Traditional Chinese medicine composition for resisting aging of stem cells as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention relates to the technical field of traditional Chinese medicines, and discloses a traditional Chinese medicine composition for resisting stem cell aging as well as a preparation method and application thereof. The traditional Chinese medicine composition comprises 30 parts of astragalus membranaceus, 15 parts of fructus psoraleae, 15 parts of codonopsis pilosula, 15 parts of roasted rhizoma atractylodis macrocephalae, 10 parts of eucommia ulmoides, 15 parts of herba epimedii, 15 parts of glossy privet fruit, 15 parts of Chinese yam, 15 parts of radix paeoniae alba, 15 parts of gynostemma pentaphylla, 6 parts of pseudo-ginseng powder, 20 parts of sculellaria barbata, 15 parts of poria cocos, 10 parts of radix bupleuri, 30 parts of raw oyster shell, 15 parts of vinegar turtle shell, 15 parts of rhizoma pinellinae praeparata, 15 parts of eclipta, 6 parts of pericarpium citri reticulatae and 6 parts of liquorice. The preparation method comprises water extraction and concentration to prepare powder. The composition can be used for inhibiting the aging of the mesenchymal stem cells and improving the cell activity, and can be combined with the mesenchymal stem cells to treat degenerative bone diseases including osteoporosis and femoral head necrosis.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and in particular to a traditional Chinese medicine composition for resisting stem cell aging, and a preparation method and application thereof. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] With the development of science and technology and the improvement of people's living standards, human life expectancy has nearly doubled in the past 200 years. However, the healthy life expectancy (healthspan) has only increased by 4.6 years. A longer life means taking on more risks of degenerative chronic diseases related to aging. Among them, degenerative bone diseases include osteoporosis, osteoarthritis and femoral head necrosis. In particular, osteoporosis is a systemic metabolic bone disease characterized by decreased bone mass and damaged normal bone tissue structure, resulting in reduced bone strength and easy fracture. It is one of the main causes of bone pain, fractures, disability and death from fractures in the elderly. The adverse consequences associated with osteoporotic fractures include functional impairment, chronic pain, decreased quality of life, loss of independence and increased mortality, which have brought huge burdens to families and society, caused a large amount of medical insurance expenditures, and have become a major public health issue.

[0004] Another typical bone metabolic disease is femoral head necrosis. Osteonecrosis of the femoral head (ONFH) is a refractory and disabling orthopedic disease that occurs in young and middle-aged people due to various reasons, which causes damage or interruption of blood supply in the femoral head, death of bone cells, and ultimately changes in the structure of the femoral head, or even collapse. Among them, those that occur in young and middle-aged people are mostly non-traumatic femoral head necrosis and hormonal or alcoholic femoral head necrosis. The early pathological changes of SONFH are a decrease in bone marrow mesenchymal stem cells, a decrease in osteogenic differentiation of stem cells, an increase in adipogenic differentiation, an increase in adipose tissue, abnormal proliferation and hypertrophy of fat cells in the femoral head bone marrow, fatty degeneration and necrosis of osteocytes, and a decrease in the number of osteocytes in trabeculae. Osteonecrosis rarely heals on its own. About 67% of asymptomatic patients and 85% of symptomatic patients will have necrotic femoral heads that progress to collapse. Once the femoral head collapses and the hip cartilage degenerates or secondary osteoarthritis occurs, total hip arthroplasty (THA) is usually the last choice. However, the wear between the prostheses after joint replacement, as well as the complications of infection and periprosthetic fractures, limit the use time of joint prostheses. In addition, the age of onset of ONFH is relatively young, and they are the main labor force in the family. If joint replacement is performed too early, multiple revision surgeries will inevitably be faced, which will bring huge economic and psychological pressure to patients and their families, and bring huge social burdens to the country, causing a series of social problems.

[0005] It can be seen that bone metabolic diseases have a common pathological mechanism, namely, the number of bone marrow mesenchymal stem cells decreases, the osteogenic differentiation ability of stem cells is weakened, the adipogenic differentiation increases, the bone mass decreases, and the normal structure of bone tissue is damaged, resulting in reduced bone strength and easy fractures. Although a large number of related drugs have been put into clinical use, there is no reliable drug treatment method so far, and this deteriorating trend has not been alleviated. It is imperative to study new effective treatment methods.

[0006] Stem cell therapy is one of the most active research fields at present. It has been widely used in tissue repair and functional reconstruction, and provides innovative technologies and methods for the treatment of refractory diseases such as femoral head necrosis, osteoporosis, osteoarthritis, nerve damage, neurodegenerative diseases, diabetes, cardiovascular and cerebrovascular diseases, and pulmonary fibrosis. This is another medical revolution after drug therapy and surgical treatment. However, in order to meet the sufficient number of cells required for clinical administration, MSCs need to be expanded and cultured in vitro for a long time, which inevitably drives the cells to replicate "aging" and leads to quality decline. This is one of the problems that need to be solved urgently to restrict the clinical transformation of stem cells.

[0007] At present, there are many traditional Chinese medicine prescriptions for the treatment of bone diseases, most of which are empirical prescriptions that have been used in clinical practice for many years. They have certain clinical efficacy, but the effect is limited, especially without considering the needs of modern mesenchymal stem cell clinical research. Based on the multi-target and safety of traditional Chinese medicine, it is necessary to develop traditional Chinese medicine prescriptions for anti-stem cell aging and their combined clinical application. Summary of the invention

[0008] The purpose of the present invention is to provide a Chinese medicine composition for resisting stem cell aging, a preparation method and an application thereof, in response to the problem of stem cell aging caused by the current continuous subculture. The present invention utilizes the multi-target and safety characteristics of Chinese medicine, and improves the original formula through improvement, so that it can inhibit the aging of mesenchymal stem cells cultured in vitro for a long time, inhibit the chronic inflammatory microenvironment of aging, and improve the effect of mesenchymal stem cells in treating degenerative bone diseases including osteoporosis and femoral head necrosis.

