An epigenetics-guided traditional Chinese medicine composition, its preparation method and application

Abstract

This invention relates to the fields of traditional Chinese medicine and anti-aging technology. The invention provides a scientifically formulated traditional Chinese medicine composition based on epigenetic guidance, its preparation method, and its application. The composition is made from the following raw materials in parts by weight: ginseng 3-12 parts, cordyceps 3-12 parts, polygonatum 2-8 parts, ganoderma lucidum 2-10 parts, mulberry fungus 2-6 parts, pine needles 1-6 parts, epimedium 2-8 parts, salvia miltiorrhiza 3-10 parts, bletilla striata 1-5 parts, cistanche deserticola 2-6 parts, rosemary 1-5 parts, and banana peel 1-6 parts. The composition is designed to comprehensively consider aging-related oxidative stress, energy metabolism regulation, and multi-component intervention needs, and is constructed in conjunction with epigenetic regulatory directions, reflecting the standardized design concept of scientifically formulated traditional Chinese medicine. The composition of this invention has the characteristics of clear composition, feasible preparation process, controllable quality parameters, and verifiable efficacy indicators.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine and anti-aging, in particular to a technology-based traditional Chinese medicine composition based on epigenetic orientation and a preparation method and application thereof. BACKGROUND

[0002] Aging is usually accompanied by increased oxidative stress, energy metabolism imbalance, decreased tissue repair capacity and various physiological function degenerative changes. Existing anti-aging related technologies are mostly focused on nutritional supplements, antioxidant intervention or single active ingredient application, and the development of traditional Chinese medicine compositions around multi-component, multi-level intervention of aging-related changes still has further research space.

[0003] Traditional Chinese medicine has a long application basis in improving constitution, regulating overall state and delaying degenerative changes of body functions. Compared with single component, traditional Chinese medicine composition has the characteristics of multi-component and multi-pathway joint action. If a traditional Chinese medicine composition with clear composition, feasible preparation, controllable quality and good experimental effect in aging model can be constructed, it is expected to provide a new technical solution for the field of anti-aging related technologies.

[0004] With the advancement of related research, comprehensive intervention ideas around the regulation network related to aging have gradually attracted attention. Epigenetic related regulation can be used as a research and development consideration factor to understand aging-related changes from the perspective of gene expression regulation, and combined with oxidative stress state, energy metabolism regulation and tissue function maintenance factors for composition design. If such regulation ideas are combined with the standardization construction path of technology-based traditional Chinese medicine, it is helpful to form a technical solution with the characteristics of traditional Chinese medicine composition and modern technology expression.

[0005] Therefore, it has good technical value to develop a traditional Chinese medicine composition based on technology-based traditional Chinese medicine standardization, which can improve the oxidative stress state related to aging and has a regulating effect on the SIRT1 / AMPK related pathway. SUMMARY

[0006] The purpose of the present application is to provide a technology-based traditional Chinese medicine composition based on epigenetic orientation and a preparation method and application thereof, which comprehensively considers the oxidative stress state related to aging, energy metabolism regulation, epigenetic related regulation factors and multi-component intervention requirements in design, and has good anti-aging related research and application value.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: This invention provides a traditional Chinese medicine composition directed on epigene repair. The composition is made from the following raw materials in parts by weight: ginseng 3-12 parts, cordyceps 3-12 parts, polygonatum 2-8 parts, ganoderma lucidum 2-10 parts, mulberry fungus 2-6 parts, pine needles 1-6 parts, epimedium 2-8 parts, salvia miltiorrhiza 3-10 parts, bletilla striata 1-5 parts, cistanche deserticola 2-6 parts, rosemary 1-5 parts, and banana peel 1-6 parts.

[0008] Preferably, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: ginseng 4-8 parts, cordyceps 4-8 parts, polygonatum 3-6 parts, ganoderma lucidum 3-6 parts, mulberry fungus 2-4 parts, pine needles 2-4 parts, epimedium 3-6 parts, salvia miltiorrhiza 4-8 parts, bletilla striata 2-4 parts, cistanche deserticola 3-5 parts, rosemary 2-4 parts, and banana peel 2-4 parts.

[0009] Preferably, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 5-7 parts ginseng, 5-7 parts cordyceps, 3-5 parts polygonatum, 3-5 parts ganoderma, 2-4 parts mulberry fungus, 2-4 parts pine needles, 3-5 parts epimedium, 5-7 parts salvia miltiorrhiza, 2-4 parts bletilla striata, 3-5 parts cistanche deserticola, 2-4 parts rosemary, and 2-4 parts banana peel.

