Composition containing polygonatum sibiricum saponin and application of composition in product for resisting immune aging and enhancing immunity

Abstract

The invention relates to the technical field of medicines, in particular to a composition containing polygonatum sibiricum saponin, a preparation method, a preparation and application in immune aging resistance. The total content of polygonatum sibiricum saponin in the polygonatum sibiricum saponin extract in the composition is 20-35%, the purity of peach gum polysaccharide is 80-95%, and the mass ratio of the polygonatum sibiricum saponin extract to the peach gum polysaccharide is (1-3): (1-2), preferably 2: 1. Animal experiments show that the composition can significantly reduce the proportion of myeloid propensity CD150hi hematopoietic stem cells, GMP, CMP and MEP in marrow of an aged mouse, improve the proportion of lymphatic propensity CD150lo hematopoietic stem cells and CLP, and correct the myeloid differentiation bias of the aged bone marrow hematopoietic stem cells; meanwhile, the proportion of bone marrow and peripheral blood myeloid cells can be reduced, and the proportion of B cells, T cells and NK cells can be increased, so that the immune aging phenotype is improved, and the method is suitable for preparing drugs or health care products for regulating differentiation of senescence bone marrow hematopoietic stem cells and resisting immune aging.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a composition containing Polygonatum saponins and its application in anti-immunoaging and immune-boosting products. Background Technology

[0002] With the increasing aging of the population, the incidence of age-related immune function decline, malignant tumors, and various chronic diseases has significantly increased, making immunosenescence a significant factor limiting the healthy lifespan of the elderly. Numerous studies have shown that hematopoietic stem cells (HSCs) in the bone marrow are the source of immune cell renewal. While the number of HSCs increases with age, their function is impaired, exhibiting a clear "myeloid differentiation bias": differentiation into myeloid progenitor cells and myeloid effector cells (neutrophils, monocytes / macrophages, etc.) is enhanced, while differentiation into lymphoid progenitor cells and lymphocytes is suppressed. This leads to an increased proportion of myeloid cells and a decreased proportion of lymphocytes, thereby inducing a decline in immune response and a chronic inflammatory state. This myeloid differentiation bias is considered an important cellular basis for immunosenescence and one of the important reasons for the increased risk of myeloid malignancies and increased susceptibility to infection in the elderly.

[0003] In existing technologies, there have been some reports on traditional Chinese medicines or health products focusing on "anti-aging, improving hematopoietic function, and enhancing immunity." Chinese patent document CN101422569A discloses a "health product preparation for improving the proliferation capacity of bone marrow hematopoietic stem cells to achieve anti-aging and anti-anemia." This preparation is composed of donkey-hide gelatin, sophora japonica flower, astragalus, polygonatum, rehmannia glutinosa, licorice, and excipients such as lactose, pregelatinized starch, and maltodextrin. It aims to enhance immunity, anti-aging, and anti-anemia through comprehensive qi-tonifying, blood-nourishing, and antioxidant effects. Although this document mentions "improving the proliferation capacity of bone marrow hematopoietic stem cells," its formula is a compound preparation of multiple medicinal herbs. It does not enrich or define the structure of any single active ingredient, nor does it target the imbalance in the myeloid / lymphatic differentiation ratio of HSCs in the aging state, and it does not establish an evaluation system centered on correcting "myeloid differentiation bias."

[0004] Chinese patent document CN1091029A discloses a "peach gum liquid for enhancing immune function and improving blood counts," using peach gum as the principal ingredient. The liquid is prepared by decocting, dissolving, filtering, concentrating, and precipitating, followed by the addition of sugar and preservatives. It is intended for use in patients with malignant tumors to enhance immune function, increase white blood cell count, and protect hematopoietic function. Pharmacological and clinical studies have shown that this peach gum preparation can increase T cell subsets, white blood cell count, and improve blood counts, and has a certain protective effect against hematopoietic suppression caused by chemotherapy and radiotherapy. However, this document uses a decoction of whole peach gum raw materials without further separation and purification of the main active ingredients such as polysaccharides. It does not clarify the structural characteristics and purity range of peach gum polysaccharides, nor does it address its targeted intervention on "myeloid differentiation bias in aging bone marrow hematopoietic stem cells," and it lacks any approach of compounding and optimizing the proportions with other active ingredients of traditional Chinese medicine (such as polygonatum saponins) to improve immune aging.

[0005] Chinese patent document CN102670978B discloses "a compound granule of Polygonatum and its preparation method." This granule is composed of Polygonatum extract, Salvia miltiorrhiza extract, hawthorn extract, Ginkgo biloba extract, Ganoderma lucidum extract, and excipients. Polygonatum and hawthorn have both medicinal and edible properties, Ganoderma lucidum and Salvia miltiorrhiza protect the liver and promote blood circulation, while Ginkgo biloba and Salvia miltiorrhiza work together to improve cardiovascular function. This compound granule is used for lowering lipids, lowering blood sugar, anti-aging, and regulating immune function, emphasizing the synergistic effect of multiple drug extracts in "delaying aging and regulating immunity." Although the document mentions that the combination of Polygonatum and Ganoderma lucidum has a better anti-aging effect, it mainly targets cardiovascular metabolic syndrome and "anti-aging" in a general sense, without focusing on bone marrow hematopoietic stem cells as the source target of immunosenescence, and does not propose a technical path to correct immunosenescence by regulating the differentiation ratio of HSCs into myeloid and lymphoid lineages. Furthermore, the document does not enrich or quantify precisely defined active ingredients such as Polygonatum saponins.

[0006] In summary, existing technologies still have significant shortcomings in the following aspects: 1. At the target level, the focus remains on the macroscopic level of "enhancing immunity" and "improving hematopoiesis." For example, CN101422569A and CN1091029A both aim to achieve anti-aging or strengthening the body by increasing the proliferation of bone marrow hematopoietic stem cells or protecting hematopoietic function after chemotherapy and improving blood counts. However, they do not provide a quantitative description or intervention for the "myeloid differentiation bias" of hematopoietic stem cells in the aging state, nor have they established an immune aging evaluation index system with myeloid / lymphatic differentiation balance as the core.

[0007] 2. At the level of active ingredients, most are still "crude extracts" or combinations of multiple medicinal materials, lacking a combination of ingredients with clear structure and controllable content. In CN1091029A, peach gum is only used in the form of decoction, and its main active ingredient "peach gum polysaccharide" has not been separated, purified, or had its content determined; in CN101422569A and CN102670978B, polygonatum is mostly used in the form of raw medicinal material or total extract, and the content range of the key active ingredient "polygonatum saponin" has not been specifically enriched and defined, resulting in an unclear effective material basis in the preparation, which is not conducive to dosage optimization and quality control.

[0008] 3. The formulation approach is mostly "multi-herb compound prescriptions," lacking targeted design of binary active ingredient combinations. Existing compound prescriptions containing Polygonatum, such as CN102670978B, are often combined with multiple herbs such as Salvia miltiorrhiza, hawthorn, ginkgo biloba, and Ganoderma lucidum. While the target points are broad, they are relatively dispersed, making it difficult to precisely regulate the specific pathological process of immune aging. On the other hand, preparations mainly based on peach gum, such as CN1091029A, do not synergize with Polygonatum-like herbs, nor do they optimize the combination based on the mechanism of saponins and polysaccharides synergistically regulating HSC differentiation.

