS), it was confirmed to be Nicotiflorine, with the IUPAC name 5,7-dihydroxy-2-(4-hydroxyphenyl)-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one. Its chemical structural formula is as follows:

[0225]

[0226] The BUF2-9 separation section was purified by reverse-phase high-efficiency liquid chromatography (methanol / water = 3 / 4, v / v) to obtain the bioactive substance TCI-AP-08. After analysis of its chemical structure by 1H-NMR and electrospray ionization mass spectrometry (ESIMS), it was confirmed to be rutin, with the IUPAC name butyl(1S,3R,4R,5R)-3-[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy-1,4,5-trihydroxycyclohexane-1-carboxylate. Its chemical structural formula is as follows:

[0227]

[0228] It is known that peach extract contains at least the following bioactive substances: hyperoside (TCI-AP-01), isoquercitrin (TCI-AP-02), safflower glycoside (TCI-AP-07), and rutin (TCI-AP-08).

[0229] Example 8: Fingerprint analysis of peach extract

[0230] Therefore, high performance liquid chromatography (HPLC) was used to perform quantitative and qualitative analysis of the bioactive substances in the peach extract prepared in Example 1.

[0231] Test process:

[0232] The peach extract prepared in Example 1 was used as a sample, with a concentration of 50 mg / mL and an injection volume of 10 μL.

[0233] The solvents used were methanol and water, with 0.1% formic acid added to both. The flow rate was set at 1 mL / min, and the extraction conditions were set as follows: methanol:water 2:98 at 0 minutes, 2:98 at 10 minutes, 70:30 at 40 minutes, 100:0 at 50 minutes, and 100:0 at 60 minutes. The column temperature was set at 40℃.

[0234] Test results:

[0235] refer to Figure 11 In the analysis, the peak of bioactive substance TCI-AP-01 was obtained around 34 minutes, and the peaks of bioactive substances TCI-AP-02, TCI-AP-08 and TCI-AP-07 were obtained sequentially between 35 and 40 minutes.

[0236] Example 9: Experiment on the resistance of bioactive substances to blue light damage

[0237] Materials and Instruments:

[0238] 1. Cell line: Human skin fibroblasts CCD-966sk, obtained from the Bioresource Conservation and Research Center (BCRC); Cat.60153, hereinafter referred to as human skin fibroblasts.

[0239] 2. Cell culture medium: Earle's balanced salt type MEM (Minimum essential medium) culture medium containing 0.1M non-essential amino acids, 1.5g / L sodium bicarbonate, 0.1M pyruvate, and 10% fetal bovine serum (purchased from Gibco) was added.

[0240] 3. Phosphate-buffered saline (PBS): purchased from Gibco, product number 10437-028.

[0241] 4. DCFH-DA solution: The fluorescent dye DCFH-DA (purchased from Sigma / SI-D6883-50MG) was prepared with dimethyl sulfoxide (DMSO) to a concentration of 5 mg / ml, which can stain reactive oxidizing agents (ROS).

[0242] 5. Trypsin-EDTA: 10X Trypsin-EDTA (purchased from Gibco) diluted 10 times with PBS.

[0243] 6. Flow cytometry, BD Accuri C6 Plus.

[0244] Experimental procedure:

[0245] The room temperature referred to in this test is 25±5℃.

[0246] 1. Add 2 mL of cell culture medium to each well of a six-well culture dish, and seed each well with 1 × 10⁶ cells / well. 5 One human skin fibroblast CCD-966sk (preservation number BCRC 60153).

[0247] 2. Place the culture dish in a 5% CO2 environment at 37°C and incubate for 24 hours.

[0248] 3. After culturing, the cells were divided into the following three groups and subjected to the corresponding treatment procedures.

[0249] Blank group: Each well was replaced with 2 mL of simple cell culture medium (i.e., without the peach extract of the present invention) and cultured at 5% CO2 and 37°C for 1 hour. Then, 2 μL of DCFH-DA solution was added to each well and incubated for 15 minutes. After that, the wells were moved to the dark and incubated for another 15 minutes (at room temperature).