[0009] The technical solution of the present invention is as follows:

[0010] A Chinese medicine composition for resisting stem cell aging comprises the following raw materials in parts by weight: 25-35 parts of astragalus, 10-20 parts of psoralea corylifolia, 10-20 parts of codonopsis pilosula, 10-20 parts of stir-fried atractylodes, 5-15 parts of eucommia, 10-20 parts of epimedium, 10-20 parts of glossy privet, 10-20 parts of yam, 10-20 parts of white peony root, 10-20 parts of gynostemma pentaphyllum, 3-9 parts of notoginseng powder, 15-25 parts of barbata scutellaria, 10-20 parts of tuckahoe, 5-15 parts of bupleurum, 25-35 parts of raw oyster, 10-20 parts of vinegar turtle shell, 10-20 parts of pinellia ternata, 10-20 parts of eclipta prostrata, 3-9 parts of tangerine peel, and 3-9 parts of liquorice.

[0011] According to a preferred embodiment, the raw materials are composed of the following parts by weight: 27-33 parts of astragalus, 12-17 parts of psoralea corylifolia, 12-17 parts of codonopsis pilosula, 12-17 parts of stir-fried atractylodes, 7-13 parts of eucommia, 12-17 parts of epimedium, 12-17 parts of glossy privet, 12-17 parts of yam, 12-17 parts of white peony root, 12-17 parts of gynostemma pentaphyllum, 5-7 parts of notoginseng powder, 17-22 parts of barbata, 12-17 parts of poria, 7-13 parts of bupleurum, 27-32 parts of raw oyster, 12-17 parts of vinegar turtle shell, 12-17 parts of French pinellia, 12-17 parts of Ecliptae, 5-7 parts of tangerine peel, and 5-7 parts of licorice.

[0012] According to a preferred embodiment, the raw materials are composed of the following parts by weight: 30 parts of astragalus, 15 parts of psoralea, 15 parts of codonopsis, 15 parts of stir-fried atractylodes, 10 parts of eucommia, 15 parts of epimedium, 15 parts of glossy privet, 15 parts of yam, 15 parts of white peony, 15 parts of gynostemma pentaphyllum, 6 parts of notoginseng powder, 20 parts of barbata, 15 parts of poria, 10 parts of bupleurum, 30 parts of raw oyster, 15 parts of vinegar turtle shell, 15 parts of French pinellia, 15 parts of Ecliptae, 6 parts of tangerine peel, and 6 parts of licorice.

[0013] This prescription is a combination of the classic prescription Chaishao Liujunzi Decoction and Erzhi Pills, combined with the experience of famous old Chinese medicine practitioners in China. Astragalus is the main ingredient in the prescription, aiming to replenish qi and raise yang, strengthen the body and consolidate the exterior, produce body fluid and nourish blood; Codonopsis, Atractylodes, Eucommia, Ligustrum lucidum, Psoralea corylifolia, Epimedium, Chinese yam, and white peony root are the assistant ingredients. Among them, Codonopsis pilosula replenishes qi and enhances the effect of Astragalus in replenishing qi; Atractylodes and Chinese yam strengthen the spleen and replenish qi, help digestion and stimulate appetite, and replenish the source of acquired metabolism; Eucommia, Ligustrum lucidum, Psoralea corylifolia, and Epimedium nourish the liver and kidney, promote yang and promote qi circulation; White peony root nourishes yin, softens the liver and relieves pain; Gynostemma pentaphyllum, Scutellaria barbata, Panax notoginseng powder, Raw oyster, vinegar turtle shell, bupleurum, poria, pinellia, and eclipta are adjuvants. Among them, Gynostemma pentaphyllum and Scutellaria barbata clear away heat and detoxify, Panax notoginseng powder promotes blood circulation and removes blood stasis, stops bleeding and reduces swelling, raw oyster and vinegar turtle shell nourish yin and suppress yang, soften hard masses and disperse nodules, bupleurum soothes the liver and relieves depression, reconciles the exterior and interior, poria and pinellia dry dampness and promote diuresis, and eliminate edema; tangerine peel and licorice are guiding drugs, which have the effects of regulating qi and strengthening the spleen, and alleviating the effects of various drugs. All the drugs are used together to achieve the effects of nourishing yin, strengthening the body, clearing toxins, strengthening the spleen, and nourishing the liver and kidneys.

[0014] Astragalus, with a sweet taste and slightly warm nature, enters the lung and spleen meridians and is an important medicine for replenishing qi. Modern pharmacological research results show that Astragalus mainly contains polysaccharides, flavonoids, saponins and other ingredients, which have the effects of regulating immunity, anti-inflammatory and antioxidant. Experiments have shown that astragaloside IV (astragaloside I) can regulate the PI3K / Akt pathway to prevent hormone-induced ONFH, astragaloside II can promote MC3T3-E1 cell proliferation and differentiation by regulating the Wnt / β-catenin signaling pathway, and astragaloside polysaccharides can regulate the SP1 / MEK / ERK axis to promote osteoblast activity and inhibit osteoblast apoptosis in the SONFH model.

[0015] Psoralea corylifolia tastes bitter and pungent, is warm in nature, and enters the kidney and spleen meridians. Modern pharmacological research results show that the main active ingredients of Psoralea corylifolia are coumarins, flavonoids and monoterpenoid phenol compounds, which have the effects of regulating bone metabolism and anti-inflammatory. Experimental research results show that the active ingredients contained in Psoralea corylifolia, such as psoralen, isopsoralen, and psoralen isoflavones, can play a role in promoting osteoblast proliferation and differentiation and inhibiting osteoclast proliferation by affecting bone metabolism-related pathways.

[0016] Codonopsis pilosula is sweet and mild in nature, and enters the spleen and lung meridians. Modern pharmacological research results show that Codonopsis pilosula mainly contains flavonoids, alkaloids, sugars and other substances, which have anti-inflammatory, antioxidant, and immune-regulating effects. Based on the diagnosis and treatment idea of ​​"the same disease of phlegm and blood stasis, treatment from the spleen", Professor Chen Weiheng formulated a prescription for strengthening the spleen and activating bones to treat ONFH with the method of "strengthening the spleen and resolving phlegm, activating blood circulation and unblocking collaterals", and Codonopsis pilosula is the main medicine of this prescription. Sun Shiyi further explained the mechanism of this prescription for treating ONFH through network pharmacology research, that is, the compounds such as quercetin, luteolin, and kaempferol contained in Codonopsis pilosula can reduce inflammation, improve microcirculation, and regulate target factors such as interleukin-6 (IL-6) and tumor necrosis factor (TNF) to achieve the effect of treating ONFH.

