Application of traditional Chinese medicine composition in preparation of anti-aging medicine

Abstract

The invention provides application of a traditional Chinese medicine composition in preparation of a medicine for delaying senescence. The traditional Chinese medicine composition is prepared from traditional Chinese medicinal materials including ginseng, rhizoma polygonati, rhizoma atractylodis, radix sophorae flavescentis, radix ophiopogonis, rehmannia, polygonum multiflorum, dogwood, poria cocos, eupatorium, coptis chinensis, rhizoma anemarrhenae, herba epimedii and the like. Experiments prove that the pharmaceutical composition can significantly reduce the expression of P16, P21, TP53BP1 and gamma-H2AX in pancreas, reduce the content of TNF-alpha, IL-6 and IL-1beta in pancreas, reduce the number of pro-inflammatory immune cells CD80 +, CD4 + and CD8 + in pancreas, increase the number of anti-inflammatory immune cells CD206 + in pancreas, and delay pancreas aging.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to an application of a traditional Chinese medicine composition in the preparation of an anti-aging drug. Background Art

[0002] Aging is a process characterized by cumulative degenerative damage. With advancing age, the risk of diabetes, cardiovascular disease, cancer, and neurodegenerative diseases increases, accompanied by multi-organ dysfunction and ultimately death. As the world's population gradually ages, aging has become a hot topic of discussion in academia. Aging not only refers to macroscopic aging of the organism but also encompasses cellular senescence. Cellular senescence occurs at any stage of life and can lead to permanent cell cycle arrest, making it a key factor in the development of many chronic diseases. The SASP, a collective term for a series of cytokine secretions, refers to the production of large amounts of extracellular secretions during cellular senescence. The SASP includes a variety of bioactive molecules, primarily composed of interleukins (ILs), chemokines, growth factors, secreted proteases, and secreted insoluble proteins / extracellular matrix (ECM). These molecules can directly or indirectly influence cellular function and participate in processes such as inflammatory responses, cell cycle arrest, and apoptosis. Therefore, the SASP, secreted by senescent cells, is a key indicator of aging and a contributing factor to many age-related diseases. Therefore, reducing SASP secretion and thereby modulating inflammatory responses in the body is an effective approach to delaying cellular senescence.

[0003] The Chinese medicine composition of the present invention is an oral hypoglycemic drug clinically used to treat type 2 diabetes. It has significant advantages in regulating glucose metabolism disorders and improving insulin resistance. However, there is currently no research on the use of this pharmaceutical composition for delaying aging. Summary of the Invention

[0004] The present invention provides a use of a traditional Chinese medicine composition in the preparation of an anti-aging drug. The technical solution adopted by the present invention is:

[0005] The traditional Chinese medicine composition of the present invention is composed of the following raw materials in parts by weight: 50-150 parts of ginseng, 60-180 parts of polygonatum, 40-90 parts of atractylodes, 30-58 parts of sophora flavescens, 60-180 parts of ophiopogon, 60-110 parts of rehmannia root, 40-90 parts of polygonatum, 60-180 parts of cornus officinalis, 40-90 parts of poria, 35-58 parts of euphratica root, 35-58 parts of coptis chinensis, 35-90 parts of anemarrhena, 35-58 parts of epimedium, 40-110 parts of salvia miltiorrhiza, 60-180 parts of kudzu root, 80-140 parts of litchi seed and 40-90 parts of lycium bark.

[0006] Preferably, the Chinese medicine composition of the present invention can delay pancreatic aging.

[0007] Preferably, the traditional Chinese medicine composition in the present application can protect the islet tissue structure, protect the ultrastructure of islet β cells, and improve the secretion function thereof.

[0008] Preferably, the traditional Chinese medicine composition in the present application can reduce the expression of the cell aging effect factor P16 in the pancreas.

[0009] Preferably, the traditional Chinese medicine composition in the present application can reduce the expression of the cell aging effect factor P21 in the pancreas.

[0010] Preferably, the traditional Chinese medicine composition in the present application can reduce the expression of the aging marker protein TP53BP1 in the pancreas.

[0011] Preferably, the traditional Chinese medicine composition in the present application can reduce the expression of the aging marker protein γ-H2AX in the pancreas.

[0012] Preferably, the traditional Chinese medicine composition in the present application can reduce the content of SA-β-Gal in the pancreas.

[0013] Preferably, the traditional Chinese medicine composition in the present application can reduce the contents of TNF-α, IL-6, and IL-1β in the pancreas.

[0014] Preferably, the traditional Chinese medicine composition in the present application can reduce the number of pro-inflammatory immune cells CD80+, CD4+, and CD8+ in the pancreas and increase the number of anti-inflammatory immune cells CD206+ in the pancreas.

[0015] Preferably, the traditional Chinese medicine composition in the present application can delay the aging of islet α cells and islet β cells and reduce the number of aged islet α cells and islet β cells.

[0016] Preferably, the proportion of raw materials of the traditional Chinese medicine composition in the present application is as follows: ginseng 50, rhizoma polygonati 180, atractylodes 40, sophora 58, ophiopogon 60, rehmannia 110, radix polygoni multiflori 40, cornus 180, poria 40, pellitory 35, coptis 58, anemarrhena 90, epimedium 35, salvia 40, pueraria 180, litchi kernel 80, psoralea 40.

[0017] Or: ginseng 150, rhizoma polygonati 60, atractylodes 90, sophora 30, ophiopogon 180, rehmannia 60, radix polygoni multiflori 90, cornus 60, poria 90, pellitory 58, coptis 35, anemarrhena 35, epimedium 58, salvia 110, pueraria 60, litchi kernel 140, psoralea 90.

[0018] Or: ginseng 102, rhizoma polygonati 136, atractylodes 68, sophora 56, poria 83, ophiopogon 136, radix polygoni multiflori 83, rehmannia 102, cornus 136, coptis 56, pellitory 56, litchi kernel 136, epimedium 56, anemarrhena 68, salvia 89, pueraria 136, psoralea 83.

[0019] Among them, the Atractylodes lancea is preferably stir-fried with bran, the Polygonum multiflorum is preferably processed Polygonum multiflorum, and the Epimedium is preferably roasted Epimedium.

