Fargesia and citrus grandis pericarp anti-aging composition for reducing methylation age and application thereof

Abstract

This invention relates to an anti-aging composition of Sophora japonica buds and dried tangerine peel that reduces methylation age and its applications. The composition comprises Sophora japonica bud powder and dried tangerine peel powder in a mass ratio of 1:(0.5-2). This invention optimizes the preparation of Sophora japonica bud powder through a process combining enzymatic hydrolysis with cellulase, pectinase, and xylanase, along with synergistic fermentation with Lactobacillus plantarum and Lactobacillus rhamnosus, significantly improving the dissolution rate and bioavailability of active ingredients. This, in conjunction with the dried tangerine peel powder, achieves bidirectional regulation of DNA methylation. The above anti-aging composition can be widely used in the preparation of products that reduce methylation age, delay aging, and improve tissue homeostasis, including health foods, dietary supplements, functional foods, skincare products, and pharmaceuticals.

Description

Technical Field

[0001] This invention relates to the field of anti-aging composition technology, specifically to an anti-aging composition of Sophora japonica buds and dried tangerine peel that reduces methylation age and its application. Background Technology

[0002] DNA methylation, as a core regulatory mechanism of epigenetics, has a level of change that is highly correlated with physiological age. The PathwayAge epigenetic clock model proposed in the paper "Decoding disease–specific aging mechanisms through pathway-level epigenetic clock: insights from multi-cohort validation" published by Pan Li et al. has confirmed that methylated physiological age can accurately reflect the true aging state of the body. Methylation age of systemic pathways (cardiovascular, immune, metabolic, etc.) accelerates aging and directly increases the risk of age-related diseases.

[0003] Existing anti-aging products often employ single mechanisms such as targeted antioxidation and anti-inflammation. For example, grape seed extract provides antioxidant protection by scavenging free radicals, and probiotics provide anti-inflammatory protection by regulating gut microbiota. However, none of these methods address the regulation of DNA methylation age. Furthermore, some products suffer from issues such as single extraction processes for active ingredients, low dissolution rates, and a lack of synergistic effects or poor synergistic effects between components. Flavonoids and polysaccharides in Sophora japonica buds and tangerine peel possess antioxidant and anti-inflammatory activities, but current technologies utilize single extraction methods for both, failing to demonstrate their combined regulatory effect on DNA methylation. Moreover, traditional single-enzymatic hydrolysis and single-bacterial fermentation processes result in low dissolution rates and poor bioavailability of active ingredients. Therefore, developing an anti-aging composition that targets and regulates DNA methylation age through innovative processes, synergistic components, and multi-layered experimental validation of efficacy has become an urgent need in the anti-aging health product field. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an anti-aging composition of Sophora japonica buds and dried tangerine peel that reduces methylation age and its application. This composition improves the dissolution rate and bioavailability of active ingredients through an innovative extraction process of compound enzymatic hydrolysis and dual-strain fermentation of Sophora japonica buds. It utilizes the synergistic effect of Sophora japonica bud powder and dried tangerine peel powder to bidirectionally regulate DNA methylation level. Its efficacy has been verified by in vitro enzyme activity and human clinical trials, and it has no obvious toxic side effects.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an anti-aging composition of Sophora japonica buds and dried tangerine peel that reduces methylation age, the anti-aging composition comprising Sophora japonica bud powder and dried tangerine peel powder in a mass ratio of 1:(0.5-2); The preparation method of the Sophora japonica powder includes the following steps: (1) Compound enzyme extraction: Powder the Sophora japonica flowers into powder, add 5-10 times the mass of citrate-sodium citrate buffer, add compound enzyme for enzymatic hydrolysis, obtain enzymatic hydrolysate, sterilize the enzymatic hydrolysate and set aside; wherein, the amount of compound enzyme added is 0.3-0.5% of the mass of Sophora japonica flowers; the compound enzyme is cellulase, pectinase and xylanase; (2) Co-fermentation of two bacteria: A compound bacterial agent is added to the enzymatic hydrolysate for anaerobic fermentation. After fermentation, the mixture is sterilized to obtain the fermentation broth. The compound bacterial agent includes Lactobacillus plantarum and Lactobacillus rhamnosus. (3) Centrifuge the fermentation broth, collect the supernatant, concentrate the supernatant, and dry it to obtain the Sophora japonica powder.