[0017] Atractylodes macrocephala tastes sweet and bitter, is warm in nature, and enters the spleen and stomach meridians. Modern pharmacological research results show that Atractylodes macrocephala mainly contains sesquiterpenes, flavonoids, polysaccharides, polyacetylene, phenolic acids, glycosides and other ingredients, which have anti-inflammatory and immune-regulating effects. Research results show that Atractylodes macrocephala can reduce the levels of IL-1β and TNF-α in macrophages, activate the AMPK / SIRT1 signaling pathway, and inhibit the activation of nuclear factor-κB (NF-κB) and the polarization of macrophages to a pro-inflammatory phenotype.

[0018] Eucommia ulmoides is bitter in taste and warm in nature. It enters the liver and kidney meridians. Modern pharmacological research results show that the main components of Eucommia ulmoides are phenylpropanoids, iridoids, flavonoids, and phenolic acids, which have anti-inflammatory, antioxidant, anti-apoptotic and bone protective effects. The results of the study showed that the use of whole Eucommia capsules to intervene in ONFH beagle dogs can improve the changes in canine ONFH tissues, increase bone density, increase the content of VEGF and bFGF in serum, increase the expression of Bcl-2, ColⅠ, and RUNX2 in bone tissue, and inhibit the expression of Bax and Caspase-3. At the same time, research results also show that Eucommia ulmoides alcohol extract can activate the RhoA / ROCK signaling pathway and promote the osteogenic differentiation of BMMSC in osteoporotic rats. Some experts have used acidic polysaccharides extracted from Eucommia ulmoides to reduce the number of osteoclasts on the cortical bone surface of dexamethasone-induced osteoporotic rats, increase the number of osteoblasts, cortical bone thickness and mineralized bone area, and have an anti-osteoporotic effect.

[0019] Epimedium, also known as Xianlingpi, is pungent and sweet in taste, warm in nature, and enters the liver and kidney meridians. It is light and strong in energy, and can rise or fall. It has the effects of tonifying the kidney and strengthening yang, dispelling wind and dampness, and strengthening muscles and bones. Epimedium activates the HIF-1α / VEGF / VEGFR-2 signaling pathway, which can reduce the inflammatory response of osteoporotic rats, regulate bone metabolism balance, and improve tibial bone microstructure. The main ingredient, icariin, has shown significant pharmacological effects in multiple biomedical fields, including anti-tumor, anti-inflammatory, immunomodulatory, anti-liver fibrosis, and anti-osteoporosis. Icariin can reduce vascular damage and protect the integrity of intraosseous blood vessels by increasing the expression of angiogenic factors and inhibiting inflammatory factors. At the same time, icariin can reduce osteocyte apoptosis, inhibit bone marrow adipocyte proliferation, promote local bone repair, and delay disease progression. Icariin can also inhibit adipose differentiation by promoting the expression of osteogenesis-related genes, and play its role in treating femoral head necrosis through epigenetic mechanisms. In addition, icariin can reverse the adverse effects caused by hypoxia and protect osteoblast function by activating related signaling pathways.

[0020] Fructus Ligustri Lucidi is sweet and bitter in taste, cool in nature, and enters the liver and kidney meridians. Fructus Ligustri Lucidi contains a variety of chemical components such as polysaccharides, triterpenes, iridoids, phenylethanoid glycosides and flavonoids. Studies have found that Fructus Ligustri Lucidi polysaccharides inhibit bone resorption and promote bone formation by inhibiting the proliferation and differentiation of bone marrow-derived monocytes into osteoclasts induced by RANKL and reducing the activity of tartrate resistant acidphosphatase (TRAP). The special ligustrin can inhibit the formation of osteoclasts induced by receptor activator of nuclear factor-κB ligand (RANKL) and thus inhibit diabetes-induced bone loss.

[0021] Chinese yam is sweet and neutral in nature. It enters the lung, spleen and kidney meridians. The main chemical components of Chinese yam include starch, protein, amino acids, saponins, mucopolysaccharides, etc. Among them, Chinese yam polysaccharides can effectively relieve inflammatory responses. Research results show that Chinese yam polysaccharides can significantly reduce the expression of inflammatory factors IL-6, IL-18 and TNF-α, and can effectively inhibit the expression of matrix metalloproteinase-13 (MMP-13) in articular cartilage, inhibit the degradation of articular cartilage collagen, and promote the synthesis of TGF-β1 and Col II. Other research results show that the compound 6,7-dihydroxy-2,4-dimethoxyphenanthrene in Chinese yam can reduce the content of TNF-α, interferon-γ (IFN-γ), IL-10, and IL-23, inhibit the NF-κB / COX-2 signaling pathway, and can also inhibit the MAPK / NF-κB signaling pathway to exert anti-inflammatory effects.

[0022] White peony root is bitter and sour, slightly cold in nature, and enters the liver and spleen meridians. White peony root contains volatile oils, monoterpenes, triterpenes and flavonoids, etc., which have antispasmodic, analgesic, anti-inflammatory, immunomodulatory, vasodilation, and antiplatelet aggregation effects. White peony total glucosides can effectively reduce the levels of TNF-α, IL-1α, and IL-6 in the serum of rats with collagen-induced arthritis (CIA) and the mRNA and protein expressions of RANKL and TRAP in the synovium, and increase the mRNA and protein expressions of osteoprotegerin (OPG) and p-AMPK in the synovium. Other research results show that white peony total glucosides have the ability to promote the proliferation, differentiation and maturation of MC3T3-E1 cells.

[0023] Gynostemma pentaphyllum tastes slightly sweet and bitter, is cool in nature, and enters the lung, spleen, and kidney meridians. Gynostemma pentaphyllum contains saponins, polysaccharides, flavonoids, amino acids, vitamins, and other ingredients. Gynostemma pentaphyllum saponins are the components with a higher content in Gynostemma pentaphyllum. They can promote the expression of molecular chaperones by regulating the Wnt / β-catenin signaling pathway in osteoblasts, regulate endoplasmic reticulum stress, and increase the rate of correct protein folding in the endoplasmic reticulum, thereby promoting the proliferation and osteogenic differentiation of osteoblasts. Gynostemma pentaphyllum saponins can inhibit the expression of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) protein and activate the BMP / Smad pathway, thereby having a certain effect on H2O2-induced oxidative stress damage to osteoblasts, and promoting the proliferation and differentiation of damaged osteoblasts.