[0009] 4. Regarding extraction and purification processes, a systematic process route specifically targeting "Polygonatum saponins + peach gum polysaccharides" has not yet been established. Although existing literature includes traditional processes such as ethanol extraction and water extraction for Polygonatum, as well as water extraction and alcohol precipitation for peach gum, systematic process combinations such as "water extraction and alcohol precipitation combined with multi-solvent gradient extraction to enrich Polygonatum saponins" and "Sevage deproteinization combined with DEAE-52 cellulose column gradient elution to obtain high-purity peach gum polysaccharides" have not been publicly disclosed or applied in the aforementioned literature.

[0010] 5. Lack of systematic pharmacological and animal experimental data on "myeloid differentiation bias in aging bone marrow hematopoietic stem cells". Existing patents related to Polygonatum sibiricum or peach gum mostly use general immune indicators (such as peripheral blood leukocyte count, immune organ weight, changes in total T / B cell count, etc.) as evaluation endpoints. There is a lack of systematic research on the fine classification of HSC subsets (LT-HSC, ST-HSC, MPP), myeloid / lymphoid progenitor cells (CMP, GMP, MEP, CLP) in bone marrow, as well as the fine classification of myeloid and lymphoid cells in bone marrow and peripheral blood. Furthermore, there is no systematic evaluation of the effect of drugs on the ratio of "myeloid-biased HSCs" and "lymphoid-biased HSCs" in aging animal models.

[0011] Therefore, starting from the differentiation balance of bone marrow hematopoietic stem cells—the source of immunosenescence—and focusing on the core scientific question of "correcting myeloid differentiation bias in aging HSCs," it remains a technical need that urgently needs to be addressed by those skilled in the art and that has not yet been met by existing technologies. This is based on the dual-source nature of traditional Chinese medicine as food and medicine, constructing a binary combination of Polygonatum saponins and peach gum polysaccharides with clearly defined components and controllable content, establishing corresponding refined extraction and purification processes, and systematically verifying its effect on improving myeloid / lymphatic differentiation balance and immunosenescence phenotype in aging models using methods such as multicolor flow cytometry. Summary of the Invention

[0012] The technical objective of this invention is to provide a composition of Polygonatum saponins and peach gum polysaccharides with clearly defined components and controllable content, and its preparation method. By specifically regulating the differentiation ratio of aging bone marrow hematopoietic stem cells into myeloid and lymphoid lines, it corrects the myeloid differentiation bias of aging hematopoietic stem cells, thereby improving the imbalance of bone marrow and peripheral immune cell composition, delaying the decline of immune function, and realizing a safe and long-term applicable anti-immunoaging intervention method, providing a new technical solution for the development of related drugs and health products.

[0013] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a composition for regulating myeloid differentiation bias in aging bone marrow hematopoietic stem cells and achieving anti-immunoaging effects, the composition comprising the following active ingredients: Polygonatum saponin extract; Peach gum polysaccharide extract; in: 1) The mass ratio of the extract of Polygonatum saponins to the extract of peach gum polysaccharides is (1-3):(1-2) based on the dry weight of the extract. 2) The total saponin content in the Polygonatum saponin extract is 20-35 wt%; 3) The polysaccharide purity of the peach gum polysaccharide extract is 80-95 wt%.

[0014] Preferably, the mass ratio of the Polygonatum saponin extract to the peach gum polysaccharide extract is (1.5-2.5):(1-1.5), with the most preferred range being 2:1.

[0015] Preferably, the Polygonatum saponin extract is derived from Polygonatum yunnanensis, a plant of the Liliaceae family. Polygonatum kingianum Coll.etH emsl.), Polygonatum ( Polygonatum sibiricum Red.) or Polygonatum multiflorum ( Polygonatum cyrtonema The dried rhizome of *Prunus chinensis* (Hua); the peach gum polysaccharide extract is derived from the resin secretions of *Prunus persica* or *Prunus serrulata*, plants of the Rosaceae family.

[0016] In a second aspect, the present invention provides an oral formulation comprising the composition described above, the oral formulation being selected from one or more of tablets, capsules, granules, oral liquids, oral solutions, or oral emulsions, and may further comprise pharmaceutically acceptable fillers, disintegrants, binders, wetting agents, sweeteners, and / or flavoring agents.

[0017] Preferably, the total mass percentage of the composition, based on the total mass of the preparation, is 1-60 wt% for the extracts of Polygonatum saponins and the extracts of peach gum polysaccharides.

[0018] Thirdly, the present invention provides a method for preparing the composition, comprising the following steps: 1) Preparation of Polygonatum saponin extract: ① Take Polygonatum sibiricum medicinal material, add 8 to 12 times its volume of ethanol with a volume fraction of 75 to 95%, soak it, and reflux extract it at 65 to 75℃ 2 to 3 times, each time for 1.5 to 2 hours, and combine the ethanol extracts; ② After the extracted residue has been allowed to stand to evaporate the ethanol residue, add 8 to 12 times the volume of water and extract with water 2 to 3 times at 65 to 75°C, each time for 1.5 to 2 hours. Combine the water extracts and concentrate them. ③ The concentrated water extract was subjected to water extraction and alcohol precipitation. 75-95% ethanol was added to adjust the pH of the solution to 5.5-6.0. After standing for 5-6 hours, the solution was filtered and the supernatant was collected and combined with the ethanol extract from step ①. The ethanol was recovered by rotary evaporation and then suspended in pure water. ④ Add dichloromethane, water-saturated ethyl acetate, water-saturated n-butanol and pure water to the suspension in a volume ratio of 1:1 for liquid-liquid extraction. Extract each liquid-liquid mixture 2 to 3 times. Collect the n-butanol phase, concentrate under reduced pressure and dry to obtain the n-butanol extract of Polygonatum sibiricum, which is used as the saponin extract of Polygonatum sibiricum. 2) Preparation of peach gum polysaccharide extract: ① Take peach gum raw material, add 12 to 15 times the volume of water, heat under reflux for 1 to 2 hours, repeat twice, filter and combine the filtrates and concentrate under reduced pressure; ② Add 1 / 4 to 1 / 3 of the volume of Sevage test solution to the concentrate, shake at room temperature for 20 to 25 minutes, centrifuge at 2000 r / min for 5 minutes, collect the supernatant, and repeat the protein removal step 3 to 4 times. ③ Add 3 to 4 times the volume of 80 to 90% ethanol to the combined supernatant, let stand overnight at 4°C, filter and collect the precipitate to obtain crude peach gum polysaccharide. ④ After dissolving the crude peach gum polysaccharide, the sample was loaded onto a DEAE-52 cellulose column and eluted sequentially with 0, 0.1 and 0.2 mol / L NaCl solutions. The absorbance of the eluent was detected online using the anthrone-sulfuric acid method. The eluent fraction with absorbance > 0.3 was collected, concentrated at 50℃ and freeze-dried to obtain the peach gum polysaccharide extract. 3) Preparation of the composition: The Polygonatum saponin extract obtained in step 1) and the peach gum polysaccharide extract obtained in step 2) are mixed in the mass ratio described above. If necessary, pharmaceutically acceptable excipients are added. The mixture is then mixed, dried, and shaped to obtain the composition.