[0006] The Sophora japonica bud powder of this invention is first extracted by a complex enzyme (cellulase, pectinase and xylanase). Under this process, the total flavonoid dissolution rate of Sophora japonica buds reaches more than 95.6%, which is significantly higher than that of single enzyme hydrolysis or dual enzyme combination. Then, the enzymatic hydrolysate is fermented by two bacteria. Lactobacillus plantarum mainly converts flavonoid glycosides into aglycones, while Lactobacillus rhamnosus further degrades the crude fiber of Sophora japonica buds and improves the polysaccharide dissolution rate. The two work synergistically to increase the bioavailability of Sophora japonica bud flavonoids by 10%-15% compared with single bacteria fermentation.

[0007] After optimization using three enzymes and two bacteria, the bioavailability of the active ingredients in Sophora japonica bud powder was significantly improved, and the regulatory effect was more stable. Specifically: 1. Sophora japonica powder inhibited DNMT3A activity in a concentration-dependent manner: the inhibition rate reached 42.6% at 50 μmol / L and 52.0% at 100 μmol / L, which is close to the inhibitory effect of the positive control 5-azacytidine; 2. Concentration-dependent activation of TET demethylase activity by tangerine peel powder: The activation rate reached 38.9% at a concentration of 20 μmol / L and 48.0% at a concentration of 40 μmol / L, which was superior to the positive control vitamin C; 3. Synergistic effect of the two: Sophora japonica powder reduces DNA methylation modification, while tangerine peel powder promotes the demethylation of methylated DNA. The bidirectional regulation restores the DNA methylation level to a youthful state, thereby reducing the physiological age of methylation and delaying the aging of various system pathways.

[0008] The synergistic effect of Sophora japonica flower powder and dried tangerine peel powder allows for bidirectional regulation of DNA methylation levels. Sophora japonica flower powder inhibits DNA methyltransferase 3A (DNMT3A) in a concentration-dependent manner, while dried tangerine peel powder activates TET demethylase in a concentration-dependent manner. In vitro enzyme activity experiments and human clinical trials have verified that this composition can significantly reduce the physiological age of DNA methylation, delay aging of cardiovascular, immune, metabolic, nervous, and reproductive pathways, improve tissue homeostasis, and enhance the body's resistance to damage and repair capabilities, with no significant toxic side effects.

[0009] More preferably, in step (1) of preparing the Sophora japonica powder, the pH of the citrate-sodium citrate buffer solution is 4.5-5.5, the amount of compound enzyme added is 0.3-0.5% of the mass of Sophora japonica, the mass ratio of cellulase, pectinase and xylanase is (1.5-2.5):1:1; the enzymatic hydrolysis temperature is 45-55℃, the time is 1-2h, and the pH is 4.5-5.5; the sterilization temperature is 85-95℃, and the time is 10-15min.

[0010] More preferably, in step (2) of preparing the Sophora japonica powder, the amount of the compound microbial agent added is 0.5-1% of the mass of the enzymatic hydrolysate, and the viable count ratio of the compound microbial agent is 1-8×10⁻⁶. 9 CFU / g, the live count ratio of Lactobacillus plantarum and Lactobacillus rhamnosus is 1:1; the fermentation temperature is 35-38℃, the time is 5-7h, and the pH is 5.5-6.5. After fermentation, the pH is adjusted to 7.0 to terminate fermentation; the sterilization temperature is 85-95℃ and the time is 10-15min.

[0011] Preferably, the *Lactiplantibacillus plantarum* 6096 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC NO:64374 and deposit date of February 5, 2024. This *Lactiplantibacillus plantarum* has been disclosed in CN202510515362.2, "Ginseng Extract Rich in Rare Saponins and its Preparation Method and its Application in Anti-aging". *Lactaseibacillus rhamnosus* YG-8 is also deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC NO:66443 and deposit date of May 30, 2025.

[0012] More preferably, in step (3) of preparing the Sophora japonica powder, the centrifugation speed is 9000-11000 r / min and the time is 15-25 min; the concentration is carried out by vacuum concentration treatment, with a vacuum degree of 0.06-0.08 MPa and a temperature of 50-60℃, and the concentration is carried out to a solid content of 8-12%; the drying is carried out by spray drying process, with an inlet air temperature of 180-200℃ and an outlet air temperature of 80-90℃.