[0010] The present invention also provides a method for preparing the traditional Chinese medicine composition, comprising the following steps: (1) Weigh out ginseng, cordyceps, polygonatum, ganoderma, mulberry fungus, pine needles, epimedium, salvia miltiorrhiza, bletilla striata, cistanche deserticola, rosemary and banana peel in proportion, and mix them to obtain a mixture; (2) Boil the mixture with water and separate the extract; (3) The extract is concentrated to obtain an extract, and the extract is dried to obtain the traditional Chinese medicine composition.

[0011] Preferably, the mass ratio of the mixture to water in step (2) is 1:6~12.

[0012] Preferably, the number of times the decoction is performed in step (2) is 1 to 3 times, and the time for each decoction is 30 to 90 minutes.

[0013] Preferably, in step (3), the concentration is carried out by reduced pressure concentration or vacuum low temperature concentration, and the drying is carried out by spray drying, vacuum drying or freeze drying; the relative density of the extract at 60°C is 1.10~1.20, and the water content of the traditional Chinese medicine composition obtained after drying is not higher than 8%.

[0014] The present invention also provides the application of the aforementioned traditional Chinese medicine composition in the preparation of a drug for improving age-related oxidative stress.

[0015] The present invention also provides the application of the aforementioned traditional Chinese medicine composition in the preparation of drugs for regulating the SIRT1 / AMPK related pathway.

[0016] The present invention also provides the application of the aforementioned traditional Chinese medicine composition in the preparation of a medicament for improving degenerative changes in skin tissue in aging model animals.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The traditional Chinese medicine composition provided by the present invention can improve oxidative stress in a D-galactose-induced aging model, increase SOD and GSH-Px activity and reduce MDA level; (2) The traditional Chinese medicine composition provided by the present invention can upregulate SIRT1 expression and increase the p-AMPK / AMPK ratio, indicating that it has a regulatory effect on SIRT1 / AMPK related pathways; (3) The traditional Chinese medicine composition provided by the present invention can improve the general condition and skin tissue degenerative changes of aging model animals, including increasing the thickness of the dermis and increasing the collagen fiber content; (4) Compared with the reduced formula, the complete formulation of the present invention shows a better comprehensive intervention effect in improving oxidative stress indicators, regulating SIRT1 / AMPK related pathways and improving skin tissue degenerative changes, indicating that the formulation of the present invention has good technical rationality. (5) The traditional Chinese medicine composition provided by the present invention can regulate some epigenetic indicators, indicating that it is related to the direction of epigenetic regulation. (6) The traditional Chinese medicine composition of the present invention can improve cell viability, reduce intracellular reactive oxygen species levels and reduce the proportion of aging-related positive cells in an in vitro cell model, further supporting its anti-aging effects; (7) The traditional Chinese medicine composition provided by the present invention has a clear composition, an implementable process, controllable quality parameters, and exhibits good safety under the experimental conditions. Detailed Implementation

[0018] This invention provides a traditional Chinese medicine composition directed on epigene repair. The composition is made from the following raw materials in parts by weight: ginseng 3-12 parts, cordyceps 3-12 parts, polygonatum 2-8 parts, ganoderma lucidum 2-10 parts, mulberry fungus 2-6 parts, pine needles 1-6 parts, epimedium 2-8 parts, salvia miltiorrhiza 3-10 parts, bletilla striata 1-5 parts, cistanche deserticola 2-6 parts, rosemary 1-5 parts, and banana peel 1-6 parts.

[0019] In this invention, the traditional Chinese medicine composition is preferably made from the following raw materials in parts by weight: ginseng 4-8 parts, cordyceps 4-8 parts, polygonatum 3-6 parts, ganoderma lucidum 3-6 parts, mulberry fungus 2-4 parts, pine needles 2-4 parts, epimedium 3-6 parts, salvia miltiorrhiza 4-8 parts, bletilla striata 2-4 parts, cistanche deserticola 3-5 parts, rosemary 2-4 parts, and banana peel 2-4 parts.

[0020] In the present invention, the traditional Chinese medicine composition is preferably prepared from the following raw materials in parts by weight: 5-7 parts of ginseng, 5-7 parts of cordyceps, 3-5 parts of polygonatum sibiricum, 3-5 parts of ganoderma lucidum, 2-4 parts of sanghuang, 2-4 parts of pine needles, 3-5 parts of epimedium, 5-7 parts of salvia miltiorrhiza, 2-4 parts of bletilla striata, 3-5 parts of cistanche deserticola, 2-4 parts of rosemary, and 2-4 parts of banana peel.