[0019] As a preferred embodiment, in step 1), the volume ratio of Polygonatum sibiricum to 95% ethanol is 1:10 to 1:12, and the ethanol extraction is performed 3 times and the water extraction is performed 3 times; in step 2), the volume ratio of dichloromethane to n-butanol in the Sevage test solution is 4:1, and the protein removal step is repeated 4 times.

[0020] Fourthly, the present invention provides the use of the composition in the preparation of a medicament for regulating myeloid differentiation bias in aging bone marrow hematopoietic stem cells, the medicament being used for: Reduce the proportion of common myeloid progenitor cells (CMP), granulocytic monolineage progenitor cells (GMP), and megakaryocyte-erythroid progenitor cells (MEP) in the bone marrow of aging patients; Increase the proportion of common lymphoid progenitor cells (CLPs); Reduce the proportion of myeloid cells in the bone marrow, including inflammatory monocytes, granulocytes, and macrophages; Increase the proportion of B cells, T cells, and NK cells; To improve age-related bone marrow hematopoietic imbalance.

[0021] Preferably, the drug is an oral formulation suitable for middle-aged and elderly individuals with signs of immunosenescence, used to improve the decline in immune function, increased susceptibility to infection, and weakened vaccine response caused by aging.

[0022] Fifthly, the present invention provides the use of the composition in the preparation of a health product that enhances immunity, the health product being used to improve or maintain immune homeostasis and delay age-related degenerative changes in immune function.

[0023] This invention achieves precise regulation of the differentiation fate of aging bone marrow hematopoietic stem cells by enriching Polygonatum saponins and high-purity peach gum polysaccharides and compounding them in a specific ratio. In a naturally aging mouse model, this composition can significantly reduce the proportion of myeloid skewed hematopoietic stem cells (CD150^hi HSCs) and their downstream myeloid progenitor cells (CMP, GMP, MEP) in the bone marrow, while increasing the proportion of lymphoid skewed hematopoietic stem cells (CD150^lo HSCs) and lymphoid common progenitor cells (CLPs), thus correcting the myeloid differentiation bias of aging hematopoietic stem cells from the source. At the same time, this composition can also reduce the proportion of myeloid cells such as inflammatory monocytes, granulocytes, and macrophages in the bone marrow and peripheral blood, while increasing the proportion of B cells, T cells, and NK cells. The composition of this invention improves the ratio of hematopoietic stem cell subsets to lymphocytes, restores the dynamic balance of myeloid / lymphoid immune cells, and improves age-related immune dysfunction and chronic inflammation. Compared with the use of Polygonatum saponins or peach gum polysaccharides alone, the composition of this invention shows a better synergistic effect in regulating the ratio of hematopoietic stem cell subsets and remodeling bone marrow and peripheral immune spectrum. Under the premise of ensuring the safety of food and medicine homology, it significantly improves the pertinence and effectiveness of anti-immunoaging intervention, and provides a reliable material basis and experimental evidence for the development of long-term oral anti-immunoaging drugs and functional health products. Attached Figure Description

[0024] Figure 1 The effect of Polygonatum sibiricum composition on hematopoietic stem cell typing in bone marrow of aging mice: G0 was the young group, G1 was the aging model group, G2 was the Polygonatum sibiricum saponin group, G3 was the peach gum polysaccharide group, G4 was Polygonatum sibiricum composition group 1 (Polygonatum sibiricum saponin: peach gum polysaccharide = 1:1), G5 was Polygonatum sibiricum composition group 2 (Polygonatum sibiricum saponin: peach gum polysaccharide = 2:1), and G6 was Polygonatum sibiricum composition group 3 (Polygonatum sibiricum saponin: peach gum polysaccharide = 1:2).

[0025] Figure 2 The effect of Polygonatum sibiricum composition on progenitor cell typing in the bone marrow of aging mice: G0 was the young group, G1 was the aging model group, G2 was the Polygonatum sibiricum saponin group, G3 was the peach gum polysaccharide group, G4 was Polygonatum sibiricum composition group 1 (Polygonatum sibiricum saponin: peach gum polysaccharide = 1:1), G5 was Polygonatum sibiricum composition group 2 (Polygonatum sibiricum saponin: peach gum polysaccharide = 2:1), and G6 was Polygonatum sibiricum composition group 3 (Polygonatum sibiricum saponin: peach gum polysaccharide = 1:2).

[0026] Figure 3 Effects of Polygonatum sibiricum composition on bone marrow lymphoid and myeloid cell typing in aging mice: G0 was the young group, G1 was the aging model group, G2 was the Polygonatum sibiricum saponin group, G3 was the peach gum polysaccharide group, G4 was Polygonatum sibiricum composition group 1 (Polygonatum sibiricum saponin: peach gum polysaccharide = 1:1), G5 was Polygonatum sibiricum composition group 2 (Polygonatum sibiricum saponin: peach gum polysaccharide = 2:1), and G6 was Polygonatum sibiricum composition group 3 (Polygonatum sibiricum saponin: peach gum polysaccharide = 1:2).

[0027] Figure 4 The effect of Polygonatum sibiricum composition on the differentiation tendency of hematopoietic stem cells in aging mice: G0 was the young group, G1 was the aging model group, G2 was the Polygonatum sibiricum saponin group, G3 was the peach gum polysaccharide group, G4 was Polygonatum sibiricum composition group 1 (Polygonatum sibiricum saponin: peach gum polysaccharide = 1:1), G5 was Polygonatum sibiricum composition group 2 (Polygonatum sibiricum saponin: peach gum polysaccharide = 2:1), and G6 was Polygonatum sibiricum composition group 3 (Polygonatum sibiricum saponin: peach gum polysaccharide = 1:2).

[0028] Figure 5 Effects of Polygonatum sibiricum composition on peripheral blood lymphocytes and myeloid cells in aging mice: G0 was the young group, G1 was the aging model group, G2 was the Polygonatum sibiricum saponin group, G3 was the peach gum polysaccharide group, G4 was Polygonatum sibiricum composition group 1 (Polygonatum sibiricum saponin: peach gum polysaccharide = 1:1), G5 was Polygonatum sibiricum composition group 2 (Polygonatum sibiricum saponin: peach gum polysaccharide = 2:1), and G6 was Polygonatum sibiricum composition group 3 (Polygonatum sibiricum saponin: peach gum polysaccharide = 1:2).

[0029] Figure 6 Effects of Polygonatum saponins and Polygonatum polysaccharides on peripheral blood lymphocytes and myeloid cells in aging mice: G0 was the young group, G1 was the aging model group, G2 was the Polygonatum saponin 400 mg / kg group, and G3 was the Polygonatum polysaccharide 400 mg / kg group. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present invention.