[0024] Panax notoginseng tastes sweet and slightly bitter, is warm in nature, and enters the liver and stomach meridians. Panax notoginseng mainly contains triterpenoid saponins, flavonoids, amino acids, polysaccharides, volatile oils and other active ingredients, which have good effects in promoting blood circulation, stopping bleeding, replenishing blood, anti-inflammatory, anti-tumor, and neuroprotection. Its main component, Panax notoginseng total saponins, can treat rabbit SONFH and has a repairing effect on the morphological structure of bone cells. Panax notoginseng saponins can regulate the Wnt / β-catenin signaling pathway, promote the bone density of the femur and spine of SONFH rats, improve the level of femoral biomechanical parameters, increase the percentage of cortical bone area, the average thickness of trabeculae, the area of ​​trabeculae, and the volume of trabeculae, increase the level of estradiol in serum, and reduce the levels of negative regulators of bone formation such as TRAP in serum; significantly increase the mRNA and protein expression levels of Wnt5a and c-Myc. There are also literatures summarizing and analyzing the relevant signal pathways of SONFH regulated by the effective ingredients of Panax notoginseng. It mainly regulates TGF-β / BMP, Wnt / β-catenin, OPG / RANKL / RANK, HIF-1α and other signal pathways, and plays the roles of promoting bone absorption, inhibiting osteoclast absorption, promoting lipid metabolism, and promoting angiogenesis.

[0025] Scutellaria barbata tastes pungent and bitter, is cold in nature, and enters the lung, liver, and kidney meridians. Scutellaria barbata mainly contains a variety of chemical components such as flavonoids, terpenes, volatile oils, polysaccharides, etc. Among them, flavonoids and diterpenes and their lactone components are the main active ingredients, which have anti-tumor, anti-inflammatory, antioxidant, and immune regulation effects. Experimental results show that hydroxyacetophenone, baicalin, luteolin, apigenin and other components were identified in the ethyl acetate component of the drug pair of Scutellaria barbata and Hedyotis diffusa. They inhibited the expression of TRAP and nuclear factor of activated Tcells cytoplasmic 1 (NFATc1) genes in osteoclasts formed by RAW264.7 cells induced by RANKL, promoted the expression of miR-155, and simultaneously inhibited the expression of NFATc1, tumor necrosis factor receptor-associated factor 6 (TNF receptor-associated factor 6, TRAF6), receptor activator of nuclear factor-κB (receptor activator of nuclear factor-κB, RANK), and protein cathepsin-K (cath-K).

[0026] Poria cocos has a sweet and mild taste and is neutral in nature. It enters the heart, lung, spleen, and kidney meridians. The main chemical components of Poria cocos include polysaccharides, triterpenes, steroids, volatile oils, choline, amino acids, etc.157. In the study of the drug pair of Atractylodes macrocephala and Poria cocos, it was found that it has a regulatory effect on multiple target signaling pathways that affect bone metabolism, such as NF-κB and PI3K / Akt.

[0027] Bupleurum, bitter and pungent in taste, slightly cold in nature, enters the liver, gallbladder, and lung meridians. The main components of Bupleurum include saikosaponin, bupleurum polysaccharide, volatile oil, kaempferol, quercetin, etc. Research results show that saikosaponin A can effectively inhibit the expression of factors such as IL-1β, MMP-1, MMP-3, MMP-13, prostaglandin E2 (PGE2), and NF-κB to exert anti-inflammatory effects.

[0028] Raw oysters are the dried shells of oysters of the family Ostrea. They are salty in taste, slightly cold in nature, and enter the liver, gallbladder, and kidney meridians. Oysters have antioxidant, anti-liver damage, sedative, anticonvulsant, anti-epileptic, analgesic, immune-enhancing, anti-tumor, antioxidant, anti-aging, and anti-gastric ulcer effects. Oysters are clinically used in traditional Chinese medicine prescriptions for the treatment of a variety of orthopedic-related diseases. However, current research on oysters is mostly focused on oyster soft bodies or extracts from whole oysters, while oyster shells are mostly used as medical biological calcium raw materials and chemical raw materials. Its related pharmacological research needs to be further deepened.

[0029] Vinegar turtle shell, salty in taste, slightly cold in nature, enters the liver and kidney meridians. Turtle shell mainly contains turtle shell polysaccharide, animal glue, keratin, vitamin D, calcium phosphate, calcium carbonate, amino acids, and a variety of trace elements, which have the effects of anti-liver fibrosis, anti-pulmonary fibrosis, anti-tumor and immune regulation. Experimental results show that turtle shell ultrafine powder has the function of increasing bone density, and is superior to calcium carbonate in terms of calcium apparent absorption rate and increasing femoral bone density and femoral bone calcium content. Other experimental results show that Rehmannia glutinosa-turtle shell-containing serum can promote ALP secretion, upregulate RUX2 mRNA expression, promote osteoblast proliferation, and Rehmannia glutinosa-turtle shell synergistically upregulates osteocalcin gene and protein expression in osteoblasts with mesangial cells.

[0030] Eclipta prostrata L. is the dried aerial part of the plant Channa prostrata of the Asteraceae family. It is an annual herb that is picked when the flowers bloom. It is sweet, sour, cold, and enters the kidney and liver meridians. The whole plant is used as medicine and is included in the first volume of the 2020 edition of the Pharmacopoeia of the People's Republic of China. It has the effects of nourishing yin and tonifying the liver, cooling blood and stopping bleeding. When the stems and leaves are broken, blue-black juice flows out, so it is named Eclipta prostrata, also known as Channa prostrata, Mocao, Jinlingcao, Motoucao, etc. It is called the natural "black hair grass". "Compendium of Materia Medica" records: "The fine fruit is quite like a lotus pod, so it is named Lian, which can blacken the hair and benefit the kidney yin." In recent years, a large number of studies have been carried out on the chemical composition and pharmacological effects of Eclipta prostrata, confirming that Eclipta prostrata contains chemical components such as triterpenes, steroids, flavonoids, coumarins, thiophenes, phenolic acids, alkaloids, etc. The plant and its extracts have pharmacological effects such as liver protection, anti-osteoporosis, anti-inflammatory, anti-tumor, antioxidant, hypoglycemic, hypolipidemic, hemostatic, and hair blackening.

