An antioxidant whitening soothing anti-aging antibacterial anti-inflammatory peony active peptide

Abstract

The purpose of this invention is to provide an antioxidant, whitening, soothing, anti-aging, antibacterial, and anti-inflammatory peony active peptide. This active peptide is prepared by enzymatic hydrolysis of peony flower extract. Various peony active peptides have been developed, all-natural and additive-free. The peony active peptide possesses antioxidant, whitening, moisturizing, pigmentation-fading, anti-aging, antibacterial, anti-inflammatory, and liver-protective effects. Its amino acid sequence is MYECCFECKCKR. This invention utilizes modern biological methods to scientifically and purposefully screen the types of enzymes used for hydrolysis, enabling the substances to be fully decomposed into small molecules, facilitating absorption and utilization. The final result is an enzymatic hydrolysate with high antioxidant and high peptide yield, as well as antioxidant and liver-protective peony active peptides.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, in particular to bioactive peptides, especially a peony active peptide with antioxidant, whitening, soothing, anti-aging, antibacterial and anti-inflammatory effects prepared by enzymatic hydrolysis of peony fresh flower extract. Background Art

[0002] Peony is known as the "King of Flowers", and its root bark is used as a traditional Chinese medicine, called Moutan Cortex, also known as tree peony bark, moutan bark, scraped moutan bark, etc. It is a commonly used traditional Chinese medicine for cooling blood and removing blood stasis, and is used for syndromes such as maculae caused by warm toxin, hematemesis and epistaxis, night fever with early coolness, hectic fever without perspiration, amenorrhea and dysmenorrhea, carbuncles and sores, traumatic injuries and pains. Modern studies have shown that Moutan Cortex has activities such as antibacterial, anti-inflammatory, anti-allergic, anti-tumor, hemostatic, blood stasis removing, heat-clearing and detoxifying, sedative, analgesic, and antispasmodic effects, can also promote the phagocytic function of monocytes, improve the specific immune function of the body, and increase the weight of immune organs. Fresh peony flowers used in skin care products have the functions of anti-wrinkle and antioxidant, whitening and moisturizing, fading pigment spots, delaying aging, antibacterial and anti-inflammatory.

[0003] Several preparation methods of fresh peony flower water have been disclosed at present, including supercritical CO2 fluid extraction, which has the disadvantages of large equipment investment and complicated operation; rotary evaporation, which has the disadvantages of complicated process, high operating conditions, low utilization rate, and inability to obtain fine products; distillation, which has the disadvantages of high energy consumption of the process method and low product grade. The disclosed preparation methods of fresh peony flower extract have low antioxidant activity, low utilization rate, and cannot obtain fine products.

[0004] Enzymatic hydrolysis can break down substances to a smaller molecular size, facilitating absorption and utilization. Current research on peony hydrolysis includes CN202111073238 20210914; CN20151097643920151221; and CN201410496566 20140925. While these studies utilize cellulase and pectinase or cellulase hydrolysis methods, they do not explain the effects of such hydrolysis. CN201410292815 20140626 describes a peony peptide beverage obtained using mixed proteases; CN201410628521 20141111 describes a method for extracting peony polysaccharides from peony cake using a biological enzymatic method; and CN202111459730 20211202 describes a method for preparing peony stamen protein powder. Furthermore, the extraction of active peptides from peony cake is often carried out in the field, which affects the use of oil. This application utilizes fresh peony petals to fully leverage the peony material, maximizing its value after its ornamental and other uses, while also preserving the uses of peony seeds. This invention, through modern biological methods, scientifically and purposefully screens the types of enzymes for enzymatic hydrolysis, enabling the substances to be fully decomposed to a small molecule level, facilitating absorption and utilization, and obtaining an enzymatic hydrolysate with a high peptide yield. This further yields peony active peptides with antioxidant, whitening and moisturizing, pigmentation-fading, anti-aging, antibacterial and anti-inflammatory, and liver-protective properties. Summary of the Invention

[0005] The purpose of this invention is to provide an antioxidant, whitening, soothing, anti-aging, antibacterial, and anti-inflammatory peony active peptide, especially one prepared by enzymatic hydrolysis of peony flower extract. Various peony active peptides have been developed, which are purely natural and additive-free, and possess antioxidant, whitening, moisturizing, pigmentation-fading, anti-aging, antibacterial, and anti-inflammatory properties.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The purpose of this invention is to provide a peony bioactive peptide with the amino acid sequence MYECCFECKCKR. The peony bioactive peptide is prepared using the following method.