[0031] I. Raw Material Sources and Terminology Explanation 1. Polygonatum medicinal material The Polygonatum described in this invention is the dried rhizome of *Polygonatum kingianum* Coll. et Hemsl., *Polygonatum sibiricum* Red., or *Polygonatum cyrtonema* Hua, all belonging to the Liliaceae family, and all meet the relevant quality standards of the Chinese Pharmacopoeia. Traditional Chinese medicine theory holds that Polygonatum has the effects of tonifying qi and nourishing yin, strengthening the spleen and moistening the lungs, and benefiting the kidneys and replenishing essence. Modern research shows that Polygonatum has a comprehensive regulatory effect on bone marrow hematopoietic function, immune function, and anti-aging.

[0032] 2. Peach gum raw material The peach gum described in this invention is a resin secreted and solidified from the bark of peach or wild peach trees, which belong to the Rosaceae family. It can be used as a natural product that is both medicinal and edible, and its main active ingredient is polysaccharide.

[0033] 3. Polygonatum saponin extract The "Polygonatum saponin extract" in this invention refers to the n-butanol fraction extract obtained from Polygonatum medicinal material through ethanol extraction, water extraction and alcohol precipitation, and multi-solvent gradient extraction processes. The extract is predominantly composed of steroidal saponins, with a preferred total saponin content of 20–35 wt%. This extract is one of the main active ingredients in the composition of this invention.

[0034] 4. Peach gum polysaccharide extract The "peach gum polysaccharide extract" in this invention refers to the polysaccharide-enriched fraction obtained from peach gum raw material through water extraction, Sevage deproteinization, ethanol precipitation, and DEAE-52 cellulose column gradient elution. The polysaccharide content determined by the anthrone-sulfuric acid method is preferably 80-95 wt%.

[0035] 5. Myeloid differentiation bias and related terminology The "myeloid differentiation bias of aging bone marrow hematopoietic stem cells" described in this invention refers to the increased tendency of hematopoietic stem cells to differentiate into myeloid progenitor cells and myeloid effector cells during natural aging, while their ability to differentiate into lymphoid cells decreases, manifested as an increased proportion of myeloid cells and a decreased proportion of lymphocytes. This invention utilizes multicolor flow cytometry to define the following cell subsets, including but not limited to: Hematopoietic stem cells (HSC, LSK): Lin⁻Sca1⁺c-Kit⁺; Long-term hematopoietic stem cells (LT-HSC): Lin⁻Sca1⁺c-Kit⁺Flk2⁻CD34⁻; Short-term hematopoietic stem cell (ST-HSC): Lin⁻Sca1⁺c-Kit⁺Flk2⁻CD34⁺; Pluripotent progenitor cells (MPP): Lin⁻Sca1⁺c-Kit⁺Flk2⁺CD34⁺; Common myeloid progenitor cells (CMP): Lin⁻CD127⁻Sca1⁻c-Kit⁺CD16 / 32⁻CD34⁺; Granulosaminous monolineage progenitor cells (GMP): Lin⁻CD127⁻Sca1⁻c-Kit⁺CD16 / 32⁺CD34⁺; Megakaryocytic-erythroid progenitor cells (MEP): Lin⁻CD127⁻Sca1⁻c-Kit⁺CD16 / 32⁻CD34⁻; Common lymphoid progenitor cells (CLP): Lin⁻CD127⁺Sca1^med c-Kit^med; Myeloid-biased HSC: CD150^hi LSK; Lymphoid-biased HSC: CD150^lo LSK; In addition, mature immune cell subsets such as B cells, T cells, NK cells, granulocytes, inflammatory monocytes, and macrophages.

[0036] II. Preparation of Polygonatum saponin extract The Polygonatum saponin extract in this invention can be prepared according to the following steps: 1. Ethanol extraction Take the pulverized Polygonatum sibiricum material and add 75-95% ethanol at 8-12 times the volume of the material. Reflux and extract 2-3 times at 65-75℃, each time for 1.5-2 hours. Combine the ethanol extracts from each extraction for later use.

[0037] 2. Water extraction and alcohol precipitation After removing residual ethanol from the ethanol-extracted residue by natural evaporation, add 8–12 times the volume of pure water and extract 2–3 times at 65–75°C, each time for 1.5–2 hours. Combine the water extracts and concentrate. Add 75–95% ethanol (by volume) to the concentrated water extract to adjust the pH to 5.5–6.0. ​​Let stand at room temperature for 5–6 hours, filter, and collect the supernatant. Combine the supernatant with the aforementioned ethanol extract, and recover the ethanol by rotary evaporation to obtain a water-ethanol mixed resinous concentrate.

[0038] 3. Multi-solvent gradient extraction Add an appropriate amount of pure water to the above concentrate to form a homogeneous suspension. Then, sequentially add dichloromethane, water-saturated ethyl acetate, water-saturated n-butanol, and pure water at a volume ratio of 1:1 for liquid-liquid extraction, preferably extracting 2-3 times with each solvent. Discard the dichloromethane and ethyl acetate phases, collect the n-butanol phase, concentrate under reduced pressure, and dry to obtain the n-butanol fraction extract of Polygonatum sibiricum, which is the Polygonatum sibiricum saponin extract described in this invention.

[0039] The total saponin content in the Polygonatum saponin extract can be controlled between 20 and 35 wt%, preferably around 27%, by ultraviolet spectrophotometry.

[0040] III. Preparation and Content Determination of Peach Gum Polysaccharide Extract 1. Extraction and deproteinization of peach gum polysaccharides Take an appropriate amount of peach gum raw material, add 12-15 times its volume of pure water, heat under reflux for 1-2 hours, repeat twice, filter, combine the filtrates, and concentrate under reduced pressure to an appropriate volume. Add 1 / 4-1 / 3 of its volume of Sevage test solution (dichloromethane: n-butanol = 4:(1-1.5)) to the concentrate, shake at room temperature for 20-25 minutes, centrifuge at 2000 r / min for 5 minutes, and collect the supernatant; repeat the above protein removal process 3-4 times, and combine all supernatants.

[0041] 2. Crude polysaccharide was obtained by alcohol precipitation. Add 3-4 times the volume of 80-90% ethanol to the combined supernatant, let stand overnight at 4°C to allow the polysaccharide to fully precipitate, filter and collect the precipitate to obtain crude peach gum polysaccharide.

[0042] 3. DEAE-52 cellulose column gradient elution enrichment of polysaccharides Crude peach gum polysaccharide was dissolved in an appropriate amount of pure water and loaded onto a DEAE-52 cellulose column (4×100cm). The column was eluted sequentially with 0, 0.1, and 0.2 mol / L NaCl solutions. The absorbance of the eluent was monitored online using the anthrone-sulfuric acid method, and the eluent fractions with absorbance greater than 0.3 were collected. The obtained fractions were concentrated under reduced pressure at 50℃ and then freeze-dried to obtain a high-purity peach gum polysaccharide extract.

