Application of a Traditional Chinese Medicine Composition in the Preparation of Drugs Regulating DNA Methylation Clock

Abstract

This invention relates to the field of traditional Chinese medicine (TCM) technology, specifically disclosing the application of a TCM composition in the preparation of drugs that regulate DNA methylation clocks. The TCM composition includes herbs such as Cuscuta chinensis, Lycium barbarum, Schisandra chinensis, and Cnidium monnieri. Animal experiments have demonstrated that the drug composition of this invention can regulate DNA methylation in mice and reduce their physiological age.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to the application of a traditional Chinese medicine composition in the preparation of drugs that regulate DNA methylation clock. Background Technology

[0002] Physiological age, also known as biological age, refers to the age characteristics of an individual's biological physiology, representing their developmental level, vitality, and health status. Throughout the life cycle, due to factors such as genetic background, environment, and drug intervention, significant differences in physiological age may exist between individuals of the same age. For example, individuals of the same age who maintain a healthy diet and regular lifestyle over a long period tend to appear significantly "younger" in terms of physical appearance, organ function, and other physiological indicators compared to those with long-term unhealthy lifestyle habits.

[0003] Currently, methods for systematically evaluating physiological age include the frailty index phenotypic scoring (which comprehensively scores individuals based on their physical characteristics and various physiological indicators), blood aging biomarker detection, and DNA methylation clock detection. Previous studies have reported that the pattern of DNA methylation in an organism's genome changes significantly with physiological age. DNA methylation clock detection refers to a method that obtains DNA methylation information from an individual's genome through sequencing and other means, and then calculates the individual's physiological age using a specific program. Due to its high accuracy and ease of detection, DNA methylation clock has become the mainstream method for physiological age assessment. The methylation clock is an intrinsic cellular property; it is more like a comprehensive biological clock than an aging clock. The biological processes involved in modules closely related to the DNA methylation clock include mitochondrial translation, immunity, histone modification, and autophagy. Six DNA methylation clocks (Horvath1, Horvath2, Hannum, Levine, Lin, and Yang clocks) with conserved transcriptomic signals in monocytes can, to some extent, distinguish tumor tissues (e.g., breast cancer, colon cancer, lung cancer, and pancreatic cancer) from normal tissues, with the Levine and Yang clocks showing the most significant effects. Methylation clocks do not reflect aging characteristics, but rather represent a unified clock for development and aging. Summary of the Invention

[0004] This invention provides the application of a traditional Chinese medicine composition in the preparation of drugs that regulate DNA methylation clocks. The composition comprises eight herb groups: Lycium barbarum and Cuscuta chinensis to tonify the kidneys and replenish essence; Schisandra chinensis, Rubus idaeus, and Rosa laevigata to tonify the kidneys and solidify essence; and Cnidium monnieri and Allium tuberosum to invigorate yang and replenish essence. Only Melia toosendan is not classified as a tonifying herb, but rather as a qi-regulating herb. Among qi-regulating herbs, Melia toosendan is particularly effective in regulating the qi of the lower jiao (liver and kidney), and when combined with kidney-tonifying herbs, it has a tonifying effect without causing stagnation.

[0005] The inventors were pleasantly surprised to find that the drug composition showed significant advantages in regulating the methylation clock and improving hereditary liver aging.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] The application of a traditional Chinese medicine composition in the preparation of a drug that regulates DNA methylation clock, the traditional Chinese medicine composition being made from the following raw materials in parts by weight: Cuscuta chinensis 150-350, Lycium barbarum 100-200, Schisandra chinensis 30-60, Cnidium monnieri 20-50, Rosa laevigata 20-50, Allium tuberosum 20-50, Morinda officinalis 20-50, Cistanche deserticola 20-50, Rehmannia glutinosa 30-60, Achyranthes bidentata 20-50, Epimedium brevicornu 40-100, Rubus idaeus 20-50, Panax ginseng 15-35, Cervus nippon antler 10-25, Hippocampus 15-35, and Melia toosendan 15-35.

[0008] The traditional Chinese medicine composition provided by this invention can be used in the preparation of drugs that reduce DNA methylation clock. The resulting drugs can regulate DNA methylation modification and reduce physiological age.

[0009] The traditional Chinese medicine composition provided by this invention can be used to regulate genes SIRT3, SIRT4, FOXO1, or SOD2. The traditional Chinese medicine composition provided by this invention can also be used to upregulate the expression of Ercc2 and Rbx1.

[0010] The traditional Chinese medicine composition provided by this invention can be applied to promote the hypomethylation of genes regulating longevity pathways.

[0011] The traditional Chinese medicine composition provided by this invention can be applied to the hypermethylation of genes in the aging pathway.

[0012] The traditional Chinese medicine composition provided by this invention can be used to slow down the aging process.

[0013] The traditional Chinese medicine composition provided by this invention can be used to regulate the genetic aging of the liver.

[0014] Furthermore, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: Cuscuta chinensis 150, Lycium barbarum 200, Schisandra chinensis 30, Cnidium monnieri 50, Rosa laevigata 20, Allium tuberosum 20, Morinda officinalis 50, Cistanche deserticola 20, Rehmannia glutinosa 30, Achyranthes bidentata 50, Epimedium brevicornu 40, Rubus idaeus 50, Panax ginseng 15, Cervus nippon antler 25, Hippocampus 15, and Melia toosendan 35.

[0015] Furthermore, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: Cuscuta chinensis 350, Lycium barbarum 100, Schisandra chinensis 60, Cnidium monnieri 20, Rosa laevigata 50, Allium tuberosum 50, Morinda officinalis 20, Cistanche deserticola 50, Rehmannia glutinosa 60, Achyranthes bidentata 20, Epimedium brevicornu 100, Rubus idaeus 20, Panax ginseng 35, Cervus nippon antler 10, Hippocampus 35, and Melia toosendan 15.

[0016] Furthermore, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: Cuscuta chinensis 250, Lycium barbarum 138, Schisandra chinensis 46, Cnidium monnieri 35, Rosa laevigata 35, Allium tuberosum 35, Morinda officinalis 35, Cistanche deserticola 35, Rehmannia glutinosa 46, Achyranthes bidentata 35, Epimedium brevicornu 70, Rubus idaeus 35, Panax ginseng 25, Cervus nippon antler 16, Hippocampus 21, and Melia toosendan 23.

[0017] Furthermore, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: Cuscuta chinensis 250, Lycium barbarum 150, Schisandra chinensis 45, Cnidium monnieri 30, Rosa laevigata 30, Allium tuberosum 30, Morinda officinalis 30, Cistanche deserticola 30, Rehmannia glutinosa 45, Achyranthes bidentata 30, Epimedium brevicornu 70, Rubus idaeus 30, Panax ginseng 20, Cervus nippon antler 19, Hippocampus 20, and Melia toosendan 20.

[0018] Furthermore, the dosage form of the drug is a decoction, capsule, tablet, granule, powder, or pill.

[0019] Furthermore, the method for preparing the capsules includes the following steps:

[0020] a. Weigh out Cnidium monnieri, Cuscuta chinensis, Schisandra chinensis and Epimedium according to the proportion, add 6-10 times the amount of 70% ethanol, reflux extract 1-3 times, 1-3 hours each time, filter, combine the filtrates, and recover the ethanol under reduced pressure until there is no alcohol taste to obtain the alcohol extract.