[0013] Preferably, the method for preparing the tangerine peel powder includes the following steps: S1. Compound Enzymatic Hydrolysis: Grind dried tangerine peel into powder, add 7-9 times its weight of deionized water to make a slurry, add a compound enzyme for enzymatic hydrolysis, obtain the hydrolysate, inactivate the enzyme, filter, and obtain the filtrate; wherein, the compound enzyme is cellulase and pectinase. S2. Concentration: The filtrate is concentrated under reduced pressure to obtain a concentrated solution with a solid content of 5-15%, which is then dried to obtain the tangerine peel powder.

[0014] More preferably, in step S1 of preparing the tangerine peel powder, the mass ratio of cellulase to pectinase is 3:1, the amount of compound enzyme added is 0.2-0.4% of the raw material mass; the enzymatic hydrolysis temperature is 45-55℃ and the time is 0.5-1.5h; the enzyme inactivation temperature is 75-90℃ and the time is 10-20min; the filtration is carried out using a plate and frame filter press at a pressure of 0.3-0.5MPa.

[0015] More preferably, in step S2 of the preparation of the tangerine peel powder, the drying step is as follows: 5-10% by mass of resistant dextrin is added to the concentrated liquid, mixed evenly, and then spray-dried, with an inlet air temperature of 170-190℃ and an outlet air temperature of 75-85℃.

[0016] In a second aspect, the present invention provides the application of the anti-aging composition of the first aspect in the preparation of dietary supplements, functional beverages, skin care products or pharmaceuticals, wherein the anti-aging composition accounts for 10-80% of the mass of the product, of which 30-60% is accounted for in dietary supplements and pharmaceuticals, 10-30% in functional beverages, and 20-50% in skin care products.

[0017] Preferably, the dosage form of the product includes powder, granules, capsules, tablets, oral liquid, cream, serum, injection, or gel.

[0018] More preferably, each capsule contains 200mg or 400mg of the core composition, each 10mL vial of the oral liquid contains 200mg of the core composition, and each 10g of the cream contains 2-5g of the core composition. The effective daily intake of the above oral products is: 100-500mg of Sophora japonica powder and 100-500mg of dried tangerine peel powder; preferably, the daily intake is 400mg of Sophora japonica powder and 400mg of dried tangerine peel powder.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention optimizes the preparation of Sophora japonica powder by combining the enzymatic hydrolysis of three enzymes, cellulase, pectinase and xylanase with the synergistic fermentation of two bacteria, Lactobacillus plantarum and Lactobacillus rhamnosus, which significantly improves the dissolution rate and bioavailability of active ingredients and achieves bidirectional regulation of DNA methylation in synergy with tangerine peel powder.

[0020] (2) The anti-aging composition of the present invention has been verified by in vitro enzyme activity experiments, mouse animal experiments and human clinical trials. It has been confirmed that the combination of Sophora japonica powder and tangerine peel powder prepared by three enzymes and two bacteria has significant effects in reducing methylation age and delaying systemic aging, and has no obvious toxic side effects. Compared with single bacteria fermentation, Sophora japonica powder prepared by dual bacteria fermentation process has an inhibitory effect on DNMT3A of about 8%-12% at the same dose. The above anti-aging composition can be widely used in the preparation of products that reduce methylation age, delay aging and improve tissue homeostasis, including health food, dietary supplements, functional food, skin care products and pharmaceuticals. Detailed Implementation

[0021] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0022] The sources of the raw materials used in the following examples and comparative examples are as follows: Cellulase: Manufacturer: Weifang Kangdian Biotechnology Co., Ltd.; Model: CO3; Pectinase: Manufacturer: Beijing Kandi Technology Co., Ltd., Model: JX03; Xylanase: Manufacturer: Qingdao Weilan Biotechnology Co., Ltd.; Specification: Trichoderma. Lactiplantibacillus plantarum 6096: Accession number GDMCC NO:64374, deposited at Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, on February 5, 2024. This Lactiplantibacillus plantarum has been disclosed in CN202510515362.2, "Ginseng Extract Rich in Rare Saponins and its Preparation Method and its Application in Anti-aging".

[0023] Lactobacillus rhamnosus YG-8: accession number GDMCC NO:66443, deposited at Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, on May 30, 2025.