[0250] Control group: Each well was replaced with 2 mL of simple cell culture medium (i.e., without the peach extract of the present invention) and cultured at 5% CO2 and 37°C for 1 hour. Then, 2 μL of DCFH-DA solution was added to each well and the mixture was incubated for 15 minutes. After that, the wells were transferred to a blue light chamber and irradiated with blue light (wavelength of 500 nm) for 15 minutes (at room temperature).

[0251] TCI-AP-01 group: Each well was replaced with cell culture medium containing 0.1 mM hyperoside (isolated in Example 7) and cultured at 5% CO2 and 37°C for 1 hour. Then, 2 μL of DCFH-DA solution was added to each well and incubated for 15 minutes. After that, the well was transferred to a blue light box and irradiated with blue light (wavelength 500 nm) for 15 minutes (at room temperature).

[0252] TCI-AP-02 group: Each well was replaced with cell culture medium containing 0.1 mM isoquercitrin (isolated in Example 7) and cultured at 5% CO2 and 37°C for 1 hour. Then, 2 μL of DCFH-DA solution was added to each well and incubated for 15 minutes. After that, the well was transferred to a blue light box and irradiated with blue light (wavelength 500 nm) for 15 minutes (at room temperature).

[0253] TCI-AP-07 group: Each well was replaced with cell culture medium containing 0.1 mM pyroside (isolated in Example 7) and cultured at 5% CO2 and 37°C for 1 hour. Then, 2 μL of DCFH-DA solution was added to each well and incubated for 15 minutes. After that, the well was transferred to a blue light box and irradiated with blue light (wavelength 500 nm) for 15 minutes (at room temperature).

[0254] TCI-AP-08 group: Each well was replaced with cell culture medium containing 0.1 mM rutin (isolated in Example 7) and cultured at 5% CO2 and 37°C for 1 hour. Then, 2 μL of DCFH-DA solution was added to each well and incubated for 15 minutes. After that, the well was transferred to a blue light box and irradiated with blue light (wavelength 500 nm) for 15 minutes (at room temperature).

[0255] 4. Then, rinse each group twice with PBS.

[0256] 5. Next, add 200 μL of trypsin to each well and react for 5 minutes in the dark. After the reaction, add cell culture medium to each well to stop the reaction.

[0257] 6. Collect the cells from each group into their respective centrifuge tubes containing cell culture medium, and centrifuge the tubes containing cells and cell culture medium at 400×g for 10 minutes.

[0258] 7. After centrifugation, remove the supernatant from each centrifuge tube, and then wash the cell pellet in each centrifuge tube with PBS.

[0259] 8. Next, centrifuge each centrifuge tube again at 400×g for 10 minutes.

[0260] 9. After centrifugation again, remove the supernatant from each centrifuge tube and add 1 ml of PBS to suspend the cell pellet in each centrifuge tube to obtain the cell solution to be tested.

[0261] 10. The fluorescence signal of DCFH-DA in the cell culture of each tube was detected using a flow cytometer (BD Accuri C6 Plus Flow Cytometer 660517) to quantify the intracellular ROS content. The excitation wavelength used for fluorescence signal detection was 450-490 nm, and the emission wavelength was 510-550 nm. Since DCFH-DA is first hydrolyzed into DCFH (dichlorodihydrofluorescein) after entering the cell, and then oxidized by ROS into DCF (dichlorofluorescein) which emits green fluorescence, the fluorescence intensity of cells treated with DCFH-DA reflects the intracellular ROS content, thereby indicating the proportion of cells with high ROS expression relative to the original cell count. Three replicates were performed for each group, and the average value of the measurements from the three replicates was rounded to obtain the ROS content.

[0262] Experimental results:

[0263] Therefore, the human skin fibroblasts in the control group were not exposed to blue light, meaning they were under normal physiological metabolic conditions; thus, their relative ROS production was assumed to be 100%. Furthermore, considering the ROS content of the control group as 100% relative ROS production, the ROS content of the other groups was converted to relative ROS production as a percentage using interpolation, as shown in Table 3. Figure 12 As shown.