[0020] The preparation of the active ingredients of the Chinese medicine composition of the present invention comprises the following steps:

[0021] a. Weigh the Chinese medicinal materials according to the weight ratio of the raw materials, select and break them;

[0022] b. Add 5-9 times the amount of water to extract the volatile oil from Perilla frutescens and Atractylodes lancea for 3-6 hours. Collect the volatile oil in a separate container and filter the aqueous solution for later use.

[0023] c. Use 5-9 times the amount of 50-90% ethanol as a solvent for the cornus officinalis, soak for 12-48 hours, perform percolation, collect the percolation liquid, recover the ethanol, and concentrate it into a thick paste with a relative density of 1.30-1.35 measured at 60°C, dry it, and set aside;

[0024] d. Add 6-10 times the amount of 50-90% ethanol to ginseng, ophiopogon japonicus, epimedium, anemarrhena, and kudzu root, and reflux extract 1-3 times, each time for 1-3 hours. Filter the extract, recover the ethanol, and concentrate it into a thick paste. Dry it and set aside.

[0025] e, polygonatum, sophora flavescens, rehmannia root, polygonum multiflorum, poria, coptis root, salvia miltiorrhiza, litchi seed, and lychee bark, add 7-11 times the amount of water, decocted 1-2 times, each time for 1-3 hours, the extract was filtered, and combined with the aqueous solution after the perilla and atractylodes oil extraction in step b, concentrated into a clear paste, and ethanol was added to adjust the alcohol concentration to 50-80%, refrigerated, filtered, the filtrate was recovered with ethanol, concentrated to a thick paste, dried, and set aside;

[0026] The cornus officinalis dry paste obtained in step c, the alcohol-extracted dry paste obtained in step d, the water-extracted and alcohol-precipitated dry paste obtained in step e and the volatile oil obtained in step b together constitute the active ingredients of the traditional Chinese medicine composition.

[0027] The dosage form of the Chinese medicine composition of the present invention is capsule, tablet or granule.

[0028] The preparation of the Chinese medicine composition granules of the present invention comprises the following steps:

[0029] a. Weigh the Chinese medicinal materials according to the weight ratio of the raw materials, clean and crush them;

[0030] b. Combine Perilla frutescens and Atractylodes lancea, add 5-9 times the amount of water, and extract the volatile oil using steam method for 3-6 hours. Collect the volatile oil in another container, and filter the aqueous solution for later use.

[0031] c. Use 5-9 times the amount of 50-90% ethanol as a solvent for the cornus officinalis, soak for 12-48 hours, perform percolation, collect the percolation liquid, recover the ethanol, and concentrate it into a thick paste with a relative density of 1.30-1.35 measured at 60°C, dry it, and set aside;

[0032] d. Add 6-10 times the amount of 50-90% ethanol to ginseng, ophiopogon japonicus, epimedium, anemarrhena, and kudzu root, and reflux extract 1-3 times, each time for 1-3 hours. Filter the extract, recover the ethanol, and concentrate it into a thick paste. Dry it and set aside.

[0033] e, polygonatum, sophora flavescens, rehmannia root, processed polygonum multiflorum, tuckahoe, coptis root, salvia miltiorrhiza, litchi seed, and lychee bark, add 7-11 times the amount of water, decocted 1-2 times, each time for 1-3 hours, the extract was filtered, combined with the aqueous solution after the perilla and atractylodes oil extraction in step b, concentrated into a clear paste, added ethanol to adjust the alcohol concentration to 50-80%, refrigerated, filtered, the filtrate was recovered with ethanol, concentrated to a thick paste, dried, and set aside;

[0034] f. Evenly mix the dried cornus officinalis paste obtained in step c, the alcohol-extracted dried cornus officinalis paste obtained in step d, and the water-extracted and alcohol-precipitated dried cornus officinalis paste obtained in step e, grind them, and add auxiliary materials to granulate them;

[0035] g. Add ethanol to dissolve the volatile oil obtained in step b, spray it into the granules obtained in step f, mix well, seal, and package.

[0036] Experiments have shown that the Chinese medicine composition of the present invention delays pancreatic aging, reduces aging beta and alpha cells in pancreatic islet tissue, reduces the expression of SASP factors P16, P21, TP53BP1, and γ-H2AX, and the levels of TNF-α, IL-6, and IL-1β in the pancreas, reduces the number of pro-inflammatory immune cells CD80+, CD4+, and CD8+ in the pancreas, and increases the number of anti-inflammatory immune cells CD206+ in the pancreas. Pancreatic aging can cause the pancreas to be unable to secrete insulin normally, which in turn prevents insulin target organs such as the liver, skeletal muscle, and fat from properly absorbing and utilizing glucose, and reduces insulin sensitivity, ultimately leading to insulin resistance in the body. This in turn further aggravates islet function damage and cell aging, forming a vicious cycle. The Chinese medicine composition of the present invention can also regulate related genes in insulin target organs (liver, skeletal muscle and fat), inhibit inflammatory signal transduction (chemokines, cytokine-cytokine receptor interactions, NF-κB, etc.) pathways, activate the PPAR signaling pathway and branched-chain amino acid degradation pathway, activate the liver TCA cycle, inhibit the AGE-RAGE signaling pathway, and achieve cross-organ anti-aging effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : The Chinese medicine composition of the present invention delays pancreatic aging.

[0038] (A) HE staining was used to observe the pathological changes of islets (size: 100 μm). (B) PCR was used to detect P16 in pancreatic tissues. (C) PCR was used to detect P21 in pancreatic tissues. (D) ILISA was used to detect TNF-α in pancreatic tissues. (E) ILISA was used to detect IL-1β in pancreatic tissues. (F) ILISA was used to detect IL-6 in pancreatic tissues. Compared with the control group, ##P < 0.01, compared with the model group, **P < 0.01.

[0039] Figure 2 The traditional Chinese medicine composition protects the ultrastructure of islet β cells and improves the secretion function of islet β cells.

[0040] (A) Transmission electron microscopy was used to observe the pathological changes of the nucleus, mitochondria, endoplasmic reticulum and secretory granules of islet β cells. Size: 1 μm (mitochondria, endoplasmic reticulum), size: 5 μm (nucleus, secretory granules). (B) Secretory granule count and statistics of islet β cells in each field of view, ##P < 0.01 vs. control group; *P < 0.05 vs. model group.