[0021] b. Combine the medicinal residue after alcohol extraction in step a with the appropriate amounts of wolfberry, rehmannia root, raspberry, rosehip, leek seed, morinda root, and Sichuan pepper, add 7-12 times the amount of water and decoct 1-3 times, 1-3 hours each time. Filter, combine the filtrates, mix with the alcohol extract obtained in step a, and concentrate under reduced pressure until the relative density is 1.25-1.30 when measured at 60℃ to obtain the extract.

[0022] c. Weigh out ginseng, deer antler, seahorse, cistanche and achyranthes according to the proportion, grind them into fine powder, mix them evenly with the extract obtained in step b, dry at 60-70℃, grind, sieve and fill into capsules. Attached Figure Description

[0023] Figure 1 This is a comparison of the relative mRNA expression levels of Sod2 in each group of mice in Experiment 3. n = 6. *p < 0.05, **p < 0.01.

[0024] Figure 2 This is a comparative graph showing the SOD enzyme activity in the liver tissues of mice in each group during Experiment 3. n = 8. *p < 0.05, **p < 0.01.

[0025] Figure 3 These are representative immunofluorescence images of γ-H2AX livers from each group of mice in Experiment 3. Scale bars = 100 μm.

[0026] Figure 4 This is a comparison of γ-H2AX expression in mice of different groups in Experiment 3, analyzed by immunofluorescence. *p<0.05,**p<0.01.

[0027] Figure 5 This is a comparison of cell viability of HUVECs from different groups of mice in Experiment 3.

[0028] Figure 6 These are typical images of HUVEC cells with normal senescence markers and after irradiation in Experiment Example 3.

[0029] Figure 7 This is a comparison of the nuclear area of ​​HUVEC cells with normal senescence markers and after irradiation in Experiment Example 3. **p<0.01.

[0030] Figure 8 These are typical immunofluorescence images of P16, a senescence marker, in normal and irradiated HUVEC cells in Experiment Example 3.

[0031] Figure 9 This is a comparison of the senescence marker P16 in normal and irradiated HUVEC cells in Experiment Example 3.

[0032] Figure 10 This is a comparison of senescence markers in normal and irradiated HUVEC cells in Experiment Example 3.

[0033] Figure 11 This is a comparison chart of methyltransferase mRNA expression levels (DNMT1) in HUVECs from different experimental groups in Experiment Example 3.

[0034] Figure 12 This is a comparison of the expression levels of methyltransferase mRNA (TET1) in HUVECs of different experimental groups in Experiment Example 3. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0036] Example 1: Formulation for preparing capsules: Cuscuta chinensis 250g, Lycium barbarum 138g, Schisandra chinensis 46g, Cnidium monnieri 35g, Rosa laevigata 35g, Allium tuberosum 35g, Morinda officinalis 35g, Cistanche deserticola 35g, Rehmannia glutinosa 46g, Achyranthes bidentata 35g, Epimedium brevicornu 70g, Rubus idaeus 35g, Panax ginseng 25g, Cervus nippon antler 16g, Hippocampus 21g, Melia toosendan 23g.

[0037] Preparation method:

[0038] a. Weigh out Cnidium monnieri, Cuscuta chinensis, Schisandra chinensis and Epimedium according to the proportion, add 3208ml of 70% ethanol, reflux extract 3 times, 2 hours each time, filter, combine the filtrates, and recover the ethanol under reduced pressure until there is no alcohol taste to obtain the alcohol extract.

[0039] b. Combine the medicinal residue after alcohol extraction in step a with the appropriate amounts of wolfberry, rehmannia root, raspberry, rosehip, leek seed, morinda root, and Sichuan pepper, add 6732ml of water and decoct twice, 2 hours each time. Filter, combine the filtrates, mix with the alcohol extract obtained in step a, and concentrate under reduced pressure until the relative density is 1.28 when measured at 60℃ to obtain the extract.

[0040] c. Weigh out ginseng, deer antler, seahorse, cistanche and achyranthes according to the proportion, grind them into fine powder, mix them evenly with the extract obtained in step b, dry at 65℃, grind, sieve, fill into capsules, and obtain 757 capsules.

[0041] Example 2: Formulation for tablet preparation: Cuscuta chinensis 250g, Lycium barbarum 150g, Schisandra chinensis 45g, Cnidium monnieri 30g, Rosa laevigata 30g, Allium tuberosum 30g, Morinda officinalis 30g, Cistanche deserticola 30g, Rehmannia glutinosa 45g, Achyranthes bidentata 30g, Epimedium brevicornu 70g, Rubus idaeus 30g, Panax ginseng 20g, Cervus nippon antler 19g, Hippocampus 20g, Melia toosendan 20g.

[0042] Preparation method:

[0043] a. Weigh out Cnidium monnieri, Cuscuta chinensis, Schisandra chinensis and Epimedium according to the proportion, add 2765ml of 70% ethanol, reflux extract 3 times, 1 hour each time, filter, combine the filtrates, and recover the ethanol under reduced pressure until there is no alcohol taste to obtain the alcohol extract.

[0044] b. Combine the medicinal residue after alcohol extraction in step a with the appropriate amounts of wolfberry, rehmannia root, raspberry, rosehip, leek seed, morinda root, and Sichuan pepper, add 5110ml of water and decoct once for 3 hours. Filter, combine the filtrates, mix with the alcohol extract obtained in step a, and concentrate under reduced pressure until the relative density is 1.30 when measured at 60℃ to obtain the extract.

[0045] c. Weigh out ginseng, deer antler, seahorse, cistanche and achyranthes according to the proportion, grind them into fine powder and mix them evenly with the extract obtained in step b, dry at 70℃, grind, sieve, add 1% magnesium stearate, compress into tablets, and obtain 760 tablets.

[0046] Example 3: Preparation of pills: Cuscuta chinensis 350g, Lycium barbarum 100g, Schisandra chinensis 60g, Cnidium monnieri 20g, Rosa laevigata 50g, Allium tuberosum 50g, Morinda officinalis 20g, Cistanche deserticola 50g, Rehmannia glutinosa 60g, Achyranthes bidentata 20g, Epimedium brevicornu 100g, Rubus idaeus 20g, Panax ginseng 35g, Cervus nippon antler 10g, Hippocampus 35g, Melia toosendan 15g.

[0047] Preparation method:

[0048] a. Weigh out Cnidium monnieri, Cuscuta chinensis, Schisandra chinensis and Epimedium according to the proportion, add 5300ml of 70% ethanol, reflux and extract once for 3 hours, filter, combine the filtrates, and recover the ethanol under reduced pressure until there is no alcohol taste to obtain the alcohol extract.

[0049] b. Combine the medicinal residue after alcohol extraction in step a with the appropriate amounts of wolfberry, rehmannia root, raspberry, rosehip, leek seed, morinda root, and Sichuan pepper, add 10140ml of water and decoct three times, one hour each time. Filter, combine the filtrates, mix with the alcohol extract obtained in step a, and concentrate under reduced pressure until the relative density is 1.25 when measured at 60℃ to obtain the extract.