[0031] Tangerine peel, bitter and pungent in taste, warm in nature, belongs to the lung and spleen meridians, and has the effects of regulating qi and strengthening the spleen, drying dampness and resolving phlegm. It mainly includes terpenes, alcohols, ketones, esters, aldehydes, ethers, acids, etc., which have anti-inflammatory, bone protection, and anti-obesity effects. Existing research results show that tangerine peel extract can reduce the increase of inflammatory cytokines, monocyte chemotactic protein-1 (MCP-1), 1L-1β, and creatine kinase (CK) caused by skeletal muscle injury, thereby playing a role in protecting bones. Other research results show that nobiletin can directly activate retinoic acid receptor-related orphan receptors α (RORα), and downregulate SOST levels and upregulate OPG levels in a RORα-dependent manner, thereby regulating osteoblast function and improving related bone loss caused by natural aging.

[0032] Another aspect of the present invention provides a method for preparing a Chinese medicine composition for resisting stem cell aging, comprising the following steps:

[0033] (1) First, grind each medicinal Chinese medicine in the above formula into coarse powder by a pulverizer;

[0034] (2) boiling the crude powder of Chinese medicine in water to extract the effective ingredients of the medicine;

[0035] (3) using a Chinese medicine concentration device to concentrate the decocted Chinese medicine decoction into an extract;

[0036] (4) Pour the extract into a drying tray and place it in a vacuum drying oven at 60°C for drying;

[0037] (5) crushing the dried extract and passing it through a 120-mesh sieve to form a powder;

[0038] (6) Weigh the prepared powder of the Chinese medicinal composition for resisting stem cell aging and prepare it into capsules, or pack it into sterile bottles for later use.

[0039] Another aspect of the present invention provides use of the aforementioned traditional Chinese medicine composition for resisting stem cell aging in combination with mesenchymal stem cells in the treatment of degenerative bone diseases.

[0040] According to a preferred embodiment, the degenerative bone disease includes osteoporosis, osteoarthritis or femoral head necrosis.

[0041] Another aspect of the present invention provides use of the aforementioned anti-stem cell aging Chinese medicinal composition in inhibiting the aging of mesenchymal stem cells cultured in vitro.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. A Chinese medicine composition for resisting stem cell aging. This formula has the pharmacological effects of promoting osteogenesis, inhibiting bone resorption, anti-inflammatory and anti-apoptotic. It can protect the activity of mesenchymal stem cells cultured in vitro for a long time and inhibit their aging by inhibiting the expression of inflammatory factors. It can promote the expression of osteogenesis-related factors, inhibit the expression of osteoclast-related factors, enhance bone metabolism, and can significantly improve the therapeutic effect of bone diseases such as osteoporosis, osteoarthritis, and femoral head necrosis by combining mesenchymal stem cell therapy.

[0044] 2. A Chinese medicine composition for resisting stem cell aging, which can be directly added to the culture medium of mesenchymal stem cells to inhibit the apoptosis of mesenchymal stem cells and enhance the activity of mesenchymal stem cells;

[0045] 3. A Chinese medicine composition for resisting stem cell aging, which can be taken orally in combination with mesenchymal stem cells to treat osteoporosis, osteoarthritis, femoral head necrosis and other bone diseases, inhibit the aging of mesenchymal stem cells and improve the treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 hucMSCs extracted, purified and cultured in Example 2;

[0047] Figure 2 The effect of the "Ziyin Fuzheng Qingdu Recipe" in Example 2 on inhibiting the apoptosis of hucMSCs;

[0048] Figure 3 The figure is a scanning result of bone tissue specimens of rats in different treatment groups in Example 3;

[0049] Figure 4 The results of HE staining of femoral head tissue sections of rats in different treatment groups in Example 3;

[0050] Figure 5 These are the immunohistochemical images of the expression of OPG (A) and RANKL (B) in the femoral head tissue of rats in different treatment groups in Example 3. DETAILED DESCRIPTION

[0051] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0053] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0054] Example 1 Preparation of a Chinese medicine composition for resisting stem cell aging

[0055] Take the following raw materials in parts by weight: 30g of Astragalus, 15g of Psoralea, 15g of Codonopsis, 15g of stir-fried Atractylodes, 10g of Eucommia, 15g of Epimedium, 15g of Ligustrum lucidum, 15g of Dioscorea batatas, 15g of White Peony, 15g of Gynostemma pentaphyllum, 6g of Panax notoginseng powder, 20g of Scutellaria barbata, 15g of Poria, 10g of Bupleurum, 30g of raw oyster, 15g of Vinegar-soaked Turtle Shell, 15g of Pinellia ternata, 15g of Eclipta prostrata, 6g of Citrus reticulata, and 6g of Licorice.

[0056] The "Ziyin Fuzheng Qingdu Prescription" powder of the present application is prepared according to the following steps:

[0057] (1) First, grind each medicinal Chinese medicine in the above formula into coarse powder by a pulverizer;

[0058] (2) boiling the crude powder of Chinese medicine in water to extract the effective ingredients of the medicine;

[0059] (3) using a Chinese medicine concentration device to concentrate the decocted Chinese medicine decoction into an extract;

[0060] (4) Pour the extract into a drying tray and place it in a vacuum drying oven at 60°C for drying;

[0061] (5) crushing the dried extract and passing it through a 120-mesh sieve to form a powder;

[0062] (6) Obtain the powder of "Ziyin Fuzheng Qingdu Prescription".

[0063] Example 2 Functional identification of a Chinese medicine composition for resisting stem cell aging

[0064] 1. Extraction and identification of human umbilical cord mesenchymal stem cells

[0065] The umbilical cord used was provided by the obstetrics department of the hospital. Approved by the ethics committee, the parturient or her family members gave informed consent and signed the informed consent form voluntarily. The parturient's prenatal examination results for hepatitis B virus, hepatitis C virus, syphilis spirochete, and human immunodeficiency virus were all negative. The umbilical cords left over from the placenta of healthy full-term newborns were collected under sterile conditions. HucMSCs were extracted, purified, and cultured in strict accordance with the aseptic operation specifications. The results are as follows Figure 1 As shown. Flow cytometry immunophenotyping, CD105 + : 99.97%, CD73 + : 99.91%, CD90 + :99.94%;CD45 + : 0.08%,CD34 + :0.11%, CD14: 0.17%, HLA-DR: 0.04%.

[0066] 2. Cell Grouping

[0067] In order to verify the anti-aging effect of "Ziyin Fuzheng Qingdu Recipe", the above hucMSCs were divided into 2 groups, namely the blank control group: no additional intervention, normal culture. Experimental group: 5g of "Ziyin Fuzheng Qingdu Recipe" powder was dissolved in 500ml of mesenchymal stem cell culture medium for intervention. All groups were cultured in a cell culture room under GMP conditions, 33.5℃, 5% CO2 fully humidified incubator.