[0041] Figure 3 The traditional Chinese medicine composition reduces the aging of islet cells.

[0042] (A) Immunofluorescence of islet insulin and P16 co-staining. Size: 100 μm. (B) Immunofluorescence of islet glucagon and P16 co-staining. Size: 100 μm. The P16 red fluorescence region is the aging cell in the pancreatic tissue, Figure 3 The green fluorescence region in A is the islet β cell, and the region where red light and green light are co-expressed (yellow) is the aging islet β cell. Figure 3 The green fluorescence region in B is the islet α cell, and the region where red light and green light are co-expressed (yellow) is the aging islet α cell.

[0043] Figure 4 The traditional Chinese medicine composition has an effect on the aging and inflammatory infiltration of the pancreas of MS mice.

[0044] a: SA-β-Gal staining of MS mouse pancreatic tissue, scale bar 100 μm;

[0045] b: p16 and MS mouse islet co-staining immunofluorescence image, stained with insulin (green), p16 (red) and DAPI (blue), scale bar 100 μm;

[0046] c: TP53BP1 and MS mouse islet co-staining immunofluorescence image, stained with insulin (red), TP53BP1 (green) and DAPI (blue), scale bar 100 μm;

[0047] d: Co-staining immunofluorescence images of γ-H2AX and MS mouse islets, stained with insulin (red), TP53BP1 (green) and DAPI (blue), scale bar 100 μm;

[0048] e: SA-β-Gal staining area statistics of MS mice (n=3);

[0049] f-h: p16, TP53BP1 and γ-H2AX and MS mouse islet co-staining positive cell statistics (n=3);

[0050] i-p: MS mouse pancreas CD4+ / CD8+ / CD80+ / CD206 immunofluorescence images and area statistics (n=5) stained with CD4+ / CD8+ / CD80+ / CD206 (green) and DAPI (blue), scale bar 100 μm;

[0051] *P<0.05**P<0.01***P<0.001 vs. MS group.

[0052] Figure 5 : Effect of the traditional Chinese medicine composition in the application on the pancreatic aging of T2DM mice.

[0053] a: SA-β-Gal staining of T2DM mouse pancreas tissue, scale bar 100 μm;

[0054] b: Co-staining immunofluorescence images of p16 and T2DM mouse islets, stained with insulin (green), p16 (red) and DAPI (blue), scale bar 100 μm;

[0055] c: Co-staining immunofluorescence images of TP53BP1 and T2DM mouse islets, stained with insulin (red), TP53BP1 (green) and DAPI (blue), scale bar 100 μm;

[0056] d: Co-staining immunofluorescence images of γ-H2AX and T2DM mouse islets, stained with insulin (red), TP53BP1 (green) and DAPI (blue), scale bar 100 μm;

[0057] e: SA-β-Gal staining area statistics of T2DM mice (n=3);

[0058] f-h: p16, TP53BP1 and γ-H2AX and T2DM mouse islet co-staining positive cell statistics (n=3);

[0059] *P<0.05**P<0.01***P<0.001 vs. T2DM group.

[0060] Figure 6: Effect of the Chinese medicine composition of the present invention on pancreatic inflammatory infiltration in T2DM mice.

[0061] ah: CD4+ / CD8+ / CD80+ / CD206 immunofluorescence images and area statistics of pancreas of T2DM mice (n=5), stained with CD4+ / CD8+ / CD80+ / CD206 (green) and DAPI (blue), scale bar 100 μm. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below with reference to specific embodiments:

[0063] Example 1

[0064] The formula of the Chinese medicine composition is as follows

[0065] Ginseng 102g, Polygonatum 136g, stir-fried Atractylodes 68g, Sophora flavescens 56g, Poria 83g, Ophiopogon japonicus 136g, processed Polygonum multiflorum 83g, Rehmannia glutinosa 102g, Cornus officinalis 136g, Coptis chinensis 56g, Cymbidium orientale 56g, Litchi seed 136g, roasted Epimedium 56g, Anemarrhena asphodeloides 68g, Salvia miltiorrhiza 89g, Pueraria lobata 136g, and Rehmannia root bark 83g.

[0066] The preparation process of the Chinese medicine composition granules is as follows:

[0067] a. Weigh the Chinese medicinal materials according to the prescribed amount, clean and crush them;

[0068] b. Add 6 times the amount of water to Perilla frutescens and Atractylodes lancea to extract the volatile oil. The extraction time is 5 hours. The volatile oil is collected in another container. The aqueous solution is filtered and set aside. The residue is discarded;

[0069] c. After soaking the cornus officinalis with 7 times the amount of 75% ethanol for 24 hours, perform percolation, collect the percolation liquid, recover the ethanol, and concentrate it into a thick paste with a relative density of 1.30 measured at 60°C, dry it at 70°C, and set aside;

[0070] d. Add 8 times the volume of 70% ethanol to ginseng, Ophiopogon japonicus, Epimedium, Anemarrhena asphodeloides, and Pueraria root, and extract under reflux three times, each time for 2 hours. Filter the extract, recover the ethanol, and concentrate to a thick paste with a relative density of 1.30 measured at 60°C. Dry at 65°C and set aside.

[0071] e, polygonatum, sophora flavescens, rehmannia root, polygonum multiflorum, tuckahoe, coptis root, salvia miltiorrhiza, litchi seed, cortex lychnophorae, add 9 times of water, decocted 2 times, each time for 2 hours, the extract was filtered, and the aqueous solution after the perilla and atractylodes lancea oil extraction was combined, and the relative density was measured at 60 ℃ when the clear paste was 1.15, 95% ethanol was added to mediate the alcohol concentration to 60%, and the mixture was refrigerated for 24 hours, filtered, and the filtrate was recovered ethanol, and the relative density was measured at 60 ℃ when the thick paste was 1.30, and the mixture was dried at 70 ℃ for standby use;

[0072] f, the dry extract of step c, the alcohol extract of step d, and the water extract of step e are mixed evenly, and then crushed;

[0073] g, the dry extract of step f is mixed with lactose and dextrin at a ratio of 4:5:1, and then granulated with 60% ethanol as a binder. The granules are dried at 60°C, sieved through a 12-60 mesh screen, and then sieved again to remove fine powder. The volatile oil of step b is sprayed onto the granules, and then mixed evenly. The mixture is sealed for half an hour to obtain the final product.