[0050] c. Weigh out ginseng, deer antler, seahorse, cistanche and achyranthes according to the proportion, grind them into fine powder, mix them evenly with the extract obtained in step b, dry at 60℃, grind, sieve, and make into pills, obtaining 320g of pills.

[0051] Example 4: Preparation of granules: Cuscuta chinensis 150g, Lycium barbarum 200g, Schisandra chinensis 30g, Cnidium monnieri 50g, Rosa laevigata 20g, Allium tuberosum 20g, Morinda officinalis 50g, Cistanche deserticola 20g, Rehmannia glutinosa 30g, Achyranthes bidentata 50g, Epimedium brevicornu 40g, Rubus idaeus 50g, Panax ginseng 15g, Cervus nippon antler 25g, Hippocampus 15g, Melia toosendan 35g.

[0052] Preparation method:

[0053] a. Weigh out Cnidium monnieri, Cuscuta chinensis, Schisandra chinensis and Epimedium according to the proportion, add 2430ml of 70% ethanol, reflux extract twice, 1.5 hours each time, filter, combine the filtrates, and recover the ethanol under reduced pressure until there is no alcohol taste to obtain the alcohol extract.

[0054] b. Combine the medicinal residue after alcohol extraction in step a with the remaining medicinal materials in the specified proportion, add 8800ml of water and decoct 3 times, 1 hour each time. Filter, combine the filtrates and mix with the alcohol extract obtained in step a. Concentrate under reduced pressure until the relative density is 1.29 when measured at 60℃ to obtain the extract.

[0055] c. Mix the extract obtained in step b with 300g of dextrin and 200g of sucrose powder evenly, dry at 65℃, pulverize, sieve, and granulate to obtain 700g of granules.

[0056] Example 5: Preparation of the powder formula: Cuscuta chinensis 250g, Lycium barbarum 150g, Schisandra chinensis 45g, Cnidium monnieri 30g, Rosa laevigata 30g, Allium tuberosum 30g, Morinda officinalis 30g, Cistanche deserticola 30g, Rehmannia glutinosa 45g, Achyranthes bidentata 30g, Epimedium brevicornu 70g, Rubus idaeus 30g, Panax ginseng 20g, Cervus nippon antler 19g, Hippocampus 20g, Melia toosendan 20g.

[0057] Preparation method: Weigh the Chinese medicinal materials in the formula according to the proportion, mix and grind them into fine powder to obtain 823g of powder.

[0058] Example 6: Preparation of the decoction: Cuscuta chinensis 250g, Lycium barbarum 150g, Schisandra chinensis 45g, Cnidium monnieri 30g, Rosa laevigata 30g, Allium tuberosum 30g, Morinda officinalis 30g, Cistanche deserticola 30g, Rehmannia glutinosa 45g, Achyranthes bidentata 30g, Epimedium brevicornu 70g, Rubus idaeus 30g, Panax ginseng 20g, Cervus nippon antler 19g, Hippocampus 20g, Melia toosendan 20g.

[0059] Preparation method: Weigh the Chinese medicinal materials according to the proportion, add water to cover the medicinal materials, soak for 15 minutes, heat to boiling, continue to simmer for 30 minutes, filter, add water to the dregs again to cover the medicinal materials, simmer for 30 minutes again, filter, combine the filtrates to obtain 4320g of decoction.

[0060] Experimental Example 1:

[0061] To elucidate the role of the drug of the present invention in regulating the DNA methylation clock, the following experiments were conducted using Example 1 (hereinafter referred to as the drug of the present invention or BZ):

[0062] Experimental methods: Age prediction was performed on 23-week-old young mice, 63-week-old mice without drug intervention, and 63-week-old old mice treated with the drug composition of the present invention.

[0063] result:

[0064] The physiological age of young mice at 23 weeks of age, predicted by DNA methylation clock, was 24.63 (mean, standard deviation 1.61). The physiological age of aged mice at 63 weeks of age without drug intervention, predicted by DNA methylation clock, was 61.54 (mean, standard deviation 6.14). The physiological age of aged mice at 63 weeks of age treated with the drug composition of this invention, predicted by DNA methylation clock, was 39.73 (mean, standard deviation 8.74). A t-test was used to detect differences in physiological age between the groups. Compared with the model group, both the young group and the drug composition group showed statistically significant differences (P < 0.01). Compared with the model group of the same physiological age, the drug composition group of this invention significantly reduced the physiological age of mice.

[0065] Table 1 DNA methylation clock data

[0066] Sample number YOUNG group age MODEL group age The age of the pharmaceutical composition group of the present invention 1 25.2 69.1 47.7 2 23.6 66.5 35.9 3 20.9 57.9 38.8 4 23.3 62.1 44.7 5 25.3 49.3 45.4 6 25 60.5 57.3 7 25.3 60.5 30.8 8 26.1 54.9 30.9 9 25.4 66.7 29.8 10 26.8 60.3 43.5 11 24 69.1 26.7 12 39.7 13 45.3 Overall average 24.63 61.54 39.73 Standard deviation 1.61 6.14 8.74

[0067] Experimental Example 2:

[0068] To elucidate the effect of the drug of the present invention in regulating hepatic epigenetic aging, the following experiments were conducted using Example 1 (hereinafter referred to as the drug of the present invention or BZ):

[0069] 1. Experimental Materials

[0070] 1.1 Test sample

[0071] 1.1.1 Name: Pharmaceutical composition of the present invention (BZ).

[0072] 1.1.2 Route and dosage: Oral administration

[0073] 1.1.3 Source and batch number: Shijiazhuang Yiling Pharmaceutical Co., Ltd., batch number is XB2103001.

[0074] 1.2 Tools, Drugs, Main Reagents and Materials

[0075] Sodium carboxymethyl cellulose (CMC-Na): Tianjin Damao Chemical Reagent Factory, batch number: 20181006, expiry date: December 30, 2021.

[0076] 1.3 Experimental System

[0077] 1.3.1 Animal strain: C57BL / 6J mouse.

[0078] 1.3.2 Animal grade: SPF grade.

[0079] 1.3.3 Animal sex and number: 30 mice were purchased for this experiment. In addition, 12 23-week-old mice of the same strain, sex, and manufacturer were selected directly as controls.

[0080] 1.3.4 Age of animals at the start of dosing: 52 weeks old.

[0081] 1.3.5 Body weight of animals at the start of dosing: actually 20 - 40 g.

[0082] 1.3.6 Source of animals: Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0083] 1.3.7 Animal certificate number, issuing unit, and receiving date: Certificate number 1100112011043405, license number SCXK(Beijing)2016 - 0006, the issuing unit is Beijing Vital River Laboratory Animal Technology Co., Ltd., and the receiving date is July 15, 2020.

[0084] 1.3.8 Rearing conditions: Mice were caged and reared in the New Drug Evaluation Center of Hebei Yiling Pharmaceutical Research Institute Co., Ltd. The light was 12 hours per day, the temperature was 20 - 26 °C, and the relative humidity was 40 - 70%. The license number for the use of experimental animals: SYXK(Hebei)2020 - 003.

[0085] 1.3.9 Quarantine process: Animals were quarantined for 3 days and adaptively reared for 2 days. During this period, the drinking water, food intake, and health status of the animals were observed, as well as the presence of signs of disease and death.