[0264] Table 3

[0265] Relative ROS generation Blank group 100％ control group 266.1％ TCI-AP-01 19.8％ TCI-AP-02 11.5％ TCI-AP-07 100.4％ TCI-AP-08 29.1％

[0266] exist Figure 12 In the above, compared with the control group, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001; compared with the control group, "#" represents a p-value less than 0.05, "##" represents a p-value less than 0.01, and "###" represents a p-value less than 0.001. The more "*" or "#" symbols present, the more statistically significant the difference.

[0267] Refer to Table 3 and Figure 12 In the control group, human skin fibroblasts produced a relative ROS level as high as 266.1% after 15 minutes of blue light irradiation. This indicates that blue light significantly promotes ROS production in human skin fibroblasts, potentially causing substantial damage to human skin.

[0268] Human skin fibroblasts in the TCI-AP-01 group, after treatment with hyperoside, showed a relative ROS production of 19.8% even under blue light irradiation. Compared to the control group or blank group, the ROS production in the TCI-AP-01 group was significantly reduced.

[0269] Human skin fibroblasts in the TCI-AP-02 group, after treatment with isoquercitrin, showed a relative ROS production of 11.5% even under blue light irradiation. Compared to the control group or blank group, the ROS production in the TCI-AP-02 group was significantly reduced.

[0270] After treatment with pyrolysis glycosides, the relative ROS production of human skin fibroblasts in the TCI-AP-07 group was 100.4% even under blue light exposure. Compared to the control group, the ROS production in the TCI-AP-07 group was significantly reduced, and almost the same as that in the control group. In other words, treatment with pyrolysis glycosides can almost offset the damage caused by blue light.

[0271] After treatment with rutin, the relative ROS production of human skin fibroblasts in the TCI-AP-08 group was 29.1% even under blue light irradiation. Compared with the relative ROS production of the control group or blank group, the ROS production of the TCI-AP-08 group was significantly reduced.

[0272] Therefore, the experimental results show that the bioactive substances in peach extract can significantly prevent the production of reactive oxidants caused by blue light.

[0273] Example 10: Antioxidant Experiment of Bioactive Substances

[0274] Reactive oxygen species (ROS) are one of the key determinants of oxidative stress in the body. This experiment measures ROS to determine whether an organism can combat oxidative stress. Many factors contribute to oxidative stress, such as chronic diseases, stress and anxiety, and being overweight. External factors should not be ignored, such as pollution, smoking, alcohol consumption, medications, sunlight, strenuous physical activity, or a sedentary lifestyle.

[0275] Materials and Instruments:

[0276] 1. Cell line: Human skin fibroblasts CCD-966sk, obtained from the Bioresource Conservation and Research Center (BCRC); Cat.60153, hereinafter referred to as human skin fibroblasts.

[0277] 2. Cell culture medium: basal medium containing 10 vol% FBS (purchased from Gibco). The basal medium was prepared by adding components to Eagle's minimum essential medium (MEM, purchased from Gibco, product number 15188-319) to include 1 mM sodium pyruvate (purchased from Gibco), 1.5 g / L sodium bicarbonate (purchased from Sigma, Cat. S5761-500G), and 0.1 mM non-essential amino acid solution (purchased from Gibco).

[0278] 3. Phosphate-buffered saline (PBS) solution: purchased from Gibco, product number 10437-028.

[0279] 4. DCFH-DA solution: Dissolve 2,7-dichloro-dihydro-fluorescein diacetate (DCFH-DA; product number SI-D6883, purchased from Sigma) in dimethyl sulfoxide (DMSO, purchased from Sigma, product number SI-D6883-50MG) to prepare a 5 μg / ml DCFH-DA solution.

[0280] 5. Flow cytometry, BD Accuri C6 Plus.