[0024] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0025] Example 1: Preparation of Sophora japonica flower powder The preparation method of the Sophora japonica powder includes the following steps: (1) Compound enzyme extraction: Take 1 kg of Sophora japonica flower raw material, crush it to 50 mesh, add 8 kg of citrate-sodium citrate buffer solution with pH 5, add 4 g of compound enzyme for enzymatic hydrolysis, and obtain the enzymatic hydrolysate. Sterilize the enzymatic hydrolysate for later use; wherein, the compound enzyme is cellulase, pectinase and xylanase, and the mass ratio of cellulase, pectinase and xylanase is 2:1:1; the enzymatic hydrolysis temperature is 50℃ and the time is 1.5 h; the sterilization temperature is 90℃ and the time is 12 min; (2) Co-fermentation by two microorganisms: 8g of compound microbial agent was added to the enzymatic hydrolysate for anaerobic fermentation. After fermentation, the mixture was sterilized to obtain the fermentation broth. The viable count ratio of the compound microbial agent was 5×10⁻⁶. 9 CFU / g, the compound microbial agent includes Lactobacillus plantarum and Lactobacillus rhamnosus, with a live count ratio of Lactobacillus plantarum to Lactobacillus rhamnosus of 1:1; the fermentation temperature is 37℃, the time is 6h, and the pH is 6. After the fermentation is completed, the pH is adjusted to 7.0 to terminate the fermentation. (3) The fermentation broth was centrifuged, the supernatant was collected, the supernatant was concentrated and dried to obtain about 155g of Sophora japonica powder. The total flavonoid content was 17.2%, the total polysaccharide content was 4.1%, the flavonoid aglycone conversion rate was 82.5%, and the bioavailability was 76.3%. The centrifugation speed was 10000r / min and the time was 20min. The concentration was carried out by vacuum concentration treatment with a vacuum degree of 0.07MPa and a temperature of 55℃, and the solid content was concentrated to 10%. The drying was carried out by spray drying process with an inlet air temperature of 190℃ and an outlet air temperature of 85℃.

[0026] Example 2 Preparation of Tangerine Peel Powder The method for preparing the tangerine peel powder includes the following steps: S1. Compound Enzymatic Hydrolysis: Take 1 kg of dried tangerine peel raw material, pulverize it to 40 mesh, add 8 kg of deionized water to make a slurry, add 3 g of compound enzyme for enzymatic hydrolysis, obtain the enzymatic hydrolysate, inactivate the enzyme, filter, and obtain the filtrate; wherein, the compound enzyme is cellulase and pectinase, the mass ratio of cellulase to pectinase is 3:1; the enzymatic hydrolysis temperature is 50℃ and the time is 1 h; the enzyme inactivation temperature is 80℃ and the time is 15 min; the filtration is carried out using a plate and frame filter press at a pressure of 0.4 MPa; S2. Concentration: The filtrate was concentrated under reduced pressure to obtain a concentrate with a solid content of 10%, which was then dried to obtain approximately 120g of dried tangerine peel powder. The drying step involved adding 80g of resistant dextrin to the concentrate, mixing thoroughly, and then spray drying at an inlet air temperature of 180℃ and an outlet air temperature of 80℃. Analysis revealed a total flavonoid content of 3.2% and a total polysaccharide content of 12.5%.

[0027] Example 3: Preparation of an anti-aging composition (capsule) Weigh the Sophora japonica powder from Example 1 and the tangerine peel powder from Example 2 in a 1:1 mass ratio, mix them evenly, add an appropriate amount of microcrystalline cellulose as a filler, and fill them with a capsule filling machine to make hard capsules. Each capsule contains 400mg of the core composition (200mg of Sophora japonica powder and 200mg of tangerine peel powder), and the core composition accounts for 50% of the mass of the capsule.

[0028] Example 4: Preparation of an anti-aging composition (oral liquid) Weigh 50g of Sophora japonica powder from Example 1 and 50g of tangerine peel powder from Example 2, add 5L of deionized water and heat to dissolve, add an appropriate amount of stevioside for flavoring, stir evenly, filter, sterilize, and fill into oral liquid, each 10mL vial containing 200mg of core composition, the core composition accounting for 20% of the mass of the oral liquid.

[0029] Example 5: Preparation of an anti-aging composition (cream) Weigh 20g of Sophora japonica powder from Example 1 and 30g of dried tangerine peel powder from Example 2, add 50g of petrolatum, 10g of lanolin, 5g of glycerin, 0.1g of ethylparaben and 80g of deionized water, emulsify, homogenize and sterilize to make 100g of cream, with the core composition accounting for 50% of the mass of the cream.