[0021] The present invention also provides a preparation method of the traditional Chinese medicine composition, comprising the following steps: (1) Weigh ginseng, cordyceps, polygonatum sibiricum, ganoderma lucidum, sanghuang, pine needles, epimedium, salvia miltiorrhiza, bletilla striata, cistanche deserticola, rosemary, and banana peel according to the ratio, and mix them to obtain a mixture; (2) Decoct the mixture with water, and separate to obtain an extract; (3) Concentrate the extract to obtain an extract paste, and dry the extract paste to obtain the traditional Chinese medicine composition.

[0022] In the present invention, the mass ratio of the mixture to water in step (2) is preferably 1:6-12, and more preferably 1:9.

[0023] In the present invention, the number of decoction times in step (2) is preferably 1-3 times, more preferably 2 times, and the single decoction time is preferably 30-90 min, more preferably 60 min.

[0024] In the present invention, during concentration in step (3), preferably vacuum concentration or vacuum low-temperature concentration is adopted, and during drying, preferably spray drying, vacuum drying or freeze drying is adopted; the relative density of the extract paste at 60 °C is preferably 1.10-1.20, more preferably 1.15, and the water content of the traditional Chinese medicine composition obtained after drying is preferably not higher than 8%.

[0025] The present invention also provides an application of the traditional Chinese medicine composition in the preparation of a drug for improving the oxidative stress state related to aging.

[0026] The present invention also provides an application of the traditional Chinese medicine composition in the preparation of a drug for regulating the SIRT1 / AMPK-related pathway.

[0027] The present invention also provides an application of the traditional Chinese medicine composition in the preparation of a drug for improving the skin tissue degenerative changes in aging model animals.

[0028] Quality control description

[0029] To further demonstrate the standardized construction characteristics of the traditional Chinese medicine composition of this invention, and to improve the stability of raw material quality, the repeatability of the preparation process, and the consistency of product quality, this invention sets quality control indicators for some representative raw materials, extraction processes, and the resulting extracts. The following quality control indicators are used to illustrate the preferred standardized implementation methods of the traditional Chinese medicine composition of this invention and are not intended to limit the scope of protection of this invention.

[0030] I. Raw Material Quality Control

[0031] 1. Ginseng: Preferably, ginseng raw materials or extracts that meet the pharmacopoeia standards or the company's internal control standards are used. Preferably, the total ginsenoside content is not less than 5%, using total ginsenosides as a quality control indicator; more preferably, it is not less than 8%.

[0032] 2. Ganoderma lucidum: Preferably, Ganoderma lucidum raw materials or extracts conforming to pharmacopoeia standards or the company's internal control standards are used. Preferably, Ganoderma lucidum triterpenes and / or Ganoderma lucidum polysaccharides are used as quality control indicators. Specifically, the content of Ganoderma lucidum triterpenes is preferably not less than 1%; more preferably, not less than 2%. The content of Ganoderma lucidum polysaccharides is preferably not less than 10%; more preferably, not less than 20%.

[0033] 3. Polygonatum: Preferably, Polygonatum raw materials or extracts that meet the pharmacopoeia standards or the company's internal control standards are used. Preferably, Polygonatum polysaccharides are used as a quality control indicator, and the content of Polygonatum polysaccharides is preferably not less than 10%; more preferably, not less than 20%.

[0034] 4. Cordyceps: Preferably, Cordyceps raw materials or extracts conforming to pharmacopoeia standards or the company's internal control standards are used. Preferably, cordycepin and / or adenosine are used as quality control indicators. The cordycepin content is preferably not less than 0.2%; more preferably, not less than 0.5%. The adenosine content is preferably not less than 0.05%.

[0035] 5. Epimedium: Preferably, Epimedium raw materials or extracts that meet the pharmacopoeia standards or the company's internal control standards are used. Preferably, icariin is used as a quality control indicator, and the icariin content is preferably not less than 5%; more preferably, not less than 10%.

[0036] 6. Salvia miltiorrhiza: Preferably, Salvia miltiorrhiza raw materials or extracts conforming to pharmacopoeia standards or enterprise internal control standards are used. Preferably, tanshinone IIA and / or salvianolic acid B are used as quality control indicators. Specifically, the content of tanshinone IIA is preferably not less than 0.2%; the content of salvianolic acid B is preferably not less than 3%.

[0037] 7. Rosemary: Rosemary raw materials or extracts that meet pharmacopoeia standards or the company's internal control standards are preferred. Preferably, rosmarinic acid is used as a quality control indicator, and the rosmarinic acid content is preferably not less than 2%; more preferably, not less than 5%.

[0038] 8. Phellinus linteus: Preferably, Phellinus linteus raw materials or its extracts that meet the pharmacopoeia standards or the company's internal control standards are used. Preferably, Phellinus linteus polysaccharides and / or characteristic phenolic components are used as quality control indicators. The content of Phellinus linteus polysaccharides is preferably not less than 10%; more preferably, not less than 20%.