[0008] Peony petals were collected, washed, and frozen for preservation. They were then further pulverized, cell-wall broken, sterilized, and heated using a microwave-ultrasonic combination device, followed by pressing. The supernatant was collected, and the pH was adjusted to 7.5-9.6 with sodium hydroxide solution for preliminary enzymatic hydrolysis. After complete enzymatic hydrolysis, the mixture was sterilized by heating, and the enzymes were inactivated. The residue and insoluble matter were removed by filtration. A staged enzymatic hydrolysis was then performed using a compound enzyme. Each enzyme addition was accompanied by stirring, and after complete hydrolysis, the next enzyme was added. The mixture was then sterilized by heating, and the residue and insoluble matter were removed by filtration. The mixture was then centrifuged, and the centrifuged liquid was filtered through a membrane to retain small peptides below 10,000 Daltons, yielding the peony petal hydrolysate, i.e., the peony flower extract. The preliminary enzymatic hydrolysis refers to the use of an enzyme containing lipase.

[0009] The preliminary enzymatic hydrolysis refers to the preliminary enzymatic hydrolysis performed using an enzyme containing cellulase.

[0010] The preliminary enzymatic hydrolysis refers to the preliminary enzymatic hydrolysis performed using an enzyme containing glycosidase.

[0011] The staged enzymatic hydrolysis refers to enzymatic hydrolysis using a complex enzyme of papain, trypsin, neutral protease, alkaline protease, subtilisin and / or chymotrypsin, in order to retain as many glutamic acid, arginine, lysine, phenylalanine, tyrosine, cysteine ​​and methionine as possible.

[0012] To extend the storage time of peony petal enzymatic hydrolysate, bergamot and / or geranium can be added as natural preservatives. The freezing temperature is -5 to -22°C; the ultrasonic frequency is 50 to 120 kHz; and the microwave heating is 35-55°C.

[0013] The enzymatic hydrolysate was vacuum dried, and the sample was sent to the testing institute for analysis and sequencing to predict its function. A peony active peptide with antioxidant and liver-protective properties was obtained, with the amino acid sequence MYECCFECKCKR.

[0014] Technical effect

[0015] The purpose of this invention is to provide an antioxidant, whitening, soothing, anti-aging, antibacterial, and anti-inflammatory peony active peptide, especially prepared by enzymatic hydrolysis of peony flower extract. This has led to the development of various peony active peptides that are purely natural and additive-free. This invention utilizes modern biological methods to scientifically and purposefully screen the types of enzymes used in the hydrolysis, enabling the substances to be fully decomposed into small molecules, facilitating absorption and utilization. The final product is an enzymatic hydrolysate with high antioxidant and high peptide yield, yielding peony active peptides with antioxidant, whitening, moisturizing, pigmentation-fading, anti-aging, antibacterial, anti-inflammatory, and liver-protective properties. Detailed Implementation

[0016] Example 1: Preparation method of peony active peptides, the specific preparation steps are as follows:

[0017] Collect the petals of Danfeng peony in the early morning, wash and freeze them at a temperature of -5 to -22℃. Then, use a microwave and ultrasonic combination device to further pulverize, break cell walls, and eliminate bacteria. Increase the temperature to eliminate oxidases that cause peony oxidation and decay. Adjust the ultrasonic frequency to 50-120kHz and the microwave temperature to 35-55℃. Press the mixture, collect the supernatant, and adjust the pH to 7.5-9.6 using sodium hydroxide solution. Perform preliminary enzymatic hydrolysis using lipase, cellulase, and glycosidase. After complete enzymatic hydrolysis, increase the temperature... Sterilization and enzyme inactivation are performed, followed by filtration to remove enzymatic hydrolysis residues and insoluble matter. Staged enzymatic hydrolysis is conducted using a complex enzyme combination (1:1:1:1:1:1:1) of papain, trypsin, neutral protease, alkaline protease, subtilisin, and chymotrypsin. Stirring is performed after each enzyme addition to ensure complete enzymatic hydrolysis. Afterward, sterilization and enzyme inactivation are achieved by heating, followed by filtration to remove enzymatic hydrolysis residues and insoluble matter. Centrifugation is then performed, and the centrifuged liquid is filtered through a membrane to retain small peptides below 10,000 Daltons, yielding peony petal enzymatic hydrolysate, i.e., peony flower extract. To extend the shelf life of the peony petal enzymatic hydrolysate, bergamot and / or geranium can be added as natural preservatives. The peptide yield, determined by the Kjeldahl method, reaches 94%.

[0018] After separating and purifying the peptides from the enzymatic hydrolysate, the samples were vacuum dried and sent to a testing institute for analysis and sequencing to predict their function. This yielded peony bioactive peptides with antioxidant and hepatoprotective properties, with the amino acid sequence MYECCFECKCKR.

[0019] Unless otherwise specified, in the experiments verifying the function of the enzymatic hydrolysate and active peptides, the concentration of the enzymatic hydrolysate used in the culture medium was 10%.

[0020] Comparative Example 1: The preliminary enzymatic hydrolysis step was omitted; other steps were the same as in Example 1, and the peptide yield reached 18%. Comparative Example 2: The combination of complex enzymes was replaced with a mixture of trypsin, papain, subtilisin, alkaline 2709 protease, and neutral Neutrase protease in a weight ratio of 1:1.2:0.8:1.2:1.2; other steps were the same as in Example 1, and the peptide yield reached 29%.

[0021] Comparative Example 3: The combination of complex enzymes was replaced with acidic protease, pectinase and cellulase in a 1:1:1 ratio, and the other steps were the same as in Example 1. The polypeptide yield reached 24%.

[0022] Comparative Example 4: Only chymotrypsin was retained in the combination of complex enzymes, and the other steps were the same as in Example 1, and the peptide yield reached 12%.

[0023] Example 2 Cytotoxicity

[0024] Keratinocyte (HaCat) seeding density 1×10 5Cells were seeded per well in 96-well plates. Enzyme digests were added to the culture medium for each group. After culturing for 24 hours, 10% EZ-Cytox was added to DMEM medium, and cell viability was measured. After 4 hours of culturing, absorbance was measured at 450 nm using a microplate reader. Each group was performed in quadruplicate.

[0025] Cell survival rate (%) = (Absorbance of each group / Absorbance of the control group) × 100%

[0026] Results: Experimental group 1 showed 2.25% cytotoxicity, with the active peptide exhibiting 2.5% cytotoxicity. Control group 1 showed 5.5% cytotoxicity, control group 2 showed 3.25% cytotoxicity, control group 3 showed 3.75% cytotoxicity, and control group 4 showed 3.25% cytotoxicity. Except for control group 1, all other groups showed low cytotoxicity (below 5.0%) and were suitable for use as cosmetic raw materials. Due to the complex composition of control group 1, it was removed in subsequent experiments.

[0027] Example 3 Antioxidant effect

[0028] Human fibroblasts were seeded in 96-well culture plates containing DMEM at a density of 1 × 10⁶ cells / well. 5 Cells per well were cultured with the enzyme digest for 24 hours after adding each group of enzyme digests to the culture medium. The medium was then replaced with serum-free DMEM and cultured for another 24 hours. After culturing, the supernatant from each well was collected, and the scavenging rates of hydroxyl (·OH), superoxide anion (O2-·), and DPPH· free radicals in the cells were measured according to the kit instructions. The results are shown in Table 1. Calculations were performed with the initial hydroxyl and superoxide anion free radical scavenging rates of each group of cells at 0%. Five replicates were performed for each group.