[0068] 3. Anti-apoptosis detection

[0069] For the above two groups of cells, change the culture medium every 2-3 days. After the cell confluence reaches 90-95%, collect them into a 15mL centrifuge tube. Centrifuge at 1200rpm for 5min to precipitate the cells. Add about 1mL of PBS pre-cooled at 4℃, mix well and resuspend the cells. Use the diluted 1×Binding Buffer to adjust the cell concentration to 1-5×10 6 cells / mL, resuspend and mix thoroughly. Pipette about 100μL of cell suspension into a flow cytometry centrifuge tube, then add 5μL Annexin V / FITC to mix the cell sample and staining solution evenly, and incubate at room temperature in the dark for 5 minutes; immediately after the incubation, add 5μL of propidium iodide solution (PI), and add 400μL PBS together to mix thoroughly, perform flow cytometry detection immediately, and then use software to analyze cell apoptosis.

[0070] The results are as follows Figure 2 As shown: the apoptosis rate of hucMSCs in the experimental group was significantly lower than that in the blank control group. There was a statistically significant difference (**P<0.001), which shows that the "Yin-nourishing and Poison-clearing Recipe" of the present application can inhibit the apoptosis of hucMSCs and is beneficial to protecting the activity of hucMSCs.

[0071] Example 3 Gene-modified mesenchymal stem cells for the treatment of femoral head necrosis model animals

[0072] 1. Experimental Animals

[0073] 75 8-week-old SPF male SD rats were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. The experimental animal production license number is SCXK (Xiang) 2019-0004. The animals were raised in the SPF animal laboratory of Guangxi Traditional Chinese Medicine Science Experiment Center according to standard feeding conditions, with a temperature of 22-26°C. Humidity is 37%-42%, and the light is alternating day and night for 12 hours. Free access to food and water, 2-3 per cage. The use license number of the experimental site is SYXK (Gui) 2019-0001, and all animal treatments in the experiment are carried out in accordance with the "Regulations on the Management of Experimental Animals". All the experimental procedures in this part have been reviewed and approved by the Experimental Animal Welfare Ethics Committee of Guangxi University of Chinese Medicine, and the ethics approval number is DW20220408-053.

[0074] 2. Animal Modeling

[0075] After the rats arrived at the SPF animal laboratory of Guangxi Traditional Chinese Medicine Science Experimental Center, they were raised according to standard feeding conditions, and the model was started after 10 days of adaptation. 11 rats were randomly selected by random number table to enter the blank group, and the remaining 74 rats were entered into the modeling group. The weight was accurately weighed and recorded before modeling. The modeling took a total of 7 days, and the procedure was as follows: On the 1st and 2nd days, the rats in the modeling group were intraperitoneally injected with lipopolysaccharide (LPS, 20μg / kg), once a day, each time with an interval of 24h; methylprednisolone sodium succinate (MPS, 40mg / kg) was injected 24h apart, that is, on the 3rd to 7th days, injected into the posterior lateral thigh muscles of the rats, once a day, each time with an interval of 24h. The blank group rats were injected with normal saline at the same time point. After modeling, they were raised normally and had free access to water.

[0076] 3. Modeling Verification

[0077] After the modeling, the rats were fed normally and had free access to water. After the modeling, the rats' diet, drinking water, excretion, mental state, and activity were observed, and they were weighed once a week and recorded. Four weeks after the modeling, one rat was randomly selected from the blank group and four rats were randomly selected from the modeling group by the random number table method. After being killed, bilateral femoral head tissues were obtained for HE staining. Under the microscope, the formation of empty bone pits, increased number of nuclear condensation, bone marrow necrosis, and trabecular fracture were observed. Any of the following conditions indicated that bone necrosis occurred and the modeling was successful.

[0078] 4. Animal Grouping

[0079] The modeling rats were randomly divided into 4 groups according to the random number table. Control group (blank control group): After the modeling was successfully established, the rats were raised normally without additional intervention. MSC group: The concentration of 2.5×10 7 cells / ml 5th generation hUCMSC suspension, injection volume was 2×10 7 cells / kg body weight, once a week for 4 times. Group Z: 2g of “Ziyin Fuzheng Qingdu Fang” powder was dissolved in 5ml of water and intragastrically administered once a day for 4 weeks. Group Z+MSC: Tail vein injection concentration was 2.5×10 7 cells / ml 5th generation hUCMSC suspension, injection volume was 2×10 7 / kg body weight, once a week, for a total of 4 times. At the same time, 2g of "Ziyin Fuzheng Qingdu Fang" powder was dissolved in 5ml of water and intragastrically administered once a day for a total of 4 weeks.

[0080] 5. Observation of the general condition of rats

[0081] After the rats began modeling intervention, their diet, drinking water, excretion, body temperature, mental state, activity status, etc. were observed, and their body weight was recorded once a week.

[0082] The results showed that hUCMSC combined with "Ziyin Fuzheng Qingdu Recipe" improved the effects of lipopolysaccharide and methylprednisolone sodium succinate on the general condition of rats. Most of the rats in the modeling group were listless, had reduced activity, decreased appetite, rough and dull hair, and lost weight during the medication modeling period. A small number of rats had loose and unformed stools. The above conditions were alleviated after the modeling was completed. After the intervention with Chinese medicine or / and hUCMSC, the general condition of the rats in each group was better than that in the model group, with no adverse symptoms such as fever, cough, allergy, and skin redness and swelling.

[0083] 6. Micro-CT detection of bone tissue related parameters

[0084] The left bone tissue specimens were fixed and preserved with 4% paraformaldehyde solution, and then grouped and marked. After fixation, they were placed in the scanning container of Micro-CT. After calibrating the instrument and container, the scanning parameters were set and the designated area was scanned. Scanning location and instrument: Micro-CT was completed at Hangzhou Yuebo Biotechnology Co., Ltd., using Micro-CT produced by SCANCO Medical AG.

[0085] The results are as follows Figure 3 As shown: Most of the femoral head cartilage in the control group disappeared after necrosis, but the femoral head cartilage in the MSC group did not disappear. Compared with the control group, the femoral head cartilage density of rats in the MSC group, Z group and Z+MSC group was significantly improved, and the bone volume (BV), bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), bone mineral content (BMC) and bone mineral density (BMD) at the femoral neck under the femoral head were significantly improved, and the Z+MSC group was better than the MSC group, with statistically significant differences among the groups (P<0.05).