[0086] 1.3.10 Feed: SPF mouse and rat maintenance feed provided by Beijing Keao Xieli Feed Co., Ltd. The feed production license number: Beijing Feed License (2018)06073, feed batch numbers: 20103213, 20113213.

[0087] 1.3.11 Drinking water: Drink clean water prepared by the ROA50 experimental animal drinking water system (instrument number YL - LE - A01). The drinking water bottles were filled for free drinking, and the water quality was tested to meet the "Hygienic Standard for Drinking Water".

[0088] 1.3.12 Litter: Ordinary - grade corn cob litter. The litter was used after being sterilized by high - temperature and high - pressure, provided by Beijing Keao Xieli Feed Co., Ltd., litter batch number: 20079811.

[0089] 1.3.13 Identification: Marked with ear tags for experimental animals.

[0090] 2 Test methods

[0091] Aged C57BL / 6J mice: The research applications include immunology, cancer, longevity intervention, and biomarker research. The balance and stability of stem cells in 13 - month - old C57 mice are disrupted, and the animals start to age rapidly. Therefore, naturally aged C57BL / 6J mice are used to simulate the premature aging state of humans.

[0092] 2.1 Dosage and Grouping

[0093] Mice were divided into a model group (equal volume of distilled water) and a group receiving the drug composition of this invention (2 g / kg / d) as intervention. Mice aged 23 weeks were designated as the young group and were directly collected without any treatment.

[0094] The daily dosage of the pharmaceutical composition of this invention for humans is 2.4g of crude drug (based on a human body weight of 60kg), and for mice, the dosage is 2g of crude drug / kg. Thirty C57 mice were randomly divided into two groups according to their body weight: a model group and a pharmaceutical composition group.

[0095] 2.2 Method of administering test sample

[0096] Gavage, consistent with the intended route.

[0097] 2.3 Preparation and storage of test samples

[0098] The pharmaceutical composition of this invention: 2g of the ultrafine powder of the pharmaceutical composition of this invention + 12ml of CMC, to be prepared and used immediately on the same day. Based on the dosage of 10ml / kg and the dosage setting, calculate the concentration (see Appendix Table 2), weigh BZ according to the number of animals, add solvent, grind and suspend, and make up to volume. The test sample should be prepared and used immediately. The solvent is 0.5% CMC-Na, which is evenly dispersed on the surface of pure water to fully swell, made up to volume, and stored at 2-8℃ until use.

[0099] Table 2 Grouping and Dosage Settings

[0100]

[0101] 2.4 Administration of the test sample

[0102] The test sample was administered using a syringe at a volume of 10 ml / kg. Animals in the model group and the group containing the drug composition of the present invention were given the corresponding volume of solvent, which was changed every 48 hours.

[0103] 2.5 Model Making

[0104] C57 mice were naturally raised to 13 months of age, which is considered a naturally aged animal.

[0105] 2.6 Detection Indicators

[0106] 2.6.1 DNA methylation analysis

[0107] After genomic DNA extraction, the following steps were performed to construct a library: DNA sequence fragmentation, end repair of fragmented DNA sequences, addition of an A base to the 3' end, adapter ligation, bisulfite conversion (using the EZ DNA Methylation Gold Kit, Zymo Research; typically, a certain proportion of negative control human mitochondrial gene sequence is added, and the conversion rate of bisulfite conversion is calculated based on this), fragment selection & PCR amplification (insertion fragment range: 200-500bp). Sequencing was then performed using the HiSeq 4000 sequencing platform with a 150PE sequencing strategy.

[0108] 2.6.1.1 Fragmentation

[0109] The gDNA was fragmented using NEB MspⅠ restriction endonuclease, added to a PCR tube (operated on ice), vortexed for 3 seconds to mix, and then briefly centrifuged.

[0110] Table 3 Reagents used for RRBS-DNA fragmentation

[0111]

[0112] Incubate at 37℃ for 18 hours (prepare one day in advance, overnight digestion); add 5 μL of 0.5M EDTA to terminate the digestion reaction. Purify DNA with LCSbeads (preparation: remove LC Beads from the 4℃ freezer in advance and incubate at room temperature for at least 30 minutes). Take the reaction product, add 45 μL of NF water to a final volume of 100 μL, then transfer to a new 1.5 mL centrifuge tube. Add 200 μL of LC beads, mix by pipetting 10 times, and incubate at room temperature for 5 min. Place the centrifuge tube on a magnetic rack for 3-5 min until the liquid is clear, then discard the supernatant. Leave the centrifuge tube on the magnetic rack and add 200 μL of freshly prepared 80% ethanol. After 30 s, discard the supernatant (be careful not to disturb the magnetic beads). Add another 200 μL of freshly prepared 80% ethanol, and after 30 s, discard the supernatant. Leave the centrifuge tube on the magnetic rack and allow it to dry at room temperature for 3-5 min. Add 21 μL of Nuclease-Free Water, mix by pipetting 10 times, and incubate at room temperature for 5 min. Place the centrifuge tube on a magnetic rack for 5 min until the liquid is clear, then transfer 20 μL of the supernatant to a new 500 μL PCR clear tube for the next step of bisulfite conversion.

[0113] 2.6.1.2 For bisulfite conversion, add 130 μL of CT conversion reagent to the 20 μL fragmentation product from the previous step, gently tap or pipette to mix, centrifuge to the bottom of the tube; place in a PCR instrument and run the following program: 98℃ for 10 min, 64℃ for 2.5 h, 4℃ up to 20 h. Add 600 μL of M-Binding Buffer to the adsorption column and place the column into a collection tube. Add the reaction mixture from the PCR instrument steps to the adsorption column and mix by inverting. Centrifuge at full speed (≥10,000 x g) for 30 s, then discard the liquid in the collection tube. Add 100 μL of M-Wash Buffer and centrifuge at full speed (≥10,000 x g) for 30 s. Add 200 μL of M-Desulphonation Buffer, incubate at room temperature for 15-20 min, then centrifuge at full speed (≥10,000 x g) for 30 s. Add 200 μL of M-Wash Buffer and centrifuge at full speed (≥10,000 x g) for 30 s. Repeat this step. Place the collection column into a new 1.5 mL centrifuge tube and add 16 μL of M-Elution. Buffer onto the filter membrane and centrifuge at full speed (≥10,000xg) for 30 seconds. Take 1 μL of the transformation product for Nanodrop ssDNA quantification to determine the cycle number for library amplification. The transformation product can be stored at -20°C overnight for short-term storage and must be stored at -80°C for long-term storage.

[0114] 2.6.1.33′ Connector Connection

[0115] Thaw reagents on ice (except enzymes, which should be removed from the -20°C freezer before use); preheat the PCR instrument to 95°C; transfer 15 μL of transformation product to a 200 μL PCR tube. After transformation, denature the DNA sample on the PCR instrument at 95°C for 2 min, then place it directly on ice for 2 min; prepare the reaction system shown in the table below, and add 25 μL of Reaction Mix and 15 μL of transformation product to a 200 μL PCR tube.

[0116] Table 4 PCR reaction system with 3′ adapter ligation

[0117]

[0118] The ligation reaction program in the PCR instrument was: 37℃ for 15 min, 95℃ for 2 min, and 4℃ Hold.