[0074] Example 2

[0075] The raw material formula is as follows:

[0076] Ginseng 50g, Huangjing 180g, Cangzhu 40g, Kushen 58g, Fuling 40g, Maidong 180g, Heshouwu 40g, Dihuang 110g, Shanzhu 60g, Huanglian 58g, Perilla 35g, Lichiguo 140g, Yinyanghuo 35g, Zhimu 90g, Danshen 40g, Gegen 180g, Digupi 40g.

[0077] The preparation method is as follows:

[0078] a, the traditional Chinese medicinal materials are weighed according to the prescription, cleaned and crushed;

[0079] b, Perilla and Cangzhu are added with 5 times the amount of water to extract volatile oil. The extraction time is 3 hours. The volatile oil is collected in a separate container, and the water solution is filtered and reserved for later use. The residue is discarded.

[0080] c, Shanzhu is soaked in 5 times the amount of 50% ethanol for 12 hours, and then subjected to percolation. The percolation liquid is collected, and the ethanol is recovered. The concentrated thick paste with a relative density of 1.30 at 60°C is dried at 65°C for later use.

[0081] d, Ginseng, Maidong, Yinyanghuo, Zhimu, and Gegen are added with 6 times the amount of 5% ethanol for reflux extraction for 2 times, each for 1 hour. The extraction liquid is filtered, and the ethanol is recovered. The concentrated thick paste with a relative density of 1.30 at 60°C is dried at 65°C for later use.

[0082] e, Huangjing, Kushen, Dihuang, Heshouwu, Fuling, Huanglian, Danshen, Lichiguo, and Digupi are added with 7 times the amount of water for boiling for 1 hour. The extraction liquid is filtered and combined with the water solution of Perilla and Cangzhu after oil extraction. The clear paste with a relative density of 1.10 at 60°C is adjusted to an alcohol concentration of 50% with 95% ethanol, and then placed in a refrigerator for 24 hours. The liquid is filtered, and the ethanol is recovered. The concentrated thick paste with a relative density of 1.30 at 60°C is dried at 65°C for later use.

[0083] f, the dry extract of step c, the alcohol extract of step d, and the water extract of step e are mixed evenly, and then crushed;

[0084] g. The volatile oil obtained in step b is added to ethanol and dissolved, and then sprayed into the granules obtained in step f. The tablet is prepared by a conventional preparation method.

[0085] Example 3

[0086] The raw material formula is

[0087] Radix ginseng 150g, Rhizoma polygonati 60g, Rhizoma atractylodis 90g, Radix sophorae flavescentis 30g, Poria cocos 90g, Radix ophiopogonis 60g, Radix polygoni multiflori 90g, Radix rehmanniae 60g, Fructus corni 180g, Rhizoma coptidis 30g, Herba pterocephali 58g, Fructus litchi 80g, Herba epimedii 58g, Rhizoma anemarrhenae 35g, Radix paeoniae alba 110g, Radix puerariae 56g, Cortex lycii 90g.

[0088] The preparation method is as follows:

[0089] a. The traditional Chinese medicinal materials are weighed according to the prescription, cleaned and broken;

[0090] b. Herba pterocephali and Rhizoma atractylodis are added with 9 times the amount of water, and the volatile oil is extracted for 6 hours. The volatile oil is collected in another container, and the water solution is filtered and reserved for later use, and the residue is discarded.

[0091] c. Fructus corni is soaked in 9 times the amount of 90% ethanol for 48 hours, and then subjected to percolation. The percolation liquid is collected, and the ethanol is recovered and concentrated into a thick paste with a relative density of 1.35 at 60°C. The thick paste is dried at 70°C and reserved for later use.

[0092] d. Radix ginseng, Radix ophiopogonis, Herba epimedii, Rhizoma anemarrhenae and Radix paeoniae alba are added with 10 times the amount of 90% ethanol, and subjected to reflux extraction for 3 times, each for 3 hours. The extraction liquid is filtered, the ethanol is recovered, and the extraction liquid is concentrated into a thick paste with a relative density of 1.35 at 60°C. The thick paste is dried at 70°C and reserved for later use.

[0093] e. Rhizoma polygonati, Radix sophorae flavescentis, Radix rehmanniae, Radix polygoni multiflori, Poria cocos, Rhizoma coptidis, Radix paeoniae alba, Fructus litchi and Cortex lycii are added with 11 times the amount of water, and subjected to decoction for 2 times, each for 3 hours. The extraction liquid is filtered, combined with the water solution of Herba pterocephali and Rhizoma atractylodis after oil extraction, and concentrated into a clear paste with a relative density of 1.15 at 60°C. The clear paste is adjusted to an alcohol concentration of 80% by adding 95% ethanol, and placed in a refrigerator for 24 hours. The clear paste is filtered, the ethanol is recovered, and the extraction liquid is concentrated into a thick paste with a relative density of 1.35 at 60°C. The thick paste is dried at 70°C, pulverized and reserved for later use.

[0094] f. The volatile oil obtained in step b is added to ethanol and dissolved, and then sprayed into the granules obtained in step f. The tablet is prepared by a conventional preparation method.

[0095] To clarify the effect of the traditional Chinese medicinal composition of the present application, the following tests were conducted on the medicine prepared in Example 1 (hereinafter referred to as the medicine of the present application or JLD).

[0096] Test Example 1

[0097] 1Materials and methods

[0098] 1.1 Test animals

[0099] SPF level 12-month-old SD male rats weighing 550-700 g, 33, purchased from Beijing Vito Lihua Experimental Animal Technology Co., Ltd., license number SCXK(Jing)2021-0011, were raised in the New Drug Safety Evaluation Research Center of Hebei Province of Traditional Chinese and Western Medicine Research Institute. Under the conditions of room temperature (23±2)℃, humidity (50±10)%, 12h light and dark alternation, free feeding and drinking water, adaptive feeding for 7d before the experiment. This study was approved by the Animal Ethics Committee of Hebei Province of Traditional Chinese and Western Medicine Research Institute (approval number N2022037).