[0043] 4. Polysaccharide content determination Using glucose as a reference, a series of standard solutions were prepared. The absorbance was measured at an appropriate wavelength using anthrone-sulfuric acid colorimetric reaction to establish a standard curve. The above-mentioned peach gum polysaccharide extract sample solution was then subjected to colorimetric analysis and absorbance measurement using the same method. The polysaccharide content was calculated by referring to the standard curve. The results showed that the polysaccharide content of the peach gum polysaccharide extract obtained under the conditions of this invention was approximately 86%, which is within the preferred range of 80–95 wt% of this invention.

[0044] IV. Preparation of Polygonatum saponins-peach gum polysaccharide composition Based on the dry weight of Polygonatum saponin extract and peach gum polysaccharide extract, the two are mixed at a mass ratio of (1-3):(1-2), preferably 2:1. Different dosage forms can be prepared as needed, for example: Powdered composition: The basic composition of the present invention is obtained by directly mixing the two extracts in a set ratio; Tablets, granules or capsules: Based on the above composition, pharmaceutically acceptable excipients such as fillers, disintegrants, and binders are added, and solid dosage forms are prepared by processes such as mixing, granulation, tableting or encapsulation. Oral liquid / solution: The composition is dissolved or dispersed in a suitable solvent, and sweeteners and flavoring agents are added as needed. After filtration, dispensing and sterilization, it is prepared into an oral liquid preparation.

[0045] The resulting composition can be further converted into a dosage concentration based on the effective ingredient dose per kilogram of body weight for use in animal experiments and subsequent preclinical studies.

[0046] V. Examples: Preparation of the Composition and Key Physicochemical Indicators 1. The preparation method of the Polygonatum saponins is as follows: 3.5 kg of Polygonatum sibiricum was soaked in 12 times its volume of 95% ethanol and extracted three times at 70℃ for 2 hours each time. The residue was allowed to stand and the ethanol was evaporated. It was then extracted three times with 12 times its volume of water for 2 hours each time. The water extract was concentrated and then subjected to water extraction and alcohol precipitation. 95% ethanol was added to adjust the pH of the solution to 6 and let it stand for 6 hours. The supernatant was collected after filtration and combined with the 95% extract obtained from the first extraction by rotary evaporation. The ethanol was then recovered and the mixture was suspended in pure water. Equal volumes of dichloromethane, water-saturated ethyl acetate, water-saturated n-butanol, and pure water were added and extracted three times each. The extracts were stored separately to obtain Polygonatum sibiricum n-butanol extract, which is Polygonatum sibiricum saponin.

[0047] 2. The preparation method of the peach gum polysaccharide is as follows: Add 15 times the volume of water to peach gum and heat under reflux for 2 hours, repeating twice. Filter and combine the filtrates. Concentrate under reduced pressure, then add 1 / 4 volume of Sevage test solution (dichloromethane: n-butanol = 4:1), shake at room temperature for 20 minutes, then centrifuge at 2000 rpm for 5 minutes. Collect the supernatant, repeating the above steps 4 times, and combine the supernatants. Add 4 times the volume of 80% ethanol to the supernatant, let stand overnight at 4°C, and filter to collect the precipitate to obtain crude peach gum polysaccharide. Separate the crude peach gum polysaccharide using a DEAE-52 cellulose column (4×100 cm) with 0, 0.1, and 0.2 mol / L NaCl solutions. Analyze the eluent using the anthrone-sulfuric acid method, collecting the eluent fractions with absorbance >0.3, concentrating at 50°C, and lyophilizing to obtain the peach gum polysaccharide.

[0048] The total saponin content of the Polygonatum n-butanol extract prepared in Example 1 was determined by ultraviolet spectrophotometry, and the content was 27.42%.

[0049] Unlike traditional water extraction, the above composition removes some water-soluble impurities from Polygonatum sibiricum, enriches the polysaccharide components of peach gum, and increases the content of active ingredients, thereby better playing the role of regulating the bias of aging bone marrow hematopoietic stem cells and anti-immunoaging; and the dosage is reduced, which can increase user compliance.

[0050] 3. The content of Polygonatum saponins was determined. 3.1 Experimental Materials 3.1.1 Ultraviolet Spectrophotometry: Accurately weigh 14 mg of ginsenoside Rb and prepare a reference solution containing 1400 μg of ginsenoside Rb per ml using methanol. Using a C18E solid-phase extraction column, load 2 ml of Polygonatum sibiricum n-butanol solution, elute with 10 ml of 75% methanol, and collect the eluent (controlling the flow rate to <1 ml / min during solid-phase extraction). Evaporate the eluent to dryness and dissolve it in methanol to obtain the test solution. Then, accurately transfer 0.1 ml of each sample solution, dry completely at 60℃, add 0.2 ml of freshly prepared 5% vanillin-glacial acetic acid solution, then add 0.8 ml of perchloric acid solution, react in a 60℃ water bath for 20 min, cool in an ice-water bath for 2 min, add 5 ml of glacial acetic acid solution, and measure the absorbance at 546 nm. Develop the color with 0.1 ml of methanol using the same method as a blank.

[0051] 3.2 Experimental Results The content of scrophularia saponins in the extract of Polygonatum sibiricum was determined to be 27.42% by ultraviolet spectrophotometry.

[0052] 4. Extraction and content determination of peach gum polysaccharides 4.1 Experimental Materials and Methods 4.1.1 Extraction of Peach Gum Polysaccharide: Peach gum was added to 15 times its volume of water and heated under reflux for 2 hours, twice. The filtrates were combined and concentrated under reduced pressure. Then, 1 / 4 volume of Sevage test solution (dichloromethane: n-butanol = 4:1) was added, and the mixture was shaken at room temperature for 20 minutes. Afterward, it was centrifuged at 2000 rpm for 5 minutes, and the supernatant was collected. This process was repeated four times, and the supernatants were combined. Four times its volume of 80% ethanol was added to the supernatant, and the mixture was allowed to stand overnight at 4°C. The precipitate was collected by vacuum filtration to obtain crude peach gum polysaccharide. The crude peach gum polysaccharide was separated using a DEAE-52 cellulose column (4 × 100 cm) eluted with 0, 0.1, and 0.2 mol / L NaCl solutions. The eluent was analyzed using the anthrone-sulfuric acid method. Fractions with absorbance > 0.3 were collected, concentrated at 50°C, and lyophilized to obtain the peach gum polysaccharide.

[0053] 4.2 Determination of polysaccharide content: 4.2.1 Preparation of reference solution: Take 10.05 mg of D-anhydrous glucose (Shanghai Ronghe Pharmaceutical Technology Co., Ltd., batch number 160109) into a 100 mL volumetric flask and dilute to volume with pure water.

[0054] 4.2.2 Preparation of test solution: Take 10.20 mg of the prepared drug powder, accurately weigh it and put it into a 100 ml volumetric flask, add pure water to dissolve it (heat it in an 80℃ water bath to dissolve it), add water to the mark, and shake well.