[0030] Example 6 Comparative Example Control group 1: Sophora japonica flower powder was prepared by fermentation with a single Lactobacillus plantarum, and the remaining processes were the same as in Example 1. Each capsule contained 400mg of Sophora japonica flower powder. Control group 2: Only the tangerine peel powder from Example 2 was used to make capsules, each containing 400mg of tangerine peel powder; Control group 3: Sophora japonica powder that has not undergone enzymatic hydrolysis and fermentation was mixed with tangerine peel powder in a 1:1 ratio and made into capsules, each containing 400mg of the core composition.

[0031] Example 7: In vitro evaluation and human clinical trials 1. In vitro enzyme activity assay Example 1: Inhibitory effect of Sophora japonica flower powder on DNMT3A enzyme activity 1) Experimental materials Reagents: DNMT3A enzyme activity assay kit (catalog number K333-100, BioVision), Sophora japonica powder from Example 1, Sophora japonica powder from Control Group 1, 5-azacytidine (positive control, purity ≥98%), ultrapure water; Instruments: Microplate reader (Bio-Rad Model 680), constant temperature water bath, pipettes, centrifuge tubes, etc.

[0032] 2) Experimental methods 2.1 Sample preparation: Sophora japonica powder from Example 1 and Sophora japonica powder from Control Group 1 were dissolved in the reagent kit buffer to prepare sample solutions with concentrations of 0, 10, 20, 50, and 100 μmol / L. The positive control 5-azacytidine was prepared at a concentration of 50 μmol / L. 2.2 Enzyme activity detection: Follow the instructions in the kit to mix the enzyme solution, substrate solution and sample solutions of different concentrations, incubate at 37℃ for 1 h, and after terminating the reaction, detect the absorbance value at a wavelength of 450 nm. 2.3 Calculation of inhibition rate: Inhibition rate (%) = (Absorbance of blank group - Absorbance of sample group) / Absorbance of blank group × 100%; 2.4 Repeated experiments: Three replicates were set up for each group, and the experiment was repeated three times. The results are expressed as "mean ± standard deviation".

[0033] 3) Experimental Results Table 1. Inhibitory effect of Sophora japonica flower powder on DNMT3A enzyme activity

[0034] 4) Experimental Conclusions Sophora japonica powder prepared by a three-enzyme + two-strain process showed a more significant concentration-dependent inhibitory effect on DNMT3A enzyme activity, with an inhibition rate 2%-3% higher than that of Sophora japonica powder fermented with a single strain. The inhibition rate reached 44.5% at 50 μmol / L and 53.9% at 100 μmol / L, both of which were superior to the single-strain fermentation group, confirming that the dual-strain fermentation process can improve the inhibitory effect of Sophora japonica powder on DNMT3A.

[0035] Example 2: Activation effect of dried tangerine peel powder on TET demethylase activity 1) Experimental materials 1.1 Reagents: TET demethylase activity assay kit (catalog number K988-100, BioVision), hesperidin monoglucoside standard (purity ≥98%), vitamin C (positive control, purity ≥99%), ultrapure water; 1.2 Instruments: Microplate reader (Bio-Rad Model 680), constant temperature water bath, pipettes, centrifuge tubes, etc.

[0036] 2) Experimental methods 2.1 Sample preparation: The tangerine peel powder from Example 2 was dissolved in the reagent kit buffer to prepare sample solutions with concentrations of 0, 5, 10, 20, and 40 μmol / L; the positive control vitamin C was prepared at 200 μmol / L. 2.2 Enzyme activity detection: Follow the instructions in the kit to mix the enzyme solution, substrate solution, and sample solutions of different concentrations, incubate at 37℃ for 1.5h, and after terminating the reaction, detect the absorbance value at a wavelength of 450nm. 2.3 Calculation of activation rate: Activation rate (%) = (Absorbance of sample group - Absorbance of blank group) / Absorbance of blank group × 100%; 2.4 Repeated experiments: Three replicates were set up for each group, and the experiment was repeated three times. The results are expressed as "mean ± standard deviation".

[0037] 3) Experimental Results Table 2. Activation effect of dried tangerine peel powder on TET demethylase activity

[0038] 4) Experimental Conclusions Tangerine peel powder exhibits a concentration-dependent activation effect on TET demethylase activity, with higher concentrations resulting in more significant activation. The activation rate reached 38.9% at 20 μmol / L and 48.0% at 40 μmol / L, which is superior to the positive control vitamin C, confirming that tangerine peel powder is a highly efficient TET demethylase activator.