[0100] 1.2 Drugs and reagents

[0101] The drug composition of the application (Shijiazhuang Yiling Pharmaceutical Co., Ltd., batch number SYB2112001); Sitagliptin phosphate (Hangzhou MSD Pharmaceutical Co., Ltd.); High-fat feed (Beijing Huafukang Biotechnology Co., Ltd., batch number SCXK(Jing)2019-0008, protein energy ratio 20%, carbohydrate energy ratio 20%, fat energy ratio 60%).

[0102] 1.3 Animal modeling and grouping

[0103] The rats were randomly divided into 5 groups according to body weight, the normal group (Control) was given standard rat feed (n=5); the model group (Model, n=5) was induced by high-fat feed to induce prediabetes model; the low-dose group of the drug of the application (L-JLD, n=7) (1.5g·kg -1 ), the high-dose group of the drug of the application (H-JLD, n=7) (3.0g·kg -1 ) and the sitagliptin group (Sitagliptin, n=7) (10mg·kg -1 ) were given drug treatment while feeding high-fat feed. Last for 25 weeks. From the beginning of modeling, we detected the changes of body weight and abdominal circumference of rats every 4 weeks. The body weight of rats induced by high-fat feed increased significantly at 24 weeks (P<0.05), and the abdominal circumference increased significantly from 8 weeks. After the animal phenotype showed statistical difference at 24 weeks, we detected the fasting blood glucose (FBG) and oral glucose tolerance test (OGTT), fasting insulin (FINS) and insulin resistance index (HOMA-IR) of rats, the results showed that the FBG, OGTT and FINS of model group rats were higher than those of control group (P<0.05), and the HOMA-IR increased significantly (P<0.01), which indicated that the prediabetes combined with natural aging rat model was successfully modeled.

[0104] 1.4 Detection indicators

[0105] 1.4.1 HE staining

[0106] The pancreatic tail tissue of each rat was fixed with 4% paraformaldehyde solution, embedded in conventional paraffin, and sectioned. The degree of islet cell damage was assessed by HE staining, and the tissue was observed and photographed under an optical microscope (Leica DM6000B, Wetzlar, Germany).

[0107] 1.4.2 RT-qPCR

[0108] Total RNA was extracted using a Trizol kit (Ambion, USA). Total RNA concentration and quality were determined, and reverse transcription was performed to generate cDNA. Reaction conditions included 40 cycles of pre-denaturation at 95°C for 4 minutes, followed by denaturation at 95°C for 30 seconds and annealing at 56°C for 30 seconds, followed by extension at 72°C for 1 minute. Relative quantification was performed using the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a standard. PCR amplification primers were designed and synthesized by Beijing Dingguo Changsheng Co., Ltd. The sequences are as follows:

[0109]

[0110] 1.4.3 ELISA

[0111] Pancreatic tissue was homogenized in PBS and centrifuged (4°C, 3000 rpm, 10 min). The supernatant was collected and the levels of TNF-α (Jianglaibio, JL13202), IL-6 (Jianglaibio, JL20896), and IL-1β (Jianglaibio, JL20884) in pancreatic tissue were detected using ELISA kits strictly following the instructions.

[0112] 1.4.4 Transmission Electron Microscopy (SEM)

[0113] Carefully separate and cut no more than 3mm 3 The large and small pancreatic tail specimens were quickly placed in electron microscopy fixative for more than 2 h, washed thoroughly with PBS, fixed in osmium phosphate, washed thoroughly with PBS, dehydrated with gradient ethanol, dehydrated with acetone, embedded in epoxy resin mixture, and polymerized. The islet cells were initially located under a light microscope and sliced ​​with a Leica ultrathin microtome to a thickness of 50 nm. The sections were double-electron stained with uranyl acetate and lead citrate, and observed and photographed with a transmission electron microscope (HITACHI, SU8100, Tokyo, Japan).

[0114] 1.4.5 Immunofluorescence co-staining (IF)

[0115] Pancreatic tissue sections were dewaxed and placed in citrate-EDTA antigen retrieval solution for microwave heating repair. They were then blocked with 3% hydrogen peroxide at 37°C in the dark for 10 minutes, and then blocked with 5% bovine serum albumin (BSA) at room temperature for 30 minutes. Rabbit anti-insulin antibody (1:200 dilution, Abcam, ab181547) and mouse anti-P16 antibody (1:250 dilution, Santa Cruz, sc-377412) were incubated at 4°C overnight; rabbit anti-insulin antibody (1:200 dilution, Abcam, ab92517) and mouse anti-P16 antibody (1:250 dilution, Santa Cruz, sc-377412) were incubated at 4°C overnight. Cruz, sc-377412) at 4°C overnight. The next day, the sections were washed three times with PBS and incubated with fluorescent secondary antibodies donkey anti-rabbit IgG (1:500 dilution, Abcam, ab150081) and goat anti-mouse IgG (1:500 dilution, Abcam, ab150116) at room temperature in the dark for 1 h. The sections were mounted with an anti-fluorescence quencher containing DAPI (Beyotime, Shanghai, China) and observed and images were captured under a fluorescence microscope (Leica DMi8 THUNDE Germany).

[0116] 2. Statistical methods

[0117] SPSS 26.0 software was used to perform statistical analysis on the experimental data. Data were first tested for normality and homogeneity of variance. If the data followed a normal distribution and homogeneous variance, one-way analysis of variance (ANOVA) was used to compare multiple groups, with P < 0.05 considered significant. If the data followed a normal distribution but heterogeneous variance, Dunnett's T3 method was used for comparative analysis. If the data did not follow a normal distribution, nonparametric statistical tests were used.

[0118] 3. Results

[0119] 3.1 JLD can protect pancreatic islet tissue structure

[0120] The results of HE staining of pancreatic tissue in each group are shown in Figure 1 As shown in A, the pancreas of prediabetic and aging rats had a small number of islets, irregular morphology, scattered distribution, uneven edges, and unclear boundaries. However, all doses of JLD and sitagliptin significantly improved the islet tissue structure.