[0039] 9. Pine needles: Pine needle raw materials or their extracts that meet pharmacopoeia standards or the company's internal control standards are preferred. Preferably, total flavonoids and / or proanthocyanidins are used as quality control indicators. The total flavonoid content is preferably not less than 2%.

[0040] 10. Bletilla striata: Preferably, Bletilla striata raw materials or extracts conforming to pharmacopoeia standards or enterprise internal control standards are used. Preferably, using Bletilla striata polysaccharide as a quality control indicator, the content of Bletilla striata polysaccharide is preferably not less than 10%; more preferably, not less than 20%.

[0041] 11. Cistanche deserticola: Preferably, Cistanche deserticola raw materials or its extracts conforming to pharmacopoeia standards or the company's internal control standards are used. Preferably, verbascoside and / or echinacoside are used as quality control indicators. Specifically, the verbascoside content is preferably not less than 1%; the echinacoside content is preferably not less than 0.5%.

[0042] 12. Banana peel: Preferably, banana peel raw materials or their extracts are sourced from bananas with clear origins and processed according to regulations. Preferably, total polyphenols and / or flavonoids are used as quality control indicators. The total polyphenol content is preferably not less than 3%.

[0043] II. Quality Control of the Extraction Process

[0044] To ensure the repeatability and stability of the preparation process of the traditional Chinese medicine composition of this invention, the following process parameters are preferably controlled: 1. The mass ratio of total raw materials to water is preferably 1:6 to 1:12; 2. The number of decoctions is preferably 1 to 3; 3. The decoction time for each decoction is preferably 30 to 90 minutes; 4. The relative density of the concentrate at 60°C is preferably 1.10 to 1.20; 5. The drying method is preferably spray drying, vacuum drying, or freeze drying; 6. The water content of the dried extract is preferably not higher than 8%; more preferably, not higher than 5%.

[0045] III. Finished Product Quality Control

[0046] The extract obtained from the traditional Chinese medicine composition of this invention is preferably a brownish-yellow to brownish-brown powder or paste, with relatively stable appearance, odor, and physicochemical properties. To improve product consistency, the following indicators are preferably controlled: 1. Appearance: brownish-yellow to brownish-brown powder or paste; 2. Moisture content: not higher than 8%; 3. Total polysaccharide content: preferably not lower than 8%; more preferably, not lower than 12%; 4. Preferably, an HPLC or UPLC characteristic fingerprint of the traditional Chinese medicine composition of this invention is established to characterize batch-to-batch consistency; 5. Microbial limits, heavy metals, and pesticide residue indicators preferably comply with relevant pharmacopoeia standards, food standards, or enterprise internal control standards.

[0047] IV. Explanation

[0048] The above-mentioned quality control indicators are used to illustrate the standardized implementation method of the traditional Chinese medicine composition of this invention, reflecting the technical characteristics of "technological traditional Chinese medicine," including controllable raw material quality, clear process parameters, and evaluable finished product indicators. These quality control indicators can be appropriately adjusted according to the source of raw materials, extraction process, and specific application scenarios, and should not be construed as limiting the scope of protection of this invention.

[0049] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] Weigh the raw materials according to the following weight proportions: 6 parts ginseng, 6 parts cordyceps, 4 parts polygonatum, 4 parts ganoderma, 3 parts mulberry fungus, 3 parts pine needles, 4 parts epimedium, 6 parts salvia miltiorrhiza, 3 parts bletilla striata, 4 parts cistanche deserticola, 3 parts rosemary, and 3 parts banana peel.

[0052] After mixing the above raw materials, purified water was added at a mass ratio of 1:10 between the total amount of raw materials and water. The mixture was heated and decocted twice, each time for 60 minutes. The decoctions were combined, filtered, and concentrated under reduced pressure to an extract with a relative density of 1.10 (60°C). The extract was then spray-dried to obtain the epigene repair-oriented traditional Chinese medicine composition extract of this invention.

[0053] The obtained extract is a brownish-yellow to brownish-brown powder with a water content not exceeding 8%. The extract can be prepared into granules, capsules, tablets, or extracts using conventional methods. The yield of the extract is 12%–28% of the total mass of the raw materials. Fingerprints were established using the corresponding chromatographic peaks of ginsenosides, ganoderma triterpenoids, tanshinones, and rosmarinic acid as characteristic peaks to evaluate the consistency of extracts from different batches.