[0281] 6. Hydrogen peroxide (H2O2): Purchased from Sigma-Aldrich, product model 95299-1L.

[0282] 7. Trypsin-EDTA: 10X Trypsin-EDTA (purchased from Gibco) diluted 10 times with PBS.

[0283] Experimental procedure:

[0284] 1. Human skin fibroblasts were cultured at a density of 1 × 10⁶ cells per well. 5 The cells were inoculated in a 6-well culture dish containing 2 mL of cell culture medium per well using a specific method.

[0285] 2. Place the culture dish in 5% CO2 at 37°C and incubate for 24 hours.

[0286] 3. After culturing, the cells were divided into the following three groups and subjected to the corresponding treatment procedures.

[0287] Blank group: Each well was replaced with 2 mL of simple cell culture medium (i.e., without the peach extract of this invention).

[0288] Control group: Each well was replaced with 2 mL of simple cell culture medium.

[0289] TCI-AP-01 group: Replace each well with 2 mL of cell culture medium containing 0.1 mM hyperoside (isolated in Example 7).

[0290] TCI-AP-02 group: Replace each well with 2 mL of cell culture medium containing 0.1 mM isoquercitrin (isolated in Example 7).

[0291] TCI-AP-07 group: Replace each well with 2 mL of cell culture medium containing 0.1 mM pyroside (isolated in Example 7).

[0292] TCI-AP-08 group: Replace each well with 2 mL of cell culture medium containing 0.1 mM rutin (isolated in Example 7).

[0293] 4. After changing the culture medium, place the culture tray in 5% CO2 at 37°C for 1 hour.

[0294] 5. Add 2 μL of DCFH-DA solution to each well and allow it to react for 15 minutes.

[0295] 6. After DCFH-DA treatment, H2O2 was added to both the experimental and control groups, and the mixture was reacted at 37°C for 1 hour. This means that 1 mM hydrogen peroxide was added to each group to simulate oxidative damage.

[0296] 7. Then, rinse each well twice with 1 mL of PBS.

[0297] 8. Add 200 μL of trypsin to each well and react in the dark for 5 minutes. After the reaction, add cell culture medium to each well to stop the reaction.

[0298] 9. Collect the cells and cell culture medium from each group into their respective centrifuge tubes, and centrifuge the tubes containing the cells and cell culture medium at 400xg for 10 minutes.

[0299] 10. After centrifugation, remove the supernatant from each centrifuge tube and then reconstitute the cell pellet in each centrifuge tube with PBS.

[0300] 11. Next, centrifuge each centrifuge tube again at 400xg for 10 minutes.

[0301] 12. After centrifugation again, remove the supernatant from each centrifuge tube and suspend the cell pellet in 1 mL of PBS in the dark to obtain the cell solution to be tested.

[0302] 13. Flow cytometry was used to detect the fluorescence signal of DCFH-DA in the cell culture of each tube to quantify the intracellular ROS content. The excitation wavelength used for fluorescence detection was 450-490 nm, and the emission wavelength was 510-550 nm. Since the experiment was performed in triplicate, the average value of the measurements from each group's triplicate experiments was used as the ROS content.

[0303] Experimental results:

[0304] Therefore, the human skin fibroblasts in the control group were not damaged by hydrogen peroxide, meaning they were under normal physiological metabolic conditions; thus, it was assumed that their relative ROS production was 100%. Furthermore, considering the ROS content of the control group as 100% relative ROS production, the ROS content of the other groups was converted to relative ROS production as a percentage using interpolation, as shown in Table 4. Figure 13 As shown.

[0305] Table 4

[0306] Relative ROS generation Blank group 100％ control group 214.5％ TCI-AP-01 128.8％ TCI-AP-02 82.2％ TCI-AP-07 182.1％ TCI-AP-08 116.3％

[0307] exist Figure 13 In the above, compared with the control group, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001; compared with the control group, "#" represents a p-value less than 0.05, "##" represents a p-value less than 0.01, and "###" represents a p-value less than 0.001. The more "*" or "#" symbols present, the more statistically significant the difference.