[0039] 2. Mouse animal experiments 1) Experimental materials Laboratory animals: SPF grade C57BL / 6 mice, young mice (3 months old, half male and half female, equivalent to 20-25 years old in humans), and old mice (20 months old, half male and half female, equivalent to 65-70 years old in humans). Experimental samples: Sophora japonica flower powder (Example 1), dried tangerine peel powder (Example 2), Sophora japonica flower-dried tangerine peel composition (Example 3) (1:1), and Sophora japonica flower powder (Control group 1), were prepared into suspensions with physiological saline. Instruments: PathwayAge epigenetic clock detection platform, ELISA kits (Abcam ab282922, ab272138), fully automated biochemical analyzer, electronic balance, gavage needle, etc. Reagents: physiological saline, tissue homogenate kit, centrifuge tubes, etc.

[0040] 2) Experimental Design Seventy 20-month-old aged C57BL / 6 mice (half male and half female) were randomly divided into four groups: a model group, a control group (Sophora japonica flower powder group), a group receiving Sophora japonica flower powder (Example 1), a group receiving dried tangerine peel powder, a low-dose combination group, and a high-dose combination group, with ten mice in each group. Ten 3-month-old young mice were used as a young control group. The dosage and method of gavage administration for each group are as follows: Young control group / model group: 10 mL / kg·d of normal saline was administered by gavage; Control group 1: Sophora japonica flower powder group: administered 500 mg / kg·d of Sophora japonica flower powder suspension fermented by a single bacterium via gavage. Example 1: Sophora japonica flower powder group: 500 mg / kg·d of bifidobacterial fermented Sophora japonica flower powder suspension was administered by gavage. Tangerine peel powder group: Tangerine peel powder suspension was administered orally at a dose of 500 mg / kg·d; Low-dose group of the composition: 500 mg / kg·d of Sophora japonica bud-Chenpi composition suspension (1:1, total dose) was administered by gavage. High-dose group of the composition: oral administration of Sophora japonica buds-tangerine peel composition suspension 1000mg / kg·d (1:1, total dose).

[0041] Mice were administered the food and water by gavage for 12 consecutive weeks, during which time they were allowed free access to food and water and kept in a normal feeding environment (temperature 22-25℃, humidity 50-60%, 12-hour light and dark alternation).

[0042] 3) Detection indicators and methods Methylation age detection: After the experiment, tail blood was collected from mice, and the methylation age was detected using the PathwayAge epigenetic clock model. The reduction value and the rejuvenation rate were calculated. Tissue enzyme activity assay: Liver tissue was collected after mice were sacrificed, tissue homogenate was prepared, and the activities of DNMT3A and TET demethylases were detected using an ELISA kit. Organ index detection: Calculate the organ indices of the heart, liver, spleen, kidney, and brain in mice (organ index = organ wet weight / body weight × 100%) to assess organ damage. Statistical analysis: SPSS 26.0 was used for statistical analysis. Results are expressed as mean ± standard deviation. One-way ANOVA was used for comparison between groups. P < 0.05 was considered statistically significant.

[0043] 4) Experimental Results 4.1. Age-related changes in methylation in mice of different groups Table 3. Changes in methylation age in mice of each group (n=10, mean ± standard deviation)

[0044] 4.2. Changes in DNMT3A and TET enzyme activities in liver tissue of mice in each group Table 4. Changes in enzyme activity in liver tissue of mice in each group (n=10, mean ± standard deviation, U / mg)

[0045] Table 5. Changes in organ indices of mice in each group

[0046] Experimental results As shown in Tables 4-5, the Sophora japonica bud-dried tangerine peel composition prepared by the three enzymes + two bacteria process can significantly reduce the methylation age of aged mice in a dose-dependent manner. The high-dose group showed a rejuvenation rate of 39.1%, which is 1.5% higher than that of the single-bacterial fermentation composition. Example 1: The Sophora japonica powder group showed better inhibitory effect on DNMT3A than the control group. The Sophora japonica powder group, the low-dose group and the high-dose group of the composition significantly downregulated the DNMT3A enzyme activity in the liver tissue of aged mice and upregulated the TET demethylase activity. The enzyme activity level of the high-dose group was close to that of young mice, which proved that the dual-strain fermentation process can enhance the bidirectional regulatory effect of the composition on DNA methylation-related enzymes. No significant abnormalities were observed in the organ indices of mice in each group, confirming that the Sophora japonica bud-dried tangerine peel composition prepared by the three enzymes + two bacteria process has no in vivo toxic side effects and is highly safe.