[0054] Example 2

[0055] Weigh the ingredients according to the following proportions by weight: 5 parts ginseng, 5 parts cordyceps, 3 parts polygonatum, 3 parts ganoderma, 2 parts mulberry fungus, 2 parts pine needles, 3 parts epimedium, 5 parts salvia miltiorrhiza, 2 parts bletilla striata, 3 parts cistanche deserticola, 2 parts rosemary, and 2 parts banana peel.

[0056] After mixing the above raw materials, purified water was added at a mass ratio of 1:8 between the total amount of raw materials and water. The mixture was heated and decocted twice, each time for 45 minutes. The decoctions were combined, filtered, concentrated under vacuum at low temperature, and then freeze-dried to obtain the extract of the traditional Chinese medicine composition of this invention.

[0057] Example 3

[0058] Weigh the raw materials according to the following weight proportions: 7 parts ginseng, 7 parts cordyceps, 5 parts polygonatum, 5 parts ganoderma, 4 parts mulberry fungus, 4 parts pine needles, 5 parts epimedium, 7 parts salvia miltiorrhiza, 4 parts bletilla striata, 5 parts cistanche deserticola, 4 parts rosemary, and 4 parts banana peel.

[0059] After mixing the above raw materials, purified water was added at a mass ratio of 1:12 between the total amount of raw materials and water. The mixture was heated and decocted three times, each time for 90 minutes. The decoctions were combined, filtered, concentrated under reduced pressure, and then vacuum dried to obtain the extract of the traditional Chinese medicine composition of this invention.

[0060] Experimental Example 1: Effects of the traditional Chinese medicine composition of this invention on D-galactose-induced oxidative stress and the SIRT1 / AMPK-related pathway in aging mice.

[0061] (a) Laboratory animals and grouping

[0062] Healthy C57BL / 6 mice were randomly divided into 5 groups of 8 mice each: 1) Normal control group; 2) Aging model group; 3) Low-dose group of the compound of the present invention; 4) Medium-dose group of the compound of the present invention; 5) High-dose group of the compound of the present invention.

[0063] (II) Modeling and Drug Administration

[0064] Except for the normal control group, mice in the other groups were subcutaneously injected with D-galactose solution daily to establish an aging model for 8 consecutive weeks. Simultaneously, mice in each treatment group were administered the extract of the traditional Chinese medicine composition prepared in Example 1 by gavage, with the following dosages: low-dose group: 200 mg / kg·d; medium-dose group: 400 mg / kg·d; high-dose group: 800 mg / kg·d. The normal control group and the aging model group were administered an equal volume of physiological saline by gavage for 8 consecutive weeks.

[0065] (III) Detection Indicators

[0066] After the drug administration cycle, mice were fasted for 12 hours, blood was collected from the orbital sinus, serum was separated, and the mice were sacrificed for liver tissue analysis. The main indicators included: 1. Oxidative stress indicators: SOD activity, GSH-Px activity, and MDA content were detected using a kit; the results are shown in Table 1. 2. Protein expression indicators: The expression levels of SIRT1, AMPK, and p-AMPK in liver tissue were detected using Western blot; the results are shown in Table 2.

[0067] Table 1 Results of oxidative stress indicators in mice of each group

[0068] Table 2. Results of SIRT1 and p-AMPK / AMPK detection in each group of mice.

[0069] The results of Experiment Example 1 show that the traditional Chinese medicine composition of the present invention can significantly improve the oxidative stress state of D-galactose-induced aging mice, increase the activity of SOD and GSH-Px, reduce the level of MDA, and regulate the SIRT1 / AMPK related pathway.

[0070] Experimental Example 2: Effects of the Traditional Chinese Medicine Composition of the Invention on Degenerative Changes in Skin Tissue of an Aging Mouse Model

[0071] (a) Laboratory animals and grouping

[0072] The experimental animals were grouped, modeled, and administered the drugs in the same manner as in Experiment 1.

[0073] (II) Observation Indicators

[0074] During the experiment, changes in mouse weight, mental state, coat color and luster, and activity level were observed and recorded regularly. Mice were euthanized after the experiment, and skin tissue was collected for histological analysis. Hematoxylin-eosin (HE) staining was used to observe skin tissue structure and measure dermal thickness; Masson staining was used to observe collagen fiber content and calculate the collagen area percentage.

[0075] The results of Experiment Example 2 show that, in an aging animal model, the traditional Chinese medicine composition of the present invention can improve the general condition related to aging and improve the degenerative changes in skin tissue, including increasing the thickness of the dermis and increasing the content of collagen fibers, showing good anti-aging effects.

[0076] Experimental Example 3: Safety Evaluation of the Traditional Chinese Medicine Composition of the Invention

[0077] (a) Laboratory animals and grouping

[0078] Healthy C57BL / 6 mice were selected as experimental animals and randomly divided into a normal control group and a high-dose compound group of the present invention, with 8 mice in each group. The normal control group was given an equal volume of physiological saline by gavage; the high-dose compound group of the present invention was given the extract of the traditional Chinese medicine composition prepared in Example 1 by gavage at a dose of 1200 mg / kg·d for 28 consecutive days.