[0029] Table 1. Results of scavenging rates of hydroxyl and superoxide anion free radicals.

[0030]

[0031] Results: The scavenging effect of hydroxyl and superoxide anion free radicals in Experiment 1 was significantly better than that in Control Groups 2-4.

[0032] Example 4: Inhibition of tyrosinase activity

[0033] Cells cultured in Example 3 were used to determine the activity of tyrosinase inhibition. The tyrosinase inhibition rate was determined using the T / SHRH 015-2018 Cosmetics - Tyrosinase Activity Inhibition Test. The results are shown in Table 2.

[0034] Table 2 Tyrosinase activity inhibition test.

[0035]

[0036] Results: The scavenging effect of hydroxyl and superoxide anion free radicals in Experiment 1 was significantly better than that in Control Groups 2-4.

[0037] Example 5 Anti-autophagy effect

[0038] Autophagy experiments were conducted using keratinocytes (HaCat) as the subject. The number of autophagosomes within HaCat was measured as a quantitative standard for autophagy to assess antioxidant and anti-UV effects. Cells were cultured at a density of 1×10⁻⁶. 5 Cells per well were transferred to 96-well plates at a density of [number] cells / well, and enzyme digests were added to the culture medium for each group. The plates were then incubated for 24 hours. The concentration was 20 mJ / cm². 2 Cells were irradiated with UVB, and 0.1 mM H2O2 was added to the culture medium. The reaction was allowed to proceed for 1 hour, followed by the addition of autophagy detection reagent working solution. After reacting for 15 minutes to 1 hour, the cells were washed with PBST. PBS was added, and absorbance was measured at 360 nm / 550 nm. A blank control group of HaCaT cells (untreated with UVB and H2O2) was used as the autophagy control, with four replicates per group.

[0039] Results: The autophagy rate of HaCaT cells treated with ultraviolet light and H2O2 reached 102.25%. The autophagy rate of cells in experimental group 1 was 125.75%, the autophagy rate of cells with active peptides was 150.25%, the autophagy rate of cells in control group 2 was 106.5%, the autophagy rate of cells in control group 3 was 104.75%, and the autophagy rate of cells in control group 4 was 105.25%.

[0040] Example 6 Antibacterial effect

[0041] Cells cultured in Example 3 (cell state before serum-free medium replacement) were used to screen antimicrobial activity using the agar plate diffusion method. The size of inhibition zones for Staphylococcus aureus and Escherichia coli was observed, and the results are shown in Table 3.

[0042] Table 3. Results of inhibition zone size determination.

[0043]

[0044] Example 7: Human Trial

[0045] One hundred participants aged 20-55 years were selected and administered the medication twice daily, morning and evening, for a 30-day observation period. Each item was scored out of 5 points, with all scores being integers. The average scores for each item are shown below. The results are presented in Table 4.

[0046] Table 4 shows the scores for application comfort, irritation, anti-spot properties, and moisturizing smoothness.

[0047] Experimental group 1 4.78 0.15 4.89 4.79 Active peptides 4.83 0.10 4.95 4.87 Compare with Example 2 4.87 0.37 4.34 4.15 Compare with Example 3 4.89 0.21 4.49 4.27 Compare with Example 4 4.77 0.17 4.42 4.36

[0048] Example 8: Liver-protective effect of active peptides

[0049] SD rat hepatocytes were isolated and cultured in DMEM suspension. Active peptides were added, and then 2 mmol / L H2O2 was added to the culture medium to induce acute hepatocyte injury. The ALT activity, NO and MDA content, SOD activity and other indicators in the cell suspension were detected according to the kit instructions.

[0050] Table 5. Results of ALT, NO, SOD, and MDA measurements.