[0047] 3. Human Experimentation 1) Experimental Design One hundred and ten healthy volunteers aged 65-70 years (mean age 67.3 ± 1.5 years) were selected, with a male-to-female ratio of 1:1, and randomly divided into the experimental group (n=23), control group 1 (n=22), control group 2 (n=22), control group 3 (n=22), and blank control group (n=21). There were no significant differences among the volunteers in initial methylation age (67.1 ± 2.3 years) and baseline health status (blood pressure, blood glucose, blood lipids, etc., were all within the normal range) (P>0.05, one-way ANOVA).

[0048] Experimental group: Take one capsule prepared in Example 3 daily (double-fermented Sophora japonica flower powder + dried tangerine peel powder). Control group 1: Take one capsule (single-strain fermented Sophora japonica powder) prepared by control group 1 daily. Control group 2: Take one capsule (tangerine peel powder only) prepared by control group 2 daily. Control group 3: Take one capsule (unenzymatically fermented Sophora japonica buds + dried tangerine peel powder) prepared by control group 3 daily. Blank control group: Received placebo capsules containing no active ingredients (ingredients: microcrystalline cellulose, magnesium stearate).

[0049] Volunteers took the medication continuously for 12 months, during which time they maintained their regular diet and exercise habits.

[0050] 2) Detection indicators and methods 2.1 Physiological age and systemic pathway age based on methylation: The PathwayAge epigenetic clock model (detection platform: Illumina Infinium HumanMethylationEPIC BeadChip chip) was used to detect the methylation level of 353 CpG sites in the genome and calculate physiological age and the pathway ages of the cardiovascular, immune, metabolic, nervous, and reproductive systems. 2.2 Enzyme activity: DNMT3A activity and TET demethylase activity in peripheral blood were detected using ELISA kits (catalog numbers: ab282922, ab272138, Abcam). 2.3 Safety indicators: A fully automated biochemical analyzer (model: Cobas 8000) was used to detect blood routine (white blood cells, red blood cells, platelets, etc.) and liver and kidney function (ALT, AST, creatinine, urea nitrogen, etc.), and adverse reactions (nausea, diarrhea, skin itching, etc.) were recorded.

[0051] 3) Experimental results (all data are expressed as mean ± standard deviation, and statistical analysis was performed using SPSS 26.0) Table 6 Results of Human Clinical Trials

[0052] Note: △ indicates P < 0.05 compared to pre-treatment levels in the same group; △△ indicates P < 0.01 compared to pre-treatment levels in the same group; # indicates P < 0.05 or P < 0.01 compared to control groups 1 / 2 / 3 after treatment. Experimental results As shown in Table 6, the methylation physiological age of the experimental group decreased from 66.8 years to 61.2 years after taking the medication, a decrease of 5.6 years. This was 3.3 years more than the control group 1, which was fermented with a single bacterium. This confirms that the combination prepared by the three enzymes and two bacteria process has a more significant effect on reducing methylation age. System pathway age: The experimental group showed a significantly younger age in all system pathways than the control group, with the cardiovascular system showing an average age of 13.2 years younger, the immune system 7.8 years younger, the metabolic and nervous systems both over 9 years younger, and the reproductive system 4.0 years younger. Enzyme activity: The DNMT3A activity in the experimental group decreased by 45.2%, and the TET demethylase activity increased by 39.5%, both significantly higher than those in the control groups, confirming that the dual-strain fermentation process enhanced the synergistic regulatory effect of Sophora japonica powder and tangerine peel powder.

[0053] Safety: No significant adverse reactions were observed in either the experimental or control groups. Blood routine tests, liver and kidney function indicators were all within the normal range, confirming the high safety of the composition of this invention in clinical application.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A sophora japonica and tangerine peel anti-aging composition for reducing methylation age, characterized in that, The anti-aging composition comprises Sophora japonica powder and dried tangerine peel powder in a mass ratio of 1:(0.5-2); The preparation method of the Sophora japonica powder includes the following steps: (1) Compound enzyme extraction: Powder the Sophora japonica flowers into powder, add 5-10 times the mass of citrate-sodium citrate buffer, add compound enzyme for enzymatic hydrolysis, obtain enzymatic hydrolysate, sterilize the enzymatic hydrolysate and set aside; wherein, the amount of compound enzyme added is 0.3-0.5% of the mass of Sophora japonica flowers; the compound enzyme is cellulase, pectinase and xylanase; (2) Co-fermentation of two bacteria: A compound bacterial agent is added to the enzymatic hydrolysate for anaerobic fermentation. After fermentation, the mixture is sterilized to obtain the fermentation broth. The compound bacterial agent includes Lactobacillus plantarum and Lactobacillus rhamnosus. (3) Centrifuge the fermentation broth, collect the supernatant, concentrate the supernatant, and dry it to obtain the Sophora japonica powder.