[0079] (II) Observation Content and Testing Methods

[0080] During the administration period, the general condition of the experimental animals was observed daily, including their mental state, activity level, feeding status, and weight changes.

[0081] After the administration of the drug, blood samples were collected from mice for serum biochemical index testing, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr).

[0082] Simultaneously, major organs such as the heart, liver, spleen, lungs, and kidneys were weighed, and organ coefficients were calculated using the following formula: Organ coefficient (%) = Organ weight / Body weight × 100% Experimental data are expressed as Mean ± SD.

[0083] (III) Experimental Results

[0084] During the 28-day continuous administration period, no adverse reactions such as significant death, abnormal behavior, reduced activity, abnormal feeding, or sudden weight loss were observed in the mice in the high-dose compound group of this invention. Their general condition was basically the same as that of the normal control group.

[0085] The results of serum biochemical indicators in each group of mice are shown in Table 3.

[0086] Table 3. Results of serum biochemical indicators in mice of each group (Mean ± SD, n=8)

[0087] Statistical analysis showed that the levels of ALT, AST, BUN, and Cr in the high-dose compound group of mice were all within the normal reference range, and there were no statistically significant differences compared with the normal control group (P>0.05).

[0088] The results of the major organ coefficients of mice in each group are shown in Table 4.

[0089] Table 4. Results of major organ coefficient detection in each group of mice (Mean ± SD, n=8)

[0090] As shown in Table 4, compared with the normal control group, no significant abnormal changes were observed in the coefficients of major organs in the high-dose compound group of the present invention.

[0091] (iv) Conclusion

[0092] The above results indicate that, under the condition of continuous oral administration of 1200 mg / kg·d for 28 days, the traditional Chinese medicine composition of the present invention did not show obvious toxic reactions and did not have significant adverse effects on liver function, kidney function and major organs, indicating that it has good safety.

[0093] Experimental Example 4: Comparative Verification of the Rationality of the Traditional Chinese Medicine Composition Formulation of the Invention

[0094] (I) Experimental Objective

[0095] To verify the rationality of the formulation design of the traditional Chinese medicine composition of the present invention, and to evaluate the differences between the complete formulation and the reduced formulation in improving age-related oxidative stress, regulating the SIRT1 / AMPK-related pathway, and improving skin tissue degenerative changes, this experiment sets up a control comparison between the complete formulation and the reduced formulation to examine the overall compatibility effect of the formulation of the present invention.

[0096] (II) Experimental Materials

[0097] 1. Test samples: (1) The complete formula is the extract of Example 1; (2) Reduced formula combination A: the combination after removing Phellinus linteus, pine needles, Bletilla striata, rosemary and banana peel; (3) Reduced formula combination B: the skeleton tonic group composed of ginseng, cordyceps, polygonatum, ganoderma, epimedium and cistanche, and the extract was prepared according to the same process as in Example 1.

[0098] 2. Experimental animals: Healthy C57BL / 6 mice, regardless of sex, weighing 18-22 g were selected.

[0099] (III) Experimental Methods

[0100] Experimental animals were randomly divided into 6 groups of 8 mice each: 1) normal control group; 2) aging model group; 3) complete formulation group of this invention; 4) modified formulation group A; 5) modified formulation group B; and 6) positive control group. Except for the normal control group, mice in the other groups were subcutaneously injected with D-galactose solution daily to establish an aging model for 8 consecutive weeks. Simultaneously with modeling, each treatment group was administered medication by gavage for 8 consecutive weeks. The dosage for the complete formulation group, modified formulation group A, and modified formulation group B was 400 mg / kg / day; the positive control group received resveratrol 100 mg / kg / day; and the normal control group and aging model group received an equal volume of physiological saline by gavage.

[0101] Table 5 Results of oxidative stress index detection in mice of Experiment 4

[0102] Table 6. Results of SIRT1 and p-AMPK / AMPK detection in mice of Experiment 4

[0103] The results of Experiment 4 show that the complete formulation, reduced formulation A, and reduced formulation B of this invention can all improve the oxidative stress state, regulate the SIRT1 / AMPK-related pathway, and improve skin tissue degenerative changes in mice with D-galactose-induced aging model to a certain extent. However, the complete formulation of this invention shows better improvement effects than reduced formulation A and reduced formulation B in multiple detection indicators, indicating that the raw material composition and compatibility of the traditional Chinese medicine composition of this invention are more reasonable, and the complete formulation shows a better comprehensive intervention effect than the reduced formulation. The complete formulation performs better than the reduced formulation in multiple indicators, indicating that the raw material composition and compatibility of this invention have substantial technical significance.