[0119] 2.6.1.4 Second-strand extension and product purification

[0120] Remove the 3′ adapter ligation product from the PCR instrument and prepare the second-strand extension reaction solution:

[0121] Table 5. Two-chain extension reaction solution

[0122]

[0123] Add the second-strand extension reaction solution to the adapter ligation system from the previous step and gently vortex to mix. Perform the following reaction program in a PCR instrument: 98℃ for 1 min, 62℃ for 2 min, 65℃ for 5 min, and 4℃ Hold.

[0124] Product purification: LC Beads 1.2, take the reaction product, add 16 μL of NF water to a final volume of 100 μL, transfer to a 1.5 mL centrifuge tube, add 120 μL of LCS beads and mix by pipetting 10 times; incubate at room temperature for 5 min; place the centrifuge tube on a magnetic rack for 5 min until the liquid is clear, discard the supernatant; leave the centrifuge tube on the magnetic rack and add 200 μL of freshly prepared 80% ethanol, discard the supernatant after 30 s (be careful not to disturb the magnetic beads); add another 200 μL of freshly prepared 80% ethanol, discard the supernatant after 30 s; leave the centrifuge tube on the magnetic rack and allow it to dry at room temperature for 5-10 min; add 16 μL of 10 mM Tris, mix by pipetting 10 times, incubate at room temperature for 2 min; place the centrifuge tube on a magnetic rack for 5 min until the liquid is clear, and transfer 15 μL of the supernatant to a new 200 μL PCR tube.

[0125] 2.6.1.5 5′ Connector Ligation and Product Purification

[0126] Prepare the reaction system listed in the table below:

[0127] Table 6 Connection Reaction System

[0128]

[0129] Add 15 μL of the adapter ligation reaction mixture. Perform the following reaction program in the PCR instrument: 25 °C for 15 min, then hold at 4 °C.

[0130] Product purification: LC Beads 1.0, take the reaction product, transfer it to a 1.5 ml centrifuge tube, add 30 μL of LCSbeads, and mix by pipetting 10 times; incubate at room temperature for 5 min; place the centrifuge tube on a magnetic rack for 5 min until the liquid is clear, and discard the supernatant; leave the centrifuge tube on the magnetic rack and add 200 μL of freshly prepared 80% ethanol, discard the supernatant after 30 s (be careful not to disturb the magnetic beads); add another 200 μL of freshly prepared 80% ethanol, discard the supernatant after 30 s; leave the centrifuge tube on the magnetic rack and dry at room temperature for 5-10 min; add 21 μL of 10 mM Tris, mix by pipetting 10 times, and incubate at room temperature for 2 min; place the centrifuge tube on a magnetic rack for 5 min until the liquid is clear, and aspirate 20 μL of the supernatant to a new 200 μL PCR tube.

[0131] 2.6.1.6 Library Amplification (Introduction of Index) and Purification

[0132] Add 5 μL of the indexed amplification primers directly to the ligation product from section 2.7.1.5; prepare the library amplification reaction system as shown in the table below:

[0133] Table 7 Library amplification reaction system

[0134]

[0135] Add 25 μL of the library amplification reaction system to the previous step and gently vortex to mix.

[0136] The following reaction procedure was performed in the PCR instrument:

[0137] 98℃ for 30s, PCR Cycles: (The number of cycles depends on the total amount of DNA in 2.7.1.3, please refer to the table below for details), 98℃ for 10s, 60℃ for 30s, 68℃ for 60s, 4℃ Hold.

[0138] Table 8 PCR instrument reaction program

[0139] Input Insert Size PCR Cycles 100ng 350bp 4-6 10ng 350bp 7-9 lng 350bp 11-13 100pg 350bp 14-16 5ng 165bp (cfDNA) 7-9 100ng 200bp 11-13 10ng 200bp 14-16 lng 200bp 17-19

[0140] Product purification: LC Beads 0.8, take the reaction product, transfer it to a 1.5 ml centrifuge tube, add 40 μL of LCSbeads, and mix by pipetting 10 times; incubate at room temperature for 5 min; place the centrifuge tube on a magnetic rack for 5 min until the liquid is clear, and discard the supernatant; leave the centrifuge tube on the magnetic rack and add 200 μL of freshly prepared 80% ethanol, discard the supernatant after 30 s (be careful not to disturb the magnetic beads); add another 200 μL of freshly prepared 80% ethanol, discard the supernatant after 30 s; leave the centrifuge tube on the magnetic rack and dry at room temperature for 5-10 min; add 16 μL of 10 mM Tris, mix by pipetting 10 times, incubate at room temperature for 2 min, place the centrifuge tube on a magnetic rack for 5 min until the liquid is clear, and aspirate 15 μL of the supernatant to a new 200 μL PCR tube.

[0141] The document was quantified using Qubit and stored in a -20°C freezer.

[0142] 2.6.1.7 DMR Analysis of Promoters, Exons, and Introns

[0143] Windowing method for calculating chromosome methylation levels

[0144] The default selection is 1000bp windows and 500bp overlap (except for specific projects). Methylation distribution within the segments is statistically analyzed, and gene features are annotated for each segment. Furthermore, the relative position and CpG content within the promoter sequence are important factors influencing the degree of promoter methylation. Therefore, we further subdivide the promoter region sequences, classifying them into three categories based on different promoter CpGO / E values: low, medium, and high (LCPs, ICPs, and HCPs); and into three categories based on their relative TSS (transcript startsite) position: proximal (-200 to +500bp), intermediate (-200 to -1000bp), and distal (-1000 to -2200bp) (Li, M., 2012; Koga, Y., 2009).

[0145] Differential methylation region analysis

[0146] DMR was analyzed using the R package methylKit, with the default selection of 1000bp windows, 500bp overlap, and a p-value < 0.05 as the differential screening threshold. Statistically, a p-value less than 0.05 indicates a significant difference in DMR between the two groups.

[0147] 2.6.1.8 Construction of the aging database and KEGG enrichment

[0148] An aging-related gene library was compiled by organizing four existing aging databases (Aging_Atlas, CSgene, GenAge, SASPAtlas) and important aging protein genes from the literature. Then, the methylation levels and expression levels of these aging genes were compared in each comparison group. KEGG enrichment analysis was performed on genes with differentially methylated DMR regions. Pathway enrichment analysis was performed on KEGG pathways, using hypergeometric tests to identify pathways significantly enriched among differentially expressed genes compared to the overall genome background. The p-value for KEGG enrichment analysis was calculated using the following formula:

[0149]

[0150] These senescence genes were screened, and heatmaps and violin plots of methylation sites in the promoter region were generated using R scripts.

[0151] 2.6.2 Methylation Clock

[0152] The mouse epigenetic clock algorithm developed by Stubbs et al. (2017) was used to assess the biological age of mouse livers. Since the original model only covered mice aged 1–41 weeks, to broaden the model's age coverage to suit the experimental sample, RRBS data from mice aged 24 weeks (n=20), 40 weeks (n=20), 48 weeks (n=20), 80 weeks (n=20), and 120 weeks (n=19) were added during training (data from a paper published by Meer et al., GEO accession number: GSE121141). Similar to the original algorithm, internal tenfold cross-validation was used, and the optimal alpha and lambda were determined to be 0.05 and 0.37, respectively. The final model selected 399 loci and their beta values. The final function used was:

[0153] log(age) = 0.022654 * x 2 +1.0829*x+3.1277

[0154] Where x is the sum of the beta scores for each sample.