[0086] 7. HE staining to observe the morphological changes of bone tissue

[0087] The femoral head tissues of rats in each group were sliced ​​and stained with HE. The specific process was as follows:

[0088] (1) Decalcification, paraffin embedding and sectioning

[0089] ① When the rats were killed, 6 left femoral head tissues were randomly selected from each group, marked and immediately fixed in 4% paraformaldehyde solution;

[0090] ② After 72 hours, take out the specimens, rinse with running water for 15 minutes, and then use sterile gauze to absorb the surface moisture of the specimens. Put the specimens into a beaker filled with 10% EDTA decalcification solution, and then put the beaker into an ultrasonic cleaner for ultrasonic decalcification (ultrasonic frequency 40KHZ). Decalcify for 5 minutes, let it stand for 2 minutes, and change the decalcification solution every 30 minutes until the ultrasonic treatment lasts for 90 minutes.

[0091] ③After the ultrasonic decalcification operation is completed, the decalcification solution is replaced every 12 hours. The decalcification of the specimen is checked each time the decalcification solution is replaced. When the 1ml syringe needle can easily penetrate the bone cortex, the specimen is completely decalcified. After complete decalcification, the tissue is removed, and the target tissue is further trimmed with a surgical blade in the fume hood, and the trimmed tissue and the corresponding label are placed in the dehydration box;

[0092] ④ Place the dehydration box in the hanging basket in the dehydrator, and dehydrate the tissue with gradient ethanol, in order: 75% ethanol for 4 hours, 85% ethanol for 2 hours, 90% ethanol for 1.5 hours, 95% ethanol for 1 hour, anhydrous ethanol I for 0.5 hours, and anhydrous ethanol II for 0.5 hours;

[0093] ⑤ After the tissue block is dehydrated with ethanol, it must be transparent. The transparent agent xylene can be mixed with the dehydrating agent and paraffin at the same time, so that paraffin can better penetrate the tissue. It is treated with anhydrous ethanol: xylene (1:1) for 10 minutes, xylene I for 10 minutes, and xylene II for 10 minutes in sequence;

[0094] ⑥ After the tissue blocks have been made transparent, they are immersed in 3 cylinders of paraffin wax (60°C) in turn: paraffin wax I (60°C) for 1 hour, paraffin wax II (60°C) for 1 hour, and paraffin wax III (60°C) for 1 hour. Embed the tissues that have been immersed in paraffin in an embedding machine. First, put the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Cool in a -20°C refrigerator. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block;

[0095] ⑦ Use a pathology slicer to slice the embedded wax block, with a slice thickness of 4μm. Put the cut tissue slices into a 40℃ water bath for spreading. Insert the anti-shedding slide obliquely into the water surface to pick up the slices, so that the slices are attached to the appropriate position on the slide, and bake in a 60℃ oven for 3h.

[0096] (2) Dyeing

[0097] ① Dewaxing the sections: the paraffin sections prepared in step (1) were sequentially placed in xylene I for 20 min, xylene II for 20 min, xylene III for 20 min, anhydrous ethanol I for 5 min, aqueous ethanol II for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and then rinsed in distilled water for 5 min;

[0098] ② Soak the dewaxed sections in Mayer's hematoxylin stain for 5 minutes, and rinse with tap water;

[0099] ③Differentiate with 1% hydrochloric acid ethanol solution for 2-3 seconds, and rinse with tap water;

[0100] ④0.6% ammonia water turns blue, rinse with tap water;

[0101] ⑤ Stain the sections with 1% water-soluble eosin solution for 5 minutes, and then wash with tap water for 30 seconds;

[0102] ⑥ Soak in 95% ethanol I for 30 seconds, 95% ethanol II for 1 minute, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes;

[0103] ⑦ After dehydration, the slices were dried in a ventilated place, sealed with neutral gum, and the tissue morphology was observed under an ordinary optical microscope.

[0104] The results are as follows Figure 4 The results showed that the cartilage surface of the control group was defective, with hemorrhagic necrosis, more inflammatory cell infiltration, granulation tissue formation, sparse arrangement of trabeculae, significantly enlarged gaps, more trabeculae fractures and absence, increased fat droplets in the bone marrow, more empty bone pits, and more nuclear pyknosis. The cartilage surface of the MSC, Z and Z+MSC groups was significantly improved compared with the control group, and the Z+MSC group was better than the MSC group, with statistically significant differences (P<0.05).

[0105] 8. Immunohistochemical staining to detect the expression of related factors

[0106] (1) Immunohistochemical staining

[0107] The tissue sections prepared in step (1) of section 7 were subjected to immunohistochemical staining to detect the expression of OPG and RANKL. The specific steps are as follows:

[0108] ① Place the paraffin sections in a 72°C oven for 25 min, dewax with xylene, hydrate with gradient ethanol, and rinse twice with distilled water;

[0109] ② Antigen repair: Take 30 ml of antigen repair solution EDTA (pH = 8.0), dilute it with distilled water at a ratio of 1:50, put it in a pressure cooker and heat it to boiling, place the dewaxed and hydrated tissue sections in the boiling repair solution, ensure that the repair solution fully soaks the slides, boil at low power (maintain the temperature at about 98°C) for 25 minutes, turn off the heat and cool to room temperature, remove the slides from the buffer solution, and rinse them twice with distilled water;

[0110] ③ Block endogenous peroxidase: add 2 drops of 3% H2O2 to each slice, incubate at room temperature for 10 minutes to block the activity of endogenous peroxidase, and rinse with PBS for 3×3 minutes;

[0111] ④ Blocking: add 1% fresh bovine serum, incubate at 37℃ for 15min, rinse with PBS 3 times × 3min;

[0112] ⑤ Add primary antibody: remove PBS solution, add 1 drop of corresponding primary antibody to each slice, incubate in 4℃ refrigerator for 14h, incubate at room temperature for 1h, and rinse with PBS 5 times × 3min;

[0113] ⑥Add secondary antibody: remove PBS solution, add enzyme-labeled anti-mouse / rabbit polymer to each section, incubate at room temperature for 30 minutes, and rinse with PBS for 3×3 minutes;

[0114] ⑦ Add color developing solution: remove PBS solution, add 1 drop of freshly prepared DAB solution (1:50 dilution) to each slice, and observe under a microscope for 5 minutes;

[0115] ⑧Cell nucleus counterstaining: counterstaining with hematoxylin, differentiation with 0.1% hydrochloric acid, washing with tap water, blueing, dehydration and drying of sections with gradient ethanol, transparentization with xylene, sealing with neutral gum, and observation after drying.