[0055] 4.2.3 Colorimetric method: Take the reference / test solution and blank into 10mL stoppered test tubes. Add 0.2, 0.4, 0.6, 0.8, 0.9, 1.0, 1.2 and 1.4mL of glucose standard solution to the 8 reference test tubes respectively. Add 1mL of peach gum polysaccharide solution to the 3 test tubes for the test sample. Do not add any solution to the 2 blank test tubes. Add water to each of the above test tubes to a volume of 2.0mL. Accurately add 1.0mL of freshly prepared 5% phenol solution, shake well, and then accurately add 5.0mL of concentrated sulfuric acid. Shake well and place in a boiling water bath for 20 minutes. Remove and cool in an ice bath for 5 minutes. Use the 2 blank test tubes as blanks and measure the absorbance.

[0056] 4.3 Experimental Results 4.3.1 Polysaccharide Extraction: Peach gum was extracted with water and precipitated with ethanol to obtain crude polysaccharide. Subsequently, it was eluted with DEAE-52 cellulose to obtain three elution fractions: water, 0.1 mol / L NaCl, and 0.2 mol / L NaCl. The polysaccharide was enriched and stored at 4℃.

[0057] 4.3.2 Determination of polysaccharide content: The standard curve is: y=5.428x+0.042R2=0.999. The amount of anhydrous glucose in the test solution is read from the standard curve and calculated to be 86.034%.

[0058] VI. Study on the effect of hematopoietic stem cell differentiation bias in bone marrow of aging mice 1. Experimental Materials 1.1 Animals: 10-month-old male C57BL / 6J mice were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.; 4-6-week-old male C57BL / 6J mice were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.

[0059] 1.2 Drugs and reagents: Polygonatum composition solution: Polygonatum saponins and peach gum polysaccharides prepared in the examples were mixed at mass ratios of 1:1, 2:1 and 1:2 respectively, and prepared into corresponding concentration solutions with pure water for later use.

[0060] 1.3 Instruments: CytoFlex S flow cytometer (Beckman Coulter), Easy Eights TM EasySep TM Magnet (Model: 1000096649) STEMCELL Technologies.

[0061] 2. Experimental Methods 2.1 Grouping and Administration: 15-month-old C57BL / 6J naturally aging mice were used as aging model animals and randomly divided into 6 groups: aged model group, Polygonatum saponin 400 mg / kg group, Peach gum polysaccharide 100 mg / kg group, Polygonatum Combination No. 1 400 mg / kg group (Polygonatum saponin: Peach gum polysaccharide = 1:1), Polygonatum Combination No. 2 400 mg / kg group (Polygonatum saponin: Peach gum polysaccharide = 2:1), and Polygonatum Combination No. 3 400 mg / kg group (Polygonatum saponin: Peach gum polysaccharide = 1:2). Six-week-old C57BL / 6J mice were used as a young control group. The young control group and aged model group were given pure water. Each administration group was given the corresponding dose of drug by gavage. The drug concentrations for Polygonatum saponin, Polygonatum Combination No. 1, Polygonatum Combination No. 2, and Polygonatum Combination No. 3 were all 40 mg / ml, and the Peach gum polysaccharide concentration was 10 mg / ml. The gavage volume was 10 mL / kg. -1 Once a day for 8 consecutive weeks.

[0062] 2.2 Indicator Testing 2.2.1 Preparation of bone marrow single-cell suspension After the last administration, mice were rapidly euthanized (cervical dislocation method), disinfected with 75% alcohol, and their bilateral hip bones, femurs, tibias, and spines were aseptically separated using ophthalmic scissors and forceps. Sterile PBS and 1640 medium containing 2% fetal bovine serum were prepared and placed in 6-well plates containing 3 mL of 1640 medium. The bone marrow cavity was repeatedly flushed with a 1 mL syringe and the cells were crushed in a mortar. Cells were gently pipetted and filtered through a 70 μm cell filter to obtain bone marrow cells, which were then collected into 5 mL centrifuge tubes. The cells were centrifuged at room temperature, the supernatant was discarded, and the bone marrow cells were resuspended in 1 mL of erythrocyte lysis buffer and incubated at room temperature for 5 minutes to lyse the erythrocytes. The reaction was terminated with 3 mL of 1640 medium, centrifuged at room temperature, and the supernatant was discarded. The cells were washed once with 1 mL of PBS, centrifuged at room temperature, and the supernatant was discarded. The cells were resuspended in an appropriate amount of PBS, counted, and stored on ice for later use.

[0063] 2.2.2 Detection of the proportion of lymphocytes and myeloid cells in bone marrow Take 2~3×10 6Each bone marrow single cell was placed in a 1.5 mL EP tube. Blank tubes, single-positive tubes, and sample tubes were prepared. No antibodies were added to the blank tubes. The corresponding antibodies were added to the single-positive tubes. The surface marker antibodies were added to the sample tubes (final volume 100 μL). The antibody usage ratios are as follows: Lineage (1:10), c-Kit (1:100), Sca1 (1:100), Flk2 (1:100), CD34 (1:100), CD150 (1:100), CD48 (1:100), CD127 (1:100), CD16 / 32 (1:100), B220 (1:100), Gr-1 (1:100), CD11b (1:100), CD3e (1:100), etc. After gently mixing, incubate at room temperature in the dark for 40 min; wash once with 1 mL PBS, centrifuge at room temperature, and discard the supernatant; repeat the steps once; resuspend in an appropriate amount of PBS and perform multicolor flow cytometry to detect the phenotypic markers of various hematopoietic progenitor cells and mature cells in the bone marrow, and analyze the proportion of "lymphoid-myeloid" cells in bone marrow hematopoietic stem cells, including 1) myeloid differentiation-related cells: common myeloid progenitor cells (CMP) and their downstream myeloid cells (inflammatory monocytes, granulocytes, and macrophages); 2) lymphoid differentiation-related cells: common lymphoid progenitor cells (CLP) and their downstream lymphocytes (B cells, plasma cells, T cells, and NK cells). The surface markers of various bone marrow cells are as follows: 1) Hematopoietic stem cells (HSC): Lin-Sca1+c-Kit+; 2) Long-term hematopoietic stem cells (LT-HSC): Lin-Sca1+c-Kit+Flk2-CD34-; 3) Short-term hematopoietic stem cell (ST-HSC): Lin-Sca1+c-Kit+Flk2-CD34+; 4) Pluripotent progenitor cells (MPP): Lin-Sca1+c-Kit+Flk2+CD34+; 5) Myeloid progenitor cells (MP): Lin-Sca1-c-Kit+; 6) Common myeloid progenitor cells (CMP): Lin-CD127-Sca1-c-Kit+CD16 / 32-CD34+; 7) Granulosaminous monolineage progenitor cells (GMP): Lin-CD127-Sca1-c-Kit+CD16 / 32+CD34+; 8) Megakaryocytic-erythroid progenitor cells (MEP): Lin-CD127-Sca1-c-Kit+CD16 / 32-CD34-; 9) Common lymphoid progenitor cells (CLP): Lin-CD127+Sca1medc-Kitmed; 10) Inflammatory mononuclear cells (iMono): CD11b+Gr1lowLy6Chigh; 11) Granulocytes: CD11b+Gr1high; 12) Macrophages (Mφ): CD11b+F4 / 80+; 13) NK cells: CD11b-Gr1-CD3e-CD19-B220-NK1.1+; 14) B cells: CD11b-Gr1-CD3e-CD19+B220+NK1.1-; 15) Plasma cells: CD11b-Ly6C-Gr1-B220-CD3-NK1.1-CD138+; 16) T cells: CD11b-Gr1-CD3e+CD19-B220-NK1.1-; 17) Myeloid cells: Gr1+CD11b+.