[0121] 3.2 JLD can significantly reduce the expression of cell senescence effector factors

[0122] The expression results of P16 and P21 in pancreatic tissue of each group are as follows Figure 1As shown in Figures B and 1C, the expressions of P16 and P21 in the pancreatic tissue of rats in the model group were significantly increased (P<0.01), while the JLD groups at different doses and the Sitagliptin group significantly downregulated these factors (P<0.01).

[0123] The results of TNF-α, IL-1β and IL-6 content in the pancreas of each group were as follows Figure 1 As shown in D-1F. TNF-α( Figure 1 D), IL-1β ( Figure 1 E), IL-6 ( Figure 1 F) were significantly increased in the pancreas (P<0.01), while the JLD groups of each dose and Sitagliptin group could reduce the contents of these factors, among which H-JLD could significantly reduce the contents of TNF-α, IL-6, and IL-1β in the pancreas (P<0.01).

[0124] 3.3 JLD can protect the ultrastructure of pancreatic β cells and improve their secretory function

[0125] Mitochondria and endoplasmic reticulum are the main organelles that change in pancreatic beta cells of prediabetic rats. The results showed that the pancreatic beta cells of prediabetic rats combined with natural aging had extensive mitochondrial swelling, vacuolar degeneration, reduced mitochondrial cristae, dilated rough endoplasmic reticulum, and mild nuclear chromosome condensation; JLD dose groups and Sitagliptin could significantly improve these structural abnormalities ( Figure 2 A). The secretory granules of rats in the model group were significantly reduced (P<0.01), while those in the high-dose JLD group were significantly increased after treatment ( Figure 2 B).

[0126] 3.4 JLD can delay the aging of pancreatic α-cells and β-cells

[0127] The immunofluorescence co-staining results of pancreatic insulin, pancreatic glucagon and P16 in the pancreatic tissues of each group are shown in Figure 2. Figure 3 As shown in Figures A and 3B, the positive areas of insulin, glucagon, and P16 immunofluorescence co-staining in the model group were significantly increased compared to the model group, indicating that pancreatic α and β cells were aging. After treatment with JLD and Sitagliptin, the number of aging β and α cells was significantly reduced.

[0128] 4. Conclusion

[0129] In the experiment, aged islet β cells and α cells were found in the islet tissue of pre-diabetic rats combined with natural aging, and the aging of β cells was more obvious. Although aging is selective, β cells are also affected at the same time as α cells, which indicates that the islet tissue has more serious dysfunction. After treatment with the traditional Chinese medicine composition of the present application, the aged β cells and α cells in the islet tissue are reduced, the expression of SASP factors in the pancreatic tissue is reduced, the aging of islet cells in the islet tissue is delayed, and the aging of the pancreas is delayed.

[0130] Test Example 2

[0131] 1. Materials and methods

[0132] 1.1 Experimental animals and feeding conditions

[0133] 100 7-8 month old SPF C57BL / 6J male mice (weight 30-35 g, Beijing Sibeifeng Company) were used, and were single-caged in the SPF laminar flow cabinet of Hebei Yiling Pharmaceutical New Drug Evaluation Center. The feeding conditions were 12 h of day-night rhythm lighting, temperature 22±2℃, humidity 60±10%, bedding changed every 48 h and autoclaved. After 7 days of adaptive feeding, the mice were randomly divided into a normal group (Control) (n=20) fed with standard maintenance feed (M18101801), a metabolic syndrome (MS) group (n=40) and a diabetes model group (T2SM) (n=40) fed with high-fat feed (D12492, protein energy ratio 20%, carbohydrate energy ratio 20%, fat energy ratio 60%). The experimental protocol was approved by the institutional ethics committee (approval number: N2023141) and complied with the NIH guidelines for experimental animal welfare.

[0134] 1.2 Experimental design and drug administration

[0135] The MS model (n=40) was randomly divided into 4 groups (10 mice in each group): after 8 weeks of high-fat diet, the mice were randomly divided into an MS group (MS), a low-dose drug group (JLD-low), a high-dose drug group (JLD-high) and a metformin group (MET). The MS group was fed with 60% high-fat feed, and the drug administration groups were administered continuously for 24 weeks. The doses of the drug administration groups were: MET group (0.265 g / kg / d), JLD-high group (3.8 g / kg / d) and JLD-low group (1.9 g / kg / d). MET (M813341) was provided by Shanghai Maikelin Biochemical Technology Co., Ltd. The T2DM model: after 4 weeks of high-fat diet, the mice were injected intraperitoneally with streptozotocin (STZ, 100 mg / kg, Sigma), and the animals with FBG>11.1 mmol / L were grouped and fed with drug-containing high-fat feed for 12 weeks.

[0136] 1.3 Detection index

[0137] 1.3.1 SA-β-gal staining

[0138] After the last administration, the pancreas was dissected under anesthesia with 2% sodium pentobarbital (0.3 ml / 100 g) after 16 h of fasting, OCT-embedded section (8 μm), 37°C incubation of staining solution (Solarbio G1580) for 16 h in the dark, and Depex mounting (positive signal: cytoplasmic blue particles).

[0139] 1.3.2 Immunofluorescence

[0140] Pancreatic tissue was fixed with 4% paraformaldehyde for 48 h, dehydrated with gradient alcohol, paraffin-embedded, and sectioned (4 μm). The pancreatic paraffin sections were subjected to microwave antigen retrieval with citric acid buffer (pH 6.0), 3% H2O2 blocking, 5% BSA blocking, overnight incubation with primary antibody (see Table 1 for antibody information), Cy3-labeled secondary antibody (see Table 2 for antibody information) incubation for 1 h in the dark, DAPI counterstaining of the nucleus, and confocal microscopy image acquisition (Leica TCS SP8).

[0141] Table 1 Information of primary antibody

[0142]

[0143] Table 2 Information of secondary antibody

[0144]

[0145] 2. Statistical analysis

[0146] The data results were analyzed using IBM SPSS 28.0 statistical software. Numerical variables were represented as mean plus or minus standard deviation . Before comparing the data of each group, normality test was performed. If the data met the normality distribution, one-way ANOVA was used for comparison between groups, and LSD test was used for the results of variance equality, and Tamheini test was used for the results of variance inequality. All hypothesis testing methods were taken as 0.05 as the significance level.