[0155] 2.6.3 Real-time quantitative PCR

[0156] Total RNA was extracted from the liver using the TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China, ER501-01) according to the manufacturer's instructions. GoScript was used. TM A reverse transcription system (Promega, Madison, A5001) was used to generate cDNA according to the manufacturer's instructions. The cDNA product was then used as a template for qPCR, following the manufacturer's instructions, using MonAmp... TM qPCR was performed using CChemHS qPCR Mix (Monad, Suzhou, China, MQ00401S). The mRNA expression of SIRT3, SIRT4, FOXO1, and SOD2 in muscle tissue was measured using a Roche LightCycler 96 qPCR Real-Time PCR system (Roche, Basel, Switzerland, LightCycler96). The mRNA expression of ERCC2, RBX1, Dnmt1, Dnmt3a, and Dnmt3b was also measured. The primer sequences used for real-time quantitative PCR are listed below.

[0157] Table 9 Primer sequences for real-time quantitative PCR

[0158]

[0159] 2.7 Instrument System

[0160] Table 10 Instrument System

[0161]

[0162] 2.8 Statistical Methods

[0163] Statistical analysis was performed using IBM SPSS 22. Normality tests (Shapiro-Wilk test) and homogeneity of variance tests (Levene's test) were performed on all data, and appropriate statistical methods were selected. To reveal differences among multiple independent samples (comparison of mouse strains), one-way ANOVA was used for normally distributed data, and the Kruskal-Wallis test was used for non-normally distributed data. A p-value less than 0.05 was considered significant in all analyses (*P≤0.05, **P≤0.01). All data are expressed as mean ± standard deviation (SD).

[0164] 3 Results

[0165] 3.1 PRBS Results

[0166] To investigate the differences in methylation levels at the CG site throughout the DNA, we used principal component analysis (PCA) to observe the clustering behavior of the YOUNG, MODEL, and BZ groups. PCA showed good separation of biological replication within the YOUNG, MODEL, and BZ groups. We performed statistical analysis on the promoters, exons, and introns of DMRs. DMRs were more abundant in promoters and introns than in exons. Hypomethylated DMRs were more significantly enriched in younger groups. Compared to 'YOUNG VS MODEL', we identified 8548 identical hypermethylated DMRs and 40893 identical hypomethylated DMRs in the promoter region, 15544 identical hypermethylated DMRs and 70431 identical hypomethylated DMRs in the intron region, and 6402 identical hypermethylated DMRs and 38603 identical hypomethylated DMRs in the exon region in 'BZ VS MODEL'. The BZ group and the younger group may have the same trend.

[0167] Table 11 PKBS Results

[0168]

[0169] Table 12. Statistics on methylation sites in samples

[0170] Samplc mCpG mCHG mCHH Total mC mCpG% mCHG% mCHH% BZH1 24240499 241174 567614 25049287 0.967712135 0.009627979 0.022659886 BZH2 24388216 229038 513956 25131210 0.970435407 0.009113688 0.020450905 BZH3 26707753 256074 574027 27537854 0.969856003 0.009298982 0.020845016 BZH4 26235037 259469 592668 27087174 0.968540941 0.009579035 0.021880023 BZH5 28209570 271425 624981 29105976 0.969201995 0.009325405 0.021472601 BZH6 22851290 219935 506860 23578085 0.969174978 0.009327942 0.021497081 BZH7 25066293 223351 475091 25764735 0.972891551 0.008668865 0.018439584 Agcd1 26645231 279102 614787 27539120 0.96754112 0.010134746 0.022324134 Agcd2 25772174 240604 507160 26519938 0.971803705 0.00907257 0.019123725 Agcd3 27153709 282605 618318 28054632 0.967886836 0.010073381 0.022039783 Agcd4 27127574 256206 551011 27934791 0.971103525 0.009171574 0.019724901 Agcd5 26542865 252663 546788 27342316 0.970761401 0.009240731 0.019997867 Agcd6 26631313 266258 573677 27471248 0.969424942 0.009692243 0.020882815 Agcd7 27218490 259316 533900 28011706 0.971682696 0.009257415 0.019059889 Young1 27841187 265030 593000 28699217 0.970102669 0.009234747 0.020662585 Young2 27907614 268347 641449 28817410 0.968428946 0.009311975 0.022259079 Young3 28611438 273013 636328 29520779 0.969196578 0.009248164 0.021555258 Young4 29455593 259749 578510 30293852 0.972329072 0.008574314 0.019096614 Young5 28955866 276098 665568 29897532 0.968503554 0.009234809 0.022261637 Young6 27570339 265240 642076 28477655 0.968139371 0.009313969 0.02254666 Young7 28454888 265040 624969 29344897 0.96967074 0.009031894 0.021297366

[0171] To further explore the anti-aging mechanism of BZ, we performed DNA methylation screening on large liver samples from three groups (aged, BZH, and YOUNG) using Illumina's HiSeq 4000 sequencing platform. Using state-of-the-art data mining techniques, we obtained data on an average of 26,837,473 methylation sites per sample (96.97% CpG sites, 0.93% CHG sites, and 2.1% CHH sites). Principal component analysis (PCA) revealed significant overall separation between groups. Significantly different overall methylation levels were observed between young and aged mice, and BZ treatment (2 g / kg / d, BZH group) reversed age-related methylation changes, particularly in promoter regions. To systematically define distinct methylation regions (DMRs), we compared samples using the Methyl Kit R package, screening for autosomal CPGS with significant p-values ​​(Bonferroni corrected) and at least 5% methylation differences. In the young and aged groups, we identified a total of 98,329 DMRs. In the aged group, 74.23% of DMRs were hypermethylated, and 25.77% were hypomethylated. BZ increased the proportion of hypomethylated CPGS in aged mice by 4.73%. Most DMRs in the “young vs. aged” comparison groups were roughly evenly distributed across different regions (except the 3'-UTR), with introns accounting for the largest proportion. However, only the promoter DMRS showed a similar distribution between the CGI and the CGI shore (a 2kb-long region located on either side of the CpG island). Interestingly, BZ treatment resulted in a methylation pattern highly similar to that of the young group, contrasting with the pattern in the aged group.

[0172] Next, we focused on promoter regions. In gene promoters, hypomethylated CpGs are generally associated with active, constitutively expressed genes, while hypermethylated CpGs are associated with low-expression / silenced genes. Therefore, we localized DMRs in promoters to the gene body. We identified 84 genes associated with hypermethylation in 'YOUNG VS MODEL' and 65 in 'BZ VS MODEL', and 80 and 104 genes associated with hypomethylated DMRs in 'YOUNG VS MODEL' and 'BZ VS MODEL', respectively. We also identified several hypomethylated DMRs localized to genes belonging to the SIRT and FOXO family in both 'YOUNG VS MODEL' and 'BZ VS MODEL'. Since the SIRT and FOXO families are closely associated with healthy lifespan and lifespan, high expression of these gene families implies increased lifespan and overall health in organisms. Furthermore, in the 'BZ VS MODEL' dataset, we identified differentially hypomethylated genes associated with DNA repair, including Ercc2, Ddb1, and Rbx1. DNA damage drives aging by disrupting transcription or DNA replication. DNA damage is a persistent threat during aging, causing widespread epigenetic alterations and disrupting cellular homeostasis. These genes are essential for DNA repair, maintaining a balance between genome integrity and cell cycle progression. Higher expression of these genes can enhance DNA repair and delay aging. We also identified differentially hypomethylated genes associated with autophagy in the 'BZ VS MODEL' dataset, including Ulk1 and Tfeb. Autophagy function deteriorates with age, and genes in the autophagy-lysosomal pathway are essential for lifespan extension through various pro-longevity pathways. Increased expression of autophagy-related genes implies promotion of longevity.