[0064] 2.2.3 Effects on differentiation-prone hematopoietic stem cells Detection of the proportion of differentiated hematopoietic stem cells: After the last administration, mouse bone marrow single-cell suspension was aseptically prepared. Multicolor flow cytometry was used as described in (3) above for specific surface labeling and detection methods. Blank tubes, single positive tubes, and sample tubes were set up. No antibody was added to the blank tubes, the corresponding antibody was added to the single positive tubes, and the corresponding monoclonal antibody fluorescent markers CD150, Lineage, c-Kit, Sca1, etc. were added to the sample tubes. After gently mixing, the cells were incubated at room temperature in the dark for 40 min. PBS was added to wash once, centrifuged at room temperature, and the supernatant was discarded. The steps were repeated once. After resuspending with an appropriate amount of PBS, multicolor flow cytometry was performed to analyze the proportion of myeloid differentiated hematopoietic stem cells (CD150) in bone marrow cells. hi HSC) and lymphoid hematopoietic stem cells (CD150) lo The proportion of HSC.

[0065] 2.2.4 Effects on the typing of lymphocytes and myeloid cells in the peripheral blood of aging mice When killing rats, they were fasted but allowed to drink water for 12 hours. Blood was collected from the fundus venous plexus and placed in EDTA anticoagulant tubes. The number and proportion of white blood cells, lymphocytes, neutrophils, eosinophils, basophils, and monocytes in whole blood were measured using a fully automated blood analyzer.

[0066] 3. Experimental Results 3.1 Effects on hematopoietic stem cell typing in bone marrow of aging mice Hematopoietic stem cells (HSCs) can include pluripotent progenitor cells (MPP, Lin-Sca1+c-Kit+Flk2+CD34+), short-term hematopoietic stem cells (ST-HSC, Lin-Sca1+c-Kit+Flk2-CD34+), and long-term hematopoietic stem cells (LT-HSC, Lin-Sca1+c-Kit+Flk2-CD34-). Flow cytometry analysis showed that, compared with the young control group, the proportion of bone marrow hematopoietic stem cells (LSK, Lin-Sca1+c-Kit+) in the aging model group mice tended to increase, the proportion of short-term hematopoietic stem cells (ST-HSC, Lin-Sca1+c-Kit+Flk2-CD34+) decreased significantly, the proportion of long-term hematopoietic stem cells (LT-HSC, Lin-Sca1+c-Kit+Flk2-CD34-) increased significantly, and the proportion of pluripotent progenitor cells (MPP, Lin-Sca1+c-Kit+Flk2+CD34+) tended to decrease. Compared with the aging model group, the proportion of LSK cells in the bone marrow of mice in the Polygonatum saponin group, Polygonatum composition group 1, group 2, and group 3 was significantly reduced after drug administration. The Polygonatum composition group showed better effects than the Polygonatum saponin group, with group 2 showing the most significant effect. The proportion of short-term hematopoietic stem cells (ST-HSCs) in the bone marrow of mice in the Polygonatum saponin group and group 3 was significantly reduced. The proportion of long-term hematopoietic stem cells (LT-HSCs) in mice in groups 1, 2, and 3 was significantly increased (P < 0.05, P < 0.01). The Polygonatum composition group 2 showed a more significant effect than groups 1 and 3. Figure 1 As shown.

[0067] 3.2 Effects on progenitor cell typing in the bone marrow of aging mice Pluripotent progenitor cells (MPPs) can differentiate into myeloid common progenitor cells (CMPs, Lin-CD127-Sca1-c-Kit+CD16 / 32-CD34+) and lymphoid common progenitor cells (CLPs, Lin-CD127+Sca1medc-Kitmed). CMPs can further differentiate into granulocytic monolineage progenitor cells (GMPs, Lin-CD127-Sca1-c-Kit+CD16 / 32+CD34+) and megakaryocyte-erythroid progenitor cells (MEPs, Lin-CD127-Sca1-c-Kit+CD16 / 32-CD34-). Flow cytometry analysis showed that compared with the young control group, the proportions of granulocytic monolineage progenitor cells (GMPs), myeloid common progenitor cells (CMPs), and megakaryocyte-erythroid progenitor cells (MEPs) in the bone marrow of the aging model group mice were significantly increased, while the proportion of lymphoid common progenitor cells (CLPs) was significantly decreased (P < 0.05, P < 0.01). Compared with the aging model group, the proportions of CMP, MEP, and GMP in the bone marrow of mice in the Polygonatum saponin group, peach gum polysaccharide group, and Polygonatum composition groups 1, 2, and 3 were significantly reduced, while the proportion of CLP was significantly increased (P < 0.05, P < 0.01). The effects of the Polygonatum compositions were better than those of the Polygonatum saponin group and the peach gum polysaccharide group. Among them, the effect of Polygonatum composition group 2 was better than that of Polygonatum composition groups 1 and 3, indicating that the combined use of Polygonatum saponin and peach gum polysaccharide in a ratio of 2:1 is the most effective. Figure 2 As shown.

[0068] 3.3 Effects on lymphoid and myeloid cell typing in bone marrow of aging mice Bone marrow contains lymphoid cells, including B lymphocytes (CD45+B220+CD3eNK1.1-F4 / 80-CD11b-Gr1-B220+), T lymphocytes (CD45+B220-CD3e+NK1.1-F4 / 80-CD11b-Gr1-), and NK natural killer cells (CD45+B220-CD3eNK1.1+). Myeloid cells include inflammatory monocytes (iMono, CD45+B220-CD3e-CD11b+Gr1lowLy6Chigh), granulocytes (Gran, CD45+B220-CD3e-CD11b+Ly6ClowGr1high), and macrophages (Mφ, CD45+B220-CD3e-CD11b+F4 / 80+). Flow cytometry analysis showed that, compared with the young control group, the proportions of macrophages, inflammatory monocytes, and granulocytes in the bone marrow of mice in the aging model group were significantly increased, while the proportions of B cells, T cells, and NK cells were significantly decreased. Compared with the aging model group, the proportions of macrophages, granulocytes, and inflammatory monocytes in the bone marrow of mice in the Polygonatum saponin group, the peach gum polysaccharide group, and the Polygonatum combination groups 1, 2, and 3 were significantly decreased; while the proportions of B cells, T cells, and NK cells were significantly increased (P < 0.05, P < 0.01). The Polygonatum saponin group showed better results than the peach gum polysaccharide group, and the combined use of the two was even more effective, especially when the ratio of Polygonatum saponin to peach gum polysaccharide was 2:1. Figure 3 As shown.