[0104] Experimental Example 5: Effects of the Traditional Chinese Medicine Composition of the Invention on Epigenetic Related Indicators in an Aging Model

[0105] (I) Experimental Objective

[0106] To investigate the correlation between the traditional Chinese medicine composition of this invention and epigenetic regulatory indicators, and to verify its effect on the expression of some epigenetic proteins under aging model conditions, this experiment detected the expression levels of DNMT1, TET2 and HDAC1 in the liver tissue of D-galactose-induced aging model mice.

[0107] (II) Experimental Methods

[0108] The experimental animals were randomly divided into four groups of eight mice each: 1) normal control group; 2) aging model group; 3) medium-dose compound preparation of the present invention group; and 4) positive control group. Except for the normal control group, mice in the other groups were subcutaneously injected with D-galactose solution daily to establish an aging model for eight consecutive weeks. Simultaneously with modeling, the medium-dose compound preparation group was given 400 mg / kg / day of the extract of the traditional Chinese medicine composition prepared in Example 1, the positive control group was given 100 mg / kg / day of resveratrol, and the normal control group and the aging model group were given an equal volume of physiological saline by gavage.

[0109] (III) Detection Methods

[0110] After the drug administration cycle was completed, the mice were sacrificed and liver tissue was isolated. The expression levels of DNMT1, TET2, and HDAC1 in the liver tissue were detected by Western blot. β-actin was used as an internal reference protein. Gray-scale analysis of the target protein bands was performed, and the relative expression levels were calculated. The results are shown in Table 7.

[0111] Table 7. Results of epigenetic-related protein expression in Experiment 5

[0112] The results of Experiment 5 show that, under D-galactose-induced aging model conditions, the traditional Chinese medicine composition of this invention can regulate some epigenetic indicators, specifically by increasing the expression levels of DNMT1 and TET2 and decreasing the expression level of HDAC1. These results suggest that the traditional Chinese medicine composition of this invention has a certain correlation with epigenetic regulation and can serve as one of the experimental bases for its related design ideas.

[0113] Experimental Example 6: Protective effect of the traditional Chinese medicine composition of the present invention on a H2O2-induced cell senescence model.

[0114] (I) Experimental Objective

[0115] To further verify the ameliorative effect of the traditional Chinese medicine composition of this invention on aging-related damage at the cellular level, and to evaluate its effects on oxidative stress and SIRT1 / AMPK-related pathways, this experiment used H2O2 to induce a cellular senescence model and investigated the effects of the traditional Chinese medicine composition of this invention on cell viability, intracellular reactive oxygen species levels, aging-related β-galactosidase staining, and SIRT1 / AMPK-related protein expression.

[0116] (II) Experimental Materials

[0117] The test sample was an extract of the traditional Chinese medicine composition prepared in Example 1 of this invention. The cell line used was the human hepatocyte HepG2 cell line. The main reagents included H2O2 solution, CCK-8 reagent kit, DCFH-DA reactive oxygen species fluorescent probe, SA-β-gal staining kit, and Western blot reagents.

[0118] (III) Experimental Methods

[0119] Cells were randomly divided into 6 groups: 1) normal control group; 2) model group; 3) low-concentration group of the compound preparation of this invention; 4) medium-concentration group of the compound preparation of this invention; 5) high-concentration group of the compound preparation of this invention; and 6) positive control group. Each treatment group was pre-treated with the extract of the traditional Chinese medicine composition of this invention for 24 hours before H2O2 treatment, with the low-concentration group receiving 50 μg / mL, the medium-concentration group receiving 100 μg / mL, and the high-concentration group receiving 200 μg / mL; the positive control group received 20 μmol / L of resveratrol. The detection indicators included cell viability, ROS level, SA-β-gal positivity rate, SIRT1, and p-AMPK / AMPK. The results are shown in Tables 8-9.

[0120] Table 8. Results of cell viability assay in Experiment 6

[0121] Table 9. Results of SIRT1 and p-AMPK / AMPK detection in cells of Experimental Example 6

[0122] The results of Experiment 6 indicate that, in an H2O2-induced cellular senescence-related model, the traditional Chinese medicine composition of this invention can improve cell viability, reduce intracellular ROS levels, decrease the proportion of SA-β-gal positive cells, and regulate the SIRT1 / AMPK pathway. These results demonstrate that the traditional Chinese medicine composition of this invention has a mitigating effect on oxidative stress-related damage and aging-related changes at the cellular level, further supporting its anti-aging applications.