[0147] 3. Results

[0148] 3.1 JLD alleviates pancreatic aging and inflammatory cell infiltration in MS mice

[0149] SA-β-gal staining found that compared with the Control group, the blue staining area of the pancreas of the Ms group mice increased significantly; compared with the Ms group, the blue staining area of the pancreas of the JLD-high group, JLD-low group, and MET group mice was significantly less Figure 4a, e). Immunofluorescence co-staining showed that the number of cells positive for insulin and P16, TP53, γ-H2AX was significantly increased in the Ms group compared with the Control group. The number of cells positive for insulin and P16, TP53, γ-H2AX was significantly decreased in the JLD-high group, JLD-low group, and MET group compared with the Ms group Figure 4 b-d, f-h), which collectively suggested the therapeutic effect of JLD on pancreatic aging.

[0150] Aged cells release a large amount of pro-inflammatory SASP to recruit immune cells. Immunofluorescence results of mouse pancreatic paraffin sections showed that Figure 4 i-p), the number of M1 macrophages (CD80+) and T cells (CD4+CD8+) was significantly increased, while the number of M2 macrophages (CD206+) was significantly decreased in the Ms group compared with the Control group. The number of M1 macrophages (CD80+) and T cells (CD4+CD8+) was significantly decreased, while the number of M2 macrophages (CD206+) was significantly increased in the JLD-high group, JLD-low group, and MET group compared with the Ms group. These results further verified the function of JLD in delaying pancreatic aging and inflammation.

[0151] 3.2 JLD alleviates pancreatic aging and inflammatory cell infiltration in T2DM mice

[0152] SA-β-gal staining results are shown in Figure 5 a, compared with the Control group, the blue-stained area of the mouse pancreas in the T2DM group was significantly increased; compared with the T2DM group, the blue-stained area of the mouse pancreas in the JLD-high group, JLD-low group, and MET group was significantly less. Figure 5 b-d, the number of cells positive for insulin and P16, TP53, γ-H2AX was significantly increased in the T2DM group compared with the Control group, and the number of cells positive for insulin and P16, TP53, γ-H2AX was significantly decreased in the JLD-high group, JLD-low group, and MET group compared with the T2DM group, which suggested that the number of aged pancreatic beta cells was reduced in the administration groups.

[0153] Immunofluorescence results of mouse pancreatic paraffin sections are shown in Figure 6a-h shows that the number of M1 macrophages (CD80+) and T cells (CD4+CD8+) in the T2DM group was significantly higher than that in the Control group, while the number of M2 macrophages (CD206+) was significantly lower. The number of M1 macrophages (CD80+) and T cells (CD4+CD8+) in the JLD-high group, JLD-low group, and MET group was significantly lower than that in the T2DM group, while the number of M2 macrophages (CD206+) was significantly higher, indicating that JLD can improve the inflammatory response in T2DM and thus delay pancreatic aging within a certain concentration range.

[0154] 4Summary

[0155] In this study, three markers were selected to identify pancreatic aging cells: increased lysosomal content (SA-β-Gal), cell cycle arrest (p16), and DNA damage (γ-H2AX and 53BP1). The expression of p16 causes various cell senescence and permanent cycle arrest, while the lysosomal content of senescent cells is related to the activity of senescence-associated β-galactosidase (SA-β-gal), and persistent DNA damage can lead to DNA damage response disorder, thereby promoting inflammation and cell senescence. Both γ-H2AX and 53BP1 contribute to the activation of DNA damage. The results of this study show that, compared with the control group, the positive staining area of β cells in MS and T2DM mice for p16, γ-H2AX, 53BP1, and SA-β-gal was significantly increased, indicating that the metabolic environment of MS and T2DM promotes pancreatic aging, while JLD can inhibit the expression of these aging markers and thus delay pancreatic aging.

[0156] SASP secreted by senescent cells can stimulate the infiltration of T cells such as CD4+ and CD8+, as well as the migration of macrophages to the tissue site where senescent cells are located, releasing more inflammatory mediators and further amplifying the inflammatory response, leading to the persistence of chronic inflammation. Effective control of aging-related inflammation may be one of the ways to intervene in aging and aging-related diseases. The results of this study show that JLD can reverse the decrease in CD206+ anti-inflammatory macrophages and the infiltration of CD4+ / CD8+ / CD80+ pro-inflammatory immune cells induced by MS and T2DM, suggesting that JLD can alleviate cell senescence by improving the inflammatory environment in MS and T2DM mice.

Claims

1. Use of a Chinese medicine composition in the preparation of an anti-aging drug, characterized in that The Chinese medicine composition is composed of the following raw materials in parts by weight: Ginseng 50-150, Polygonatum 60-180, Atractylodes 40-90, Sophora flavescens 30-58, Ophiopogon japonicus 60-180, Rehmannia glutinosa 60-110, Polygonum multiflorum 40-90, Cornus officinalis 60-180, Poria 40-90, Cymbopogon citratus 35-58, Coptis chinensis 35-58, Anemarrhena asphodeloides 35-90, Epimedium 35-58, Salvia miltiorrhiza 40-110, Pueraria lobata 60-180, Litchi seed 80-140, Cortex Lycii 40-90.

2. The use according to claim 1, characterized in that The Chinese medicine composition is used in preparing a drug for delaying pancreatic aging.

3. The use according to claim 2, characterized in that The Chinese medicine composition is used in preparing medicines for improving pancreatic islet tissue structure, pancreatic islet β cell ultrastructure and secretory function.

4. The use according to claim 2, characterized in that The traditional Chinese medicine composition is used in preparing a medicine for reducing the expression of P16, P21, TP53BP1, γ-H2AX and the content of TNF-α, IL-6 and IL-1β in the pancreas.

5. The use according to claim 2, characterized in that The Chinese medicine composition is used in preparing medicines for delaying the aging of pancreatic α cells and pancreatic β cells and reducing the number of aged pancreatic α cells and pancreatic β cells.

6. The use according to claim 2, characterized in that The traditional Chinese medicine composition is used in preparing a medicine for reducing the SA-β-Gal content in the pancreas.