[0051] H2O2 damage group 170.58±4.35 30.57±0.49 31.71±0.40 4.75±0.47 Blank group 60.41±1.73 3.75±0.41 45.53±0.51 1.85±0.10 Active peptides 42.75±2.86 4.43±0.56 43.67±0.17 2.17±0.24

[0052] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A peony active peptide with antioxidant, whitening, soothing, anti-aging, antibacterial, and anti-inflammatory properties, characterized in that: The amino acid sequence of the peony active peptide is MYECCFECKCKR.

2. The peony active peptide with antioxidant, whitening, soothing, anti-aging, antibacterial, and anti-inflammatory properties according to claim 1, characterized in that: The peony active peptides are small molecule peptides with a molecular weight of less than 10,000 Daltons, obtained by enzymatic hydrolysis of peony petals and filtration through a filter membrane.

3. The peony active peptide with antioxidant, whitening, soothing, anti-aging, antibacterial, and anti-inflammatory properties according to claim 2, characterized in that: The method for enzymatic hydrolysis of peony petals includes: first, preliminary enzymatic hydrolysis; after complete enzymatic hydrolysis, sterilization and enzyme inactivation by heating; and filtration to remove enzymatic hydrolysis residue and insoluble matter. Then, staged enzymatic hydrolysis is performed using a compound enzyme. Each enzyme is stirred before adding it, and after complete enzymatic hydrolysis, the next enzyme is added. The mixture is then sterilized and inactivated by heating, and filtration is performed to remove enzymatic hydrolysis residue and insoluble matter. The mixture is then centrifuged, and the centrifuged liquid is filtered through a membrane to retain small molecule peptides below 10,000 Daltons, yielding peony petal enzymatic hydrolysate. The hydrolysate is then vacuum-dried, and the sample is sent to a testing institute for analysis and sequencing to predict its function, yielding peony active peptides with antioxidant and liver-protective properties.

4. The peony active peptide with antioxidant, whitening, soothing, anti-aging, antibacterial, and anti-inflammatory properties according to claim 2, characterized in that: Peony petals need to be pretreated. The specific pretreatment methods include: collecting Danfeng peony petals, washing and freezing them, further crushing and sterilizing them using microwave and ultrasonic equipment, pressing them, collecting the supernatant, and adjusting the pH value to 7.5-9.6 using sodium hydroxide solution.

5. The peony active peptide with antioxidant, whitening, soothing, anti-aging, antibacterial, and anti-inflammatory properties according to claim 3, characterized in that: The preliminary enzymatic hydrolysis refers to the preliminary enzymatic hydrolysis performed using an enzyme containing lipase.

6. The peony active peptide with antioxidant, whitening, soothing, anti-aging, antibacterial, and anti-inflammatory properties according to claim 3, characterized in that: The preliminary enzymatic hydrolysis refers to the preliminary enzymatic hydrolysis performed using an enzyme containing cellulase.

7. The peony active peptide with antioxidant, whitening, soothing, anti-aging, antibacterial, and anti-inflammatory properties according to claim 3, characterized in that: The preliminary enzymatic hydrolysis refers to the preliminary enzymatic hydrolysis performed using an enzyme containing glycosidase.

8. The peony active peptide with antioxidant, whitening, soothing, anti-aging, antibacterial, and anti-inflammatory properties according to claim 3, characterized in that: The staged enzymatic hydrolysis refers to enzymatic hydrolysis using a complex enzyme of papain, trypsin, neutral protease, alkaline protease, subtilisin, and / or chymotrypsin.

9. The peony active peptide with antioxidant, whitening, soothing, anti-aging, antibacterial, and anti-inflammatory properties according to claim 3, characterized in that: The staged enzymatic hydrolysis refers to enzymatic hydrolysis using a complex enzyme consisting of papain, trypsin, neutral protease, alkaline protease, subtilisin, and chymotrypsin.

10. The peony active peptide according to any one of claims 1-9, characterized in that: The peony active peptides are used for anti-oxidation, whitening, soothing, anti-aging, antibacterial, anti-inflammatory, and liver protection.

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