[0116] (2) Calculation of immunohistochemical staining results

[0117] Under an optical microscope (200×), 10 fields of view were randomly selected from the immunohistochemical staining sections of the femoral heads of rats in each group. After saving the pictures, image J software was used for data analysis to calculate the mean optical density (MOD) of the immunohistochemical staining sections of the femoral heads of rats in each group. The process was as follows:

[0118] ①Open Image J software;

[0119] ②File-Open to open the image to be analyzed

[0120] ③Colour-Colour Deconvolution-H DAB, preliminary separation of image colors;

[0121] ④Image-Type-8-bit;

[0122] ⑤Analyze-Calibrate-Uncalibrated OD;

[0123] ⑥Image-Adjust-Threshold, adjust the slider in the pop-up box to mark the positive part, and compare it with the original image to avoid non-specific staining interference:

[0124] ⑦Analyze-Set Measurement, select Area, Min & max gray value, Center of mass integrated density, Area Fraction, Limit to threshold, Mean gray value, Perimeter, Display label in the pop-up window;

[0125] ⑧Analyze-Measure;

[0126] ⑨ The average optical density value obtained is: MOD (Mean OD) = IOD (Integrated Density) / Area

[0127] The results are as follows Figure 5 The results showed that compared with the control group, the expression of OPG in the femoral head tissue of rats in the MSC group and the Z+MSC group was enhanced, and the expression in the Z+MSC group was stronger than that in the MSC group, with a statistically significant difference (P<0.05). Compared with the control group, the expression of RANKL in the femoral head tissue of rats in the MSC group and the Z+MSC group was weakened, and the expression in the Z+MSC group was weaker than that in the MSC group, with a statistically significant difference (P<0.05).

[0128] In summary, the "Ziyin Fuzheng Qingdu Recipe" used in the present invention can effectively inhibit the "aging" of mesenchymal stem cells cultured in vitro. The mesenchymal stem cells cultured in the culture medium with the "Ziyin Fuzheng Qingdu Recipe" powder added to the infusion of the present invention have strong activity, improve the aging microenvironment of the body, and delay the aging process; and are conducive to promoting the differentiation of mesenchymal stem cells into osteoblasts, enhancing bone metabolism, improving bone quality, and improving the therapeutic effect of mesenchymal stem cells in treating osteoporosis, femoral head necrosis and other bone diseases.

[0129] The embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A Chinese medicine composition for resisting stem cell aging, characterized in that: The invention comprises the following raw materials in parts by weight: 25-35 parts of astragalus, 10-20 parts of psoralea, 10-20 parts of codonopsis, 10-20 parts of stir-fried atractylodes, 5-15 parts of eucommia, 10-20 parts of epimedium, 10-20 parts of glossy privet, 10-20 parts of yam, 10-20 parts of white peony, 10-20 parts of gynostemma pentaphyllum, 3-9 parts of notoginseng powder, 15-25 parts of barbata, 10-20 parts of poria, 5-15 parts of bupleurum, 25-35 parts of raw oyster, 10-20 parts of vinegar turtle shell, 10-20 parts of pinellia, 10-20 parts of eclipta, 3-9 parts of tangerine peel and 3-9 parts of liquorice.

2. A Chinese medicine composition for resisting stem cell aging according to claim 1, characterized in that: The invention comprises the following raw materials in parts by weight: 27-33 parts of astragalus, 12-17 parts of psoralea corylifolia, 12-17 parts of codonopsis pilosula, 12-17 parts of stir-fried atractylodes, 7-13 parts of eucommia, 12-17 parts of epimedium, 12-17 parts of glossy privet, 12-17 parts of yam, 12-17 parts of white peony root, 12-17 parts of gynostemma pentaphyllum, 5-7 parts of notoginseng powder, 17-22 parts of barbata scutellaria, 12-17 parts of poria, 7-13 parts of bupleurum, 27-32 parts of raw oyster, 12-17 parts of vinegar turtle shell, 12-17 parts of pinellia ternata, 12-17 parts of eclipta prostrata, 5-7 parts of tangerine peel and 5-7 parts of liquorice.

3. A Chinese medicine composition for resisting stem cell aging according to claim 1, characterized in that: The invention comprises the following raw materials in parts by weight: 30 parts of astragalus, 15 parts of psoralea, 15 parts of codonopsis, 15 parts of stir-fried atractylodes, 10 parts of eucommia, 15 parts of epimedium, 15 parts of glossy privet, 15 parts of yam, 15 parts of white peony, 15 parts of gynostemma pentaphyllum, 6 parts of notoginseng powder, 20 parts of barbata, 15 parts of poria, 10 parts of bupleurum, 30 parts of raw oyster, 15 parts of vinegar turtle shell, 15 parts of pinellia, 15 parts of eclipta, 6 parts of tangerine peel and 6 parts of liquorice.

4. A method for preparing a Chinese medicine composition for resisting stem cell aging according to any one of claims 1 to 3, characterized in that: The steps include: (1) First, each drug in the above-mentioned formula is made into coarse powder; (2) boiling the crude powder of Chinese medicine in water to extract the effective ingredients of the medicine; (3) using a Chinese medicine concentration device to concentrate the decocted Chinese medicine decoction into an extract; (4) Pour the extract into a drying tray and place it in a vacuum drying oven at 60°C for drying; (5) crushing the dried extract and passing it through a 120-mesh sieve to form a powder; (6) Weigh the prepared powder of the Chinese medicinal composition for resisting stem cell aging and prepare it into capsules, or pack it into sterile bottles for later use.

5. Use of a Chinese medicinal composition for resisting stem cell aging as claimed in any one of claims 1 to 3 in the treatment of degenerative bone diseases in combination with mesenchymal stem cells.

6. The use of a Chinese medicine composition for resisting stem cell aging according to claim 5, characterized in that: The degenerative bone disease includes osteoporosis, osteoarthritis or femoral head necrosis.

7. Use of the anti-stem cell aging Chinese medicinal composition according to any one of claims 1 to 3 in inhibiting the aging of mesenchymal stem cells cultured in vitro.

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