[0069] 3.4 Effects on the differentiation tendency of hematopoietic stem cells in aging mice Compared with the young control group, the proportion of myeloid-predisposing HSCs (CD150hi) cells and the proportion of lymphoid-predisposing HSCs (CD150low) cells in the bone marrow of mice in the aging model group were significantly increased. Compared with the aging model group, the proportion of myeloid-predisposing HSCs (CD150hi) cells in the bone marrow of mice in the Polygonatum saponin group, and the Polygonatum composition groups 1, 2, and 3 was significantly decreased; while the proportion of lymphoid-predisposing HSCs (CD150low) cells in the mouse bone marrow of mice in the Polygonatum saponin group, the peach gum polysaccharide group, and the Polygonatum composition groups 1, 2, and 3 was significantly increased (P < 0.05, P < 0.01), thus maintaining a relative balance in the proportion of hematopoietic stem cells in middle-aged and elderly mice. Among them, the proportion of hematopoietic stem cells in the Polygonatum composition group 2 was better than that in other treatment groups. Figure 4 As shown.

[0070] 3.5 Effects on the typing of lymphocytes and myeloid cells in the peripheral blood of aging mice Compared with the young control group, the peripheral blood levels of NEUT, NEUT% and MONO% in the aging model group mice were significantly increased (P<0.05, P<0.01), while LYMPH and LYMPH% were significantly decreased (P<0.01). Compared with the aging model group, the peripheral blood levels of LYMPH and LYMPH% in the Polygonatum sibiricum composition groups 1 and 2 were significantly increased (P<0.05, P<0.01), with the increase being more significant in the Polygonatum sibiricum composition group 2; the peripheral blood levels of NEUT% in the Polygonatum sibiricum saponin group, Polygonatum sibiricum composition groups 1 and 2 were significantly decreased (P<0.05); the peripheral blood levels of MONO% in the Polygonatum sibiricum composition groups 1, 2 and 3 were significantly decreased (P<0.05, P<0.01). Compared with other groups of Polygonatum sibiricum composition, the Polygonatum sibiricum composition group 2 showed better efficacy. Figure 5 As shown.

[0071] The above results indicate that Polygonatum saponins, peach gum polysaccharides, and their combination significantly regulate the myeloid differentiation bias of bone marrow hematopoietic stem cells in aging mice, thereby achieving an anti-immunoaging effect. Among them, the combination group (Polygonatum saponins: peach gum polysaccharides = 2:1) has the most significant effect. Its anti-immunoaging effect is related to regulating the ratio of myeloid cells and lymphocytes in the bone marrow of aging mice and the differentiation tendency of hematopoietic stem cells.

[0072] VII. Effects of Polygonatum saponins and Polygonatum polysaccharides on peripheral blood lymphocytes and myeloid cells in aging mice 1. Experimental Materials 1.1 Grouping and Administration: 15-month-old C57BL / 6J naturally aging mice were used as aging model animals and randomly divided into three groups: aged model group, Polygonatum saponin 400 mg / kg group, and Polygonatum polysaccharide 400 mg / kg group. Six-week-old C57BL / 6J mice were used as a young control group. The young control group and aged model group were given pure water, while the drug administration groups were administered the corresponding dose of drug by gavage at a volume of 10 mL / kg. -1 Once a day for four consecutive weeks.

[0073] 1.2 Instruments: CytoFlex S flow cytometer (Beckman Coulter), fully automated blood analyzer (XT-2000i).

[0074] 2. Experimental Methods 2.1 Effects on the typing of lymphocytes and myeloid cells in the peripheral blood of aging mice After fasting for 12 hours but not drinking water, blood was drawn from the fundus venous plexus and placed in an EDTA anticoagulant tube. The number and proportion of lymphocytes, neutrophils, etc. in the whole blood were measured using a fully automated blood analyzer.

[0075] 2.2 Effects on the proportion of peripheral blood lymphocytes and their subsets in aging mice Blood was drawn from the posterior orbital venous plexus into an anticoagulant tube containing EDTA. 50 μl of anticoagulated blood was transferred to a flow cytometry tube. Blank, single-positive, and sample tubes were prepared. The blank tube contained no antibody. The single-positive tube contained the corresponding antibody. The sample tubes contained PE-Cy™ 7Hamster Anti-Mouse CD3e, FITC Rat Anti-Mouse CD45R / B220, PE Hamster Anti-Mouse CD49b, APC Rat Anti-Mouse CD4, BV510 Rat Anti-Mouse CD8a, PE-CF594 Rat Anti-Mouse CD44, and Percp / Cyanine 5.5 anti-mouse CD62L. The mixture was incubated in the dark, then 1×Lysing Buffer was added. The mixture was incubated in the dark for 20 min, centrifuged at 1100 r / min for 5 min, and the supernatant was discarded. 350 μl of the sample was then used. After resuspending in PBS, the proportions of memory helper T cells (THM) (CD4+CD44+CD62L+) and effector helper T cells (THE) (CD4+CD44+CD62L-) in TH cells were measured; the proportions of naïve killer T cells (TCN) (CD8+CD44+CD62L+) and effector killer T cells (TCE) (CD8+CD44+CD62L-) in TC cells were also measured.

[0076] 3. Experimental Results 3.1 Effects on the typing of lymphocytes and myeloid cells in the peripheral blood of aging mice like Figure 6 As shown, compared with the young control group, the peripheral blood LYMPH and LYMPH% of the aging model group mice were significantly decreased, while NEUT% was significantly increased. P <0.01). Compared with the aging model group, the peripheral blood LYMPH and LYMPH% were significantly increased, and NEUT% was significantly decreased in the 400 mg / kg group of Polygonatum saponins. P <0.05; In the group with Polygonatum polysaccharide 400mg / kg, only LYMPH was significantly elevated in peripheral blood ( P <0.05).

[0077] Compared with the young control group, the proportion of TCN cells and THM cells in the peripheral blood of mice in the aging model group was significantly reduced; while the proportion of THE cells was significantly increased. P <0.05, P <0.01. Compared with the aging model group, the proportion of TCE cells and THE cells in the peripheral blood of mice in the Polygonatum saponin 400 mg / kg group was significantly decreased, while the proportion of THM cells and TCN cells was significantly increased.P <0.05, P <0.01); In the group of mice with Polygonatum saponins 400 mg / kg, only the proportion of TCN cells in peripheral blood was significantly increased ( P <0.05); compared with the Polygonatum polysaccharide group, the Polygonatum saponin 400mg / kg group showed better efficacy in regulating the above indicators, such as Figure 6 As shown.

[0078] The above results indicate that Polygonatum saponins can increase the number and proportion of lymphocytes in the peripheral blood of aging mice and decrease the proportion of neutrophils, thereby regulating the ratio of myeloid to lymphocytes in peripheral blood. Furthermore, Polygonatum saponins can also regulate the distribution of T lymphocyte subsets, increasing the proportion of naive T cells