7. The use according to claim 2, characterized in that The Chinese medicine composition is used in preparing a drug for reducing the number of pro-inflammatory immune cells CD80+, CD4+, and CD8+ in the pancreas and / or increasing the number of anti-inflammatory immune cells CD206+ in the pancreas.

8. The use according to any one of claims 1 to 7, characterized in that The Chinese medicine composition is composed of the following raw materials in parts by weight: Ginseng 50, Polygonatum 180, Atractylodes lancea 40, Sophora flavescens 58, Ophiopogon japonicus 60, Rehmannia glutinosa 110, Polygonum multiflorum 40, Cornus officinalis 180, Poria 40, Cymbopogon citratus 35, Coptis chinensis 58, Anemarrhena asphodeloides 90, Epimedium 35, Salvia miltiorrhiza 40, Pueraria lobata 180, Litchi seed 80, Lycium bark 40.

9. The use according to any one of claims 1 to 7, characterized in that: The Chinese medicine composition is composed of the following raw materials in parts by weight: Ginseng 150, Polygonatum 60, Atractylodes 90, Sophora flavescens 30, Ophiopogon 180, Rehmannia glutinosa 60, Polygonum multiflorum 90, Cornus officinalis 60, Poria 90, Cymbopogon citratus 58, Coptis chinensis 35, Anemarrhena asphodeloides 35, Epimedium 58, Salvia miltiorrhiza 110, Pueraria lobata 60, Litchi seed 140, Lycium bark 90.

10. The use according to any one of claims 1 to 7, characterized in that The Chinese medicine composition is composed of the following raw materials in parts by weight: Ginseng 102, Polygonatum 136, Atractylodes 68, Sophora flavescens 56, Poria 83, Ophiopogon 136, Polygonum multiflorum 83, Rehmannia glutinosa 102, Cornus officinalis 136, Coptis chinensis 56, Cymbopogon citratus 56, Litchi seed 136, Epimedium 56, Anemarrhena asphodeloides 68, Salvia miltiorrhiza 89, Pueraria lobata 136, and Lycium bark 83.

11. The use according to any one of claims 1 to 7, characterized in that In the traditional Chinese medicine composition, the atractylodes is stir-fried with bran, the polygonum multiflorum is processed polygonum multiflorum, and the epimedium is roasted epimedium.

12. The use according to any one of claims 1 to 7, characterized in that The preparation of the active ingredient of the Chinese medicine composition consists of the following steps: a. Weigh the Chinese medicinal materials according to the weight ratio of the raw materials, select and break them; b. Add 5-9 times the amount of water to extract the volatile oil from Perilla frutescens and Atractylodes lancea for 3-6 hours. Collect the volatile oil in a separate container and filter the aqueous solution for later use. c. Use 5-9 times the amount of 50-90% ethanol as a solvent for the cornus officinalis, soak for 12-48 hours, perform percolation, collect the percolation liquid, recover the ethanol, and concentrate it into a thick paste with a relative density of 1.30-1.35 measured at 60°C, dry it, and set aside; d. Add 6-10 times the amount of 50-90% ethanol to ginseng, ophiopogon japonicus, epimedium, anemarrhena, and kudzu root, and reflux extract 1-3 times, each time for 1-3 hours. Filter the extract, recover the ethanol, and concentrate it into a thick paste. Dry it and set aside. e, polygonatum, sophora flavescens, rehmannia root, polygonum multiflorum, poria, coptis root, salvia miltiorrhiza, litchi seed, and lychee bark, add 7-11 times the amount of water, decocted 1-2 times, each time for 1-3 hours, the extract was filtered, and combined with the aqueous solution after the perilla and atractylodes oil extraction in step b, concentrated into a clear paste, and ethanol was added to adjust the alcohol concentration to 50-80%, refrigerated, filtered, the filtrate was recovered with ethanol, concentrated to a thick paste, dried, and set aside; The cornus officinalis dry paste obtained in step c, the alcohol-extracted dry paste obtained in step d, the water-extracted and alcohol-precipitated dry paste obtained in step e and the volatile oil obtained in step b together constitute the active ingredients of the traditional Chinese medicine composition.

13. The use according to any one of claims 1 to 7, characterized in that: The preparation dosage form of the traditional Chinese medicine composition is capsule, tablet or granule.

14. The use according to claim 13, characterized in that The preparation method of the Chinese medicine composition granules comprises the following steps: a. Weigh the Chinese medicinal materials according to the weight ratio of the raw materials, select and break them; b. Combine Perilla frutescens and Atractylodes lancea, add 5-9 times the amount of water, and extract the volatile oil using steam method for 3-6 hours. Collect the volatile oil in another container, and filter the aqueous solution for later use. c. Use 5-9 times the amount of 50-90% ethanol as a solvent for the cornus officinalis, soak for 12-48 hours, perform percolation, collect the percolation liquid, recover the ethanol, and concentrate it into a thick paste with a relative density of 1.30-1.35 measured at 60°C, dry it, and set aside; d. Add 6-10 times the amount of 50-90% ethanol to ginseng, ophiopogon japonicus, epimedium, anemarrhena, and kudzu root, and reflux extract 1-3 times, each time for 1-3 hours. Filter the extract, recover the ethanol, and concentrate it into a thick paste. Dry it and set aside. e, polygonatum, sophora flavescens, rehmannia root, processed polygonum multiflorum, tuckahoe, coptis root, salvia miltiorrhiza, litchi seed, and lychee bark, add 7-11 times the amount of water, decocted 1-2 times, each time for 1-3 hours, the extract was filtered, combined with the aqueous solution after the perilla and atractylodes oil extraction in step b, concentrated into a clear paste, added ethanol to adjust the alcohol concentration to 50-80%, refrigerated, filtered, the filtrate was recovered with ethanol, concentrated to a thick paste, dried, and set aside; f. Evenly mix the dried cornus officinalis paste obtained in step c, the alcohol-extracted dried cornus officinalis paste obtained in step d, and the water-extracted and alcohol-precipitated dried cornus officinalis paste obtained in step e, grind them, and add auxiliary materials to granulate them; g. Add ethanol to dissolve the volatile oil obtained in step b, spray it into the granules obtained in step f, mix well, seal, and package.

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