[0308] like Figure 13 As shown in the comparison of the blank group and the control group, it can be seen that after hydrogen peroxide treatment, the relative amount of ROS (high fluorescence performance) increased by more than two times, and reached a statistical significance level (p value less than 0.001). This shows that hydrogen peroxide treatment does indeed promote the production of intracellular ROS, thereby causing damage to skin fibroblasts.

[0309] Continue to refer to Table 4 and Figure 13 In the TCI-AP-01 group, human skin fibroblasts treated with hypericin showed a decreasing trend in relative ROS production, even when subjected to hydrogen peroxide to simulate strong oxidative damage. This means that compared to the control group, the ROS production in the TCI-AP-01 group was significantly reduced to 128.8%.

[0310] In the TCI-AP-02 group, human skin fibroblasts treated with isoquercitrin showed a decreasing trend in relative ROS production, even when subjected to hydrogen peroxide to simulate strong oxidative damage. This means that compared to the control group (100% relative ROS production), the TCI-AP-02 group, even with hydrogen peroxide to simulate strong oxidative damage, exhibited 82.2% less ROS production than under normal physiological metabolism.

[0311] After treatment with pyrolysis glycosides, the relative ROS production of human skin fibroblasts in the TCI-AP-07 group showed a decreasing trend, even when simulated with strong oxidative damage using hydrogen peroxide. This means that compared to the control group, the ROS production in the TCI-AP-07 group was significantly reduced by 182.1%.

[0312] In the TCI-AP-08 group, human skin fibroblasts, after treatment with rutin, showed a decreasing trend in relative ROS production even when subjected to hydrogen peroxide to simulate strong oxidative damage. This means that compared to the control group, the ROS production in the TCI-AP-08 group was significantly reduced to 116.3%.

[0313] The above experimental results show that the bioactive substances in the peach extract of this embodiment can effectively reduce the production or accumulation of ROS in cells.

[0314] In summary, the peach extract according to any embodiment of the present invention can be used to prepare compositions that improve skin condition. In other words, the aforementioned compositions have one or more of the following functions: resisting blue light damage, enhancing the antioxidant capacity of skin fibroblasts, enhancing mitochondrial activity, promoting the production of skin elastin, reducing skin texture, reducing skin wrinkles, minimizing skin pores, increasing heme content, and increasing the iron content in the blood.

[0315] Of course, the present invention may have other various embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and modifications according to the present invention, but these corresponding changes and modifications should all fall within the protection scope of the claims of the present invention.

Claims

1. Use of a peach extract, characterized in that, For use in the preparation of a composition for improving skin, and the peach extract is extracted from the red flesh of a peach.

2. The use of claim 1, wherein the peach extract is used to combat blue light damage.

3. The use of claim 1, wherein the peach extract is used to boost mitochondrial activity.

4. The use of claim 1, wherein the peach extract is used to promote skin elastin production.

5. The use of claim 1, wherein the peach extract is used to reduce skin texture.

6. The use of claim 1, wherein the peach extract is used to reduce skin wrinkles.

7. The use of claim 1, wherein the peach extract is used to shrink skin pores.

8. The use of any one of claims 1 to 7, wherein the peach extract is used to boost skin fibroblast antioxidant capacity.

9. The use of claim 1, wherein the peach extract comprises at least hyperoside, isoquercitrin, patuletin, and rutin.

10. The use of claim 9, wherein the hyperoside, isoquercitrin, patuletin, and rutin are used to combat blue light damage.

11. The use of claim 9, wherein the hyperoside, isoquercitrin, patuletin, and rutin are used to boost skin fibroblast antioxidant.

12. Use of a peach extract, characterized in that, For use in the preparation of a composition for improving anemia, and the peach extract is extracted from the red flesh of a peach.

13. The use of claim 12, wherein the peach extract is used to boost hemoglobin content.

14. The use of claim 12, wherein the peach extract is used to boost iron content in blood.

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