[0173] KEGG enrichment analysis revealed that the p53, TNF, and NF-κB signaling pathways play important roles in the hypermethylated DMRs of "YOUNG VS MODEL" and "BZ VS MODEL". The FoxO signaling pathway plays a crucial role in the hypomethylated DMRs of "YOUNG VS MODEL" and "BZ VS MODEL". p53 is a key trigger for cellular senescence, and the NF-κB signaling pathway is involved in the induction of the senescence-associated secretory phenotype (SASP). The TNF signaling pathway plays a vital role in inducing age-related chronic inflammation. The FoxO signaling pathway is thought to improve healthy lifespan and prolong lifespan. These results suggest that BZ can reverse age-related epigenetic changes associated with liver aging.

[0174] Table 13. Genes in the "BZH VS Older Group" promoter DMR reflected in the aging database

[0175]

[0176]

[0177] 3.2 Methylation clock age and aging rate

[0178] Compared with the model group of the same actual age (CA), the drug composition group of the present invention significantly reduced the epigenetic age of mice and reduced the aging rate of mice.

[0179] Table 14 Results of DNA methylation clock age detection (x±s)

[0180]

[0181] **** This indicates that, compared to the model group, P < 0.0001. ΔΔΔΔ This indicates that compared with the age group, P < 0.0001.

[0182] 3.3 Real-time quantitative PCR results

[0183] SIRT3 expression level: Compared with the young group, the model group showed a statistically significant difference (P < 0.05); compared with the model group, the pharmaceutical composition group of the present invention showed a statistically significant difference (P < 0.01). SIRT4 expression level: Compared with the young group, the model group showed a statistically significant difference (P < 0.01); compared with the model group, the pharmaceutical composition group of the present invention showed a statistically significant difference (P < 0.01). FOXO1 expression level: Compared with the model group, the pharmaceutical composition group of the present invention showed a statistically significant difference (P < 0.05). SOD2 expression level: Compared with the young group, the model group (P < 0.01) and the pharmaceutical composition group of the present invention (P < 0.05) showed statistically significant differences; compared with the model group, the pharmaceutical composition group of the present invention showed a statistically significant difference (P < 0.01).

[0184] Table 15 PCR Detection Results

[0185]

[0186] ** Compared with the model group, P < 0.01. * Compared with the model group, P < 0.05; compared with the young group, P < 0.01; compared with the young group, P < 0.05.

[0187] In summary, the BZ provided in this embodiment of the invention can mitigate the degree of aging by increasing genes related to longevity and mitochondrial homeostasis, such as SIRT3, SIRT4, FOXO1, and SOD2. The BZ provided in this embodiment of the invention can upregulate DNA repair-related genes, including Ercc2 and Rbx1, with significant upregulation in both the BZBS and younger groups compared to the older group (P<0.01). There was no statistically significant difference in DNMT3a / DNMT3b; however, compared to the older group, both the BZBS and younger groups significantly upregulated ERCC2 and RBX1 (P<0.01). This demonstrates the potential methylation regulatory function of BZ.

[0188] Experimental Example 3

[0189] To clarify the effects of the drug of the present invention, the inventors also conducted the following experiments using Example 1 (hereinafter referred to as the drug of the present invention or BZ):

[0190] The experimental materials and methods are the same as in Example 2.

[0191] I. Detection Indicators

[0192] 1. Immunofluorescence

[0193] Liver tissue was embedded in Sakura Tissue-Tek OCT Compound and cut into 8 μm thick sections. The sections were then fixed in 4% paraformaldehyde for 10 minutes, washed three times with PBS for 5 minutes each time, and blocked with Triton X-100 for 60 minutes. The blocking solution was then removed, and the sections were co-stained with γ-H2AX antibody and α-Tubulin antibody (1:1000 dilution) and incubated overnight at 4°C. The sections were washed three times with PBST for 5 minutes each time, with the corresponding fluorescent secondary antibody added, and incubated in the dark at room temperature for 40 minutes. The washing was repeated three times with PBST. Finally, the sections were mounted with DAPI-containing mounting medium. High-resolution images were acquired using confocal microscopy and analyzed using ImageJ.

[0194] 2. SOD Measurement

[0195] Liver tissue was homogenized in physiological saline and centrifuged (4℃, 3000 rpm, 10 min), and the supernatant was collected for analysis. Protein concentration was determined by BCA assay, and SOD was detected and calculated using a superoxide dismutase (SOD) assay kit.

[0196] 3. Cell Culture

[0197] Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC). Cells were cultured in DMEM containing 10% FBS in a 5% CO2 incubator at 37°C.

[0198] 4. MTS Measurement

[0199] Take HUVECs that have grown to approximately 80% during the logarithmic growth phase, and administer at a rate of 6 × 10⁻⁶ mg / mL. 4 Cells were seeded at a density of [number] cells per well in 96-well plates. Different concentrations of BZBS extract were added to the cells, and the cells were incubated at 37°C for 72 hours in a 5% CO2 incubator. CellTiter [data missing] was used. The AQuious One Solution Cell Proliferation Assay was used to determine the activity of HUVECs. Absorbance was measured using a microplate reader.

[0200] 5. Cellular radiation

[0201] HUVECs were pretreated with different concentrations of BZBS extract for 3 days, followed by irradiation with 6 Gy for 5 min. Cells were cultured at 37°C and 5% CO2 for 5 days. The culture medium containing BZBS extract was changed every two days. The normal control group received no irradiation treatment.

[0202] II. Results

[0203] 1. Liver's antioxidant capacity

[0204] Foxo1 can induce the expression of downstream antioxidant genes such as Catalase and Sod2. Sirtuin-3 can enhance SOD2 activity through deacetylation. Consistent with the upregulation of Sirt3 and Foxo1, the Sod2 mRNA level in BZ-treated aged mice (see attached image) Figure 1 Increased total SOD activity (see appendix) Figure 2 Table 16 shows the comparison results of the antioxidant capacity indicators of the livers of mice in each group.

[0205] Table 16 Comparison of antioxidant capacity in the livers of mice in different groups

[0206]

[0207] Note: *: p<0.05 compared with the older group; **: p<0.01 compared with the older group; #: p<0.05 compared with the younger group.

[0208] The results showed that BZ could enhance the antioxidant capacity of the liver in naturally aging mice.

[0209] 2. DNA damage

[0210] To confirm the downstream effects of the identified DNA repair genes, the levels of γ-H2AX, a marker of double-strand DNA breaks, in mouse livers were detected by immunofluorescence staining. The results showed that γ-H2AX levels were significantly elevated in the livers of older mice compared to younger mice, and that BZ could reduce γ-H2AX levels in older mice (see Table 17, Appendix). Figure 3 Appendix Figure 4 (As shown).