2. The anti-aging composition of Sophora japonica buds and dried tangerine peel for reducing methylation age as described in claim 1, characterized in that, In step (1) of preparing the Sophora japonica powder, the pH of the citrate-sodium citrate buffer solution is 4.5-5.5, the amount of compound enzyme added is 0.3-0.5% of the mass of Sophora japonica, and the mass ratio of cellulase, pectinase and xylanase is (1.5-2.5):1:1; the enzymatic hydrolysis temperature is 45-55℃, the time is 1-2h, and the pH is 4.5-5.5; the sterilization temperature is 85-95℃, and the time is 10-15min.

3. The anti-aging composition of Sophora japonica buds and dried tangerine peel for reducing methylation age as described in claim 1, characterized in that, In step (2) of preparing the Sophora japonica powder, the amount of the compound microbial agent added is 0.5-1% of the mass of the enzymatic hydrolysate, and the viable count ratio of the compound microbial agent is 1-8×10⁻⁶. 9 CFU / g, the live count ratio of Lactobacillus plantarum and Lactobacillus rhamnosus is 1:1; the fermentation temperature is 35-38℃, the time is 5-7h, and the pH is 5.5-6.

5. After fermentation, the pH is adjusted to 7.0 to terminate fermentation; the sterilization temperature is 85-95℃ and the time is 10-15min.

4. The anti-aging composition of Sophora japonica buds and dried tangerine peel for reducing methylation age as described in claim 1, characterized in that, In step (3) of preparing the Sophora japonica powder, the centrifugation speed is 9000-11000 r / min and the time is 15-25 min; the concentration is carried out by vacuum concentration treatment, with a vacuum degree of 0.06-0.08 MPa and a temperature of 50-60℃, until the solid content is 8-12%; the drying is carried out by spray drying process, with an inlet air temperature of 180-200℃ and an outlet air temperature of 80-90℃.

5. The anti-aging composition of Sophora japonica buds and dried tangerine peel for reducing methylation age as described in claim 1, characterized in that, The method for preparing the tangerine peel powder includes the following steps: S1. Compound Enzymatic Hydrolysis: Grind dried tangerine peel into powder, add 7-9 times its weight of deionized water to make a slurry, add a compound enzyme for enzymatic hydrolysis, obtain the hydrolysate, inactivate the enzyme, filter, and obtain the filtrate; wherein, the compound enzyme is cellulase and pectinase. S2. Concentration: The filtrate is concentrated under reduced pressure to obtain a concentrated solution with a solid content of 5-15%, which is then dried to obtain the tangerine peel powder.

6. The anti-aging composition of Sophora japonica buds and dried tangerine peel for reducing methylation age as described in claim 5, characterized in that, In step S1 of the preparation of the tangerine peel powder, the mass ratio of cellulase to pectinase is 3:1, and the amount of compound enzyme added is 0.2-0.4% of the raw material mass; the enzymatic hydrolysis temperature is 45-55℃ and the time is 0.5-1.5h; the enzyme inactivation temperature is 75-90℃ and the time is 10-20min; filtration is carried out using a plate and frame filter press at a pressure of 0.3-0.5MPa.

7. The anti-aging composition of Sophora japonica buds and dried tangerine peel for reducing methylation age as described in claim 5, characterized in that, In step S2 of the preparation of the tangerine peel powder, the drying step is as follows: 5-10% by mass of resistant dextrin is added to the concentrated liquid, mixed evenly, and then spray-dried with an inlet air temperature of 170-190℃ and an outlet air temperature of 75-85℃.

8. The use of the anti-aging composition according to any one of claims 1-7 in the preparation of dietary supplements, functional beverages, skin care products, or pharmaceuticals, characterized in that, The anti-aging composition accounts for 10-80% of the product by weight, of which it accounts for 30-60% in dietary supplements and pharmaceuticals, 10-30% in functional beverages, and 20-50% in skin care products.

9. The application as described in claim 8, characterized in that, The dosage forms of the products include powders, granules, capsules, tablets, oral liquids, creams, serums, injections, or gels.

Images