[0123] in conclusion

[0124] As can be seen from the above embodiments and experimental examples, the present invention provides a scientifically formulated traditional Chinese medicine composition based on epigenetic guidance, its preparation method, and its application. The traditional Chinese medicine composition is made from the following raw materials in parts by weight: ginseng 3-12 parts, cordyceps 3-12 parts, polygonatum 2-8 parts, ganoderma lucidum 2-10 parts, mulberry fungus 2-6 parts, pine needles 1-6 parts, epimedium 2-8 parts, salvia miltiorrhiza 3-10 parts, bletilla striata 1-5 parts, cistanche deserticola 2-6 parts, rosemary 1-5 parts, and banana peel 1-6 parts. The composition is designed to comprehensively consider aging-related oxidative stress, energy metabolism regulation, and multi-component intervention needs, and is constructed in conjunction with epigenetic regulatory directions, reflecting the standardized design concept of scientifically formulated traditional Chinese medicine. It has the characteristics of clear composition, feasible process, and controllable quality. Experimental results show that the traditional Chinese medicine composition can improve the oxidative stress state of D-galactose-induced aging model animals, increase SOD and GSH-Px activities, reduce MDA levels, and regulate the SIRT1 / AMPK-related pathway. Simultaneously, the composition can also improve the general condition and skin tissue degenerative changes in the aging model animals, and exhibits good safety under the experimental conditions. Further formulation control experiments show that the complete formulation of this invention has a better comprehensive intervention effect than the reduced formulation. Epigenetic index experiments show that the traditional Chinese medicine composition of this invention can regulate some epigenetic indexes. Cell experiments further show that the traditional Chinese medicine composition of this invention can also improve oxidative stress-related damage and aging-related changes in in vitro models, indicating a certain correlation with epigenetic regulation.

[0125] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine composition guided by epigene repair, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: ginseng 3-12 parts, cordyceps 3-12 parts, polygonatum 2-8 parts, ganoderma lucidum 2-10 parts, mulberry fungus 2-6 parts, pine needles 1-6 parts, epimedium 2-8 parts, salvia miltiorrhiza 3-10 parts, bletilla striata 1-5 parts, cistanche deserticola 2-6 parts, rosemary 1-5 parts, and banana peel 1-6 parts.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: ginseng 4-8 parts, cordyceps 4-8 parts, polygonatum 3-6 parts, ganoderma lucidum 3-6 parts, mulberry fungus 2-4 parts, pine needles 2-4 parts, epimedium 3-6 parts, salvia miltiorrhiza 4-8 parts, bletilla striata 2-4 parts, cistanche deserticola 3-5 parts, rosemary 2-4 parts, and banana peel 2-4 parts.

3. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 5-7 parts ginseng, 5-7 parts cordyceps, 3-5 parts polygonatum, 3-5 parts ganoderma, 2-4 parts mulberry fungus, 2-4 parts pine needles, 3-5 parts epimedium, 5-7 parts salvia miltiorrhiza, 2-4 parts bletilla striata, 3-5 parts cistanche deserticola, 2-4 parts rosemary, and 2-4 parts banana peel.

4. A method for preparing the traditional Chinese medicine composition according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Weigh out ginseng, cordyceps, polygonatum, ganoderma, mulberry fungus, pine needles, epimedium, salvia miltiorrhiza, bletilla striata, cistanche deserticola, rosemary and banana peel in proportion, and mix them to obtain a mixture; (2) Boil the mixture with water and separate the extract; (3) The extract is concentrated to obtain an extract, and the extract is dried to obtain the traditional Chinese medicine composition.

5. The preparation method according to claim 4, characterized in that, The mass ratio of the mixture to water in step (2) is 1:6~12.

6. The preparation method according to claim 4, characterized in that, The number of times to decoct in step (2) is 1 to 3, and the time for each decoction is 30 to 90 minutes.

7. The preparation method according to claim 4, characterized in that, In step (3), the concentration is carried out by vacuum concentration or vacuum low temperature concentration, and the drying is carried out by spray drying, vacuum drying or freeze drying; the relative density of the extract at 60°C is 1.10~1.20, and the water content of the traditional Chinese medicine composition obtained after drying is not higher than 8%.

8. The use of the traditional Chinese medicine composition according to any one of claims 1 to 3 in the preparation of a medicament for improving age-related oxidative stress.

9. The use of the traditional Chinese medicine composition according to any one of claims 1 to 3 in the preparation of a drug for regulating the SIRT1 / AMPK related pathway.

10. The use of the traditional Chinese medicine composition according to any one of claims 1 to 3 in the preparation of a medicament for improving degenerative changes in skin tissue of aging model animals.

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