[0211] Table 17 Comparison of antioxidant capacity in the liver of mice in each group (

[0212]

[0213] Note: *: p<0.05 compared with the elderly group; **: p<0.01 compared with the elderly group.

[0214] 3. Regulation of the role of methyltransferases in aging HUVECs

[0215] To investigate whether the drug of this invention has a direct effect on the regulation of DNA methylation during aging, this study prepared an ethanol extract of BZ and examined its regulatory effect on methyltransferases in HUVECs. The cytotoxicity of the BZ extract was detected by the MTS assay; none of the concentrations caused a significant decrease in cell viability (see attached figure). Figure 5 (As shown). This study established a radiation-induced HUVEC aging model. Short-term irradiation of 6 Gy significantly altered the cell morphology of HUVEC cells (increased nuclear area) and induced the upregulation of HUVEC cell aging markers (as shown in the attached figure). Figure 6-10 As shown in the figure, similar to what was observed in the livers of aged mice, the expression of DNMT1 and TET1 in senescent HUVECs was also significantly lower than in unirradiated cells (as shown in the figure). Figure 11 Appendix Figure 12 (As shown in the attached image). After treatment with 250 μg / ml BZ, DNMT1 expression increased (as shown in the attached image). Figure 11 (As shown). Regulation of TET1 expression appears to be more sensitive to BZ extract. 25 μg / ml of BZ extract was sufficient to rescue TET1 expression, and 250 μg / ml of BZ extract even increased TET1 expression (as shown in the attached figure). Figure 12(As shown). Although the ethanol extract contains only a portion of the chemical substances in the whole formula, these results still demonstrate the direct regulatory effect of the pharmaceutical composition of the present invention on DNA methylation. More importantly, HUVECs belong to human endothelial cells, a type of cell that constitutes blood vessels and are widely distributed throughout the body, including the liver. In vitro experiments on senescent HUVECs show that the pharmaceutical composition of the present invention has a potentially conserved methylation regulatory effect in multiple senescent human tissues. Table 18 shows the comparison results of methyltransferase mRNA expression levels in HUVECs of each experimental group.

[0216] Table 18 Comparison of methyltransferase mRNA expression levels in HUVECs

[0217]

[0218] Note: *: p<0.05 compared with the elderly group; **: p<0.01 compared with the elderly group.

[0219] #: Compared with the younger group, P<0.05; ##: Compared with the younger group, P<0.01.

[0220] Preparation of BZ extract:

[0221] BZ ultrafine powder was mixed with 50% ethanol (ratio 1:15) in an ultrasonic apparatus. The mixture was centrifuged at 3500 rpm for 10 min, and the supernatant was collected and freeze-dried to prepare the BZ extract. The maximum solubility concentration of the BZ extract was 250 mg / mL, and the working solutions were 250 μg / mL, 25 μg / mL, and 2.5 μg / mL.

[0222] The pharmaceutical composition of the present invention can reduce the DNA methylation age of the liver by promoting the hypomethylation of genes in the longevity regulatory pathway and the hypermethylation of genes in the aging pathway, thereby alleviating the epigenetic aging of the liver; the pharmaceutical composition of the present invention significantly reduces the physiological age of mice.

[0223] The drugs provided in Examples 2 to 6 of this invention have essentially the same effects as the capsules in Example 1.

[0224] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a traditional Chinese medicine composition in the preparation of a drug for alleviating epigenetic aging of the liver, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: Cuscuta chinensis 150-350, Lycium barbarum 100-200, Schisandra chinensis 30-60, Cnidium monnieri 20-50, Rosa laevigata 20-50, Allium tuberosum 20-50, Morinda officinalis 20-50, Cistanche deserticola 20-50, Rehmannia glutinosa 30-60, Achyranthes bidentata 20-50, Epimedium brevicornu 40-100, Rubus idaeus 20-50, Panax ginseng 15-35, Cervus nippon antler 10-25, Hippocampus 15-35, and Melia toosendan 15-35. The traditional Chinese medicine composition reduces the DNA methylation age of the liver.

2. The application as described in claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: Cuscuta chinensis 150, Lycium barbarum 200, Schisandra chinensis 30, Cnidium monnieri 50, Rosa laevigata 20, Allium tuberosum 20, Morinda officinalis 50, Cistanche deserticola 20, Rehmannia glutinosa 30, Achyranthes bidentata 50, Epimedium brevicornu 40, Rubus idaeus 50, Panax ginseng 15, Cervus nippon antler 25, Hippocampus 15, and Melia toosendan 35.

3. The application as described in claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: Cuscuta chinensis 350, Lycium barbarum 100, Schisandra chinensis 60, Cnidium monnieri 20, Rosa laevigata 50, Allium tuberosum 50, Morinda officinalis 20, Cistanche deserticola 50, Rehmannia glutinosa 60, Achyranthes bidentata 20, Epimedium brevicornu 100, Rubus idaeus 20, Panax ginseng 35, Cervus nippon antler 10, Hippocampus 35, and Melia toosendan 15.

4. The application as described in claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: Cuscuta chinensis 250, Lycium barbarum 138, Schisandra chinensis 46, Cnidium monnieri 35, Rosa laevigata 35, Allium tuberosum 35, Morinda officinalis 35, Cistanche deserticola 35, Rehmannia glutinosa 46, Achyranthes bidentata 35, Epimedium brevicornu 70, Rubus idaeus 35, Panax ginseng 25, Cervus nippon antler 16, Hippocampus 21, and Melia toosendan 23.

5. The application as described in claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: Cuscuta chinensis 250, Lycium barbarum 150, Schisandra chinensis 45, Cnidium monnieri 30, Rosa laevigata 30, Allium tuberosum 30, Morinda officinalis 30, Cistanche deserticola 30, Rehmannia glutinosa 45, Achyranthes bidentata 30, Epimedium brevicornu 70, Rubus idaeus 30, Panax ginseng 20, Cervus nippon antler 19, Hippocampus 20, and Melia toosendan 20.

6. The application as described in any one of claims 1 to 5, characterized in that, The dosage form of the drug is decoction, capsule, tablet, granule, powder or pill.

7. The application as described in claim 6, characterized in that, The method for preparing the capsules includes the following steps: a. Weigh out Cnidium monnieri, Cuscuta chinensis, Schisandra chinensis and Epimedium according to the proportion, add 6-10 times the amount of 70% ethanol, reflux extract 1-3 times, 1-3 hours each time, filter, combine the filtrates, and recover the ethanol under reduced pressure until there is no alcohol taste to obtain the alcohol extract. b. Combine the medicinal residue after alcohol extraction in step a with the appropriate amounts of wolfberry, rehmannia root, raspberry, rosehip, leek seed, morinda root, and Sichuan pepper, add 7-12 times the amount of water and decoct 1-3 times, 1-3 hours each time. Filter, combine the filtrates, mix with the alcohol extract obtained in step a, and concentrate under reduced pressure until the relative density is 1.25-1.30 when measured at 60℃ to obtain the extract. c. Weigh out ginseng, deer antler, seahorse, cistanche and achyranthes according to the proportion, grind them into fine powder, mix them evenly with the extract obtained in step b, dry at 60-70℃, grind, sieve and fill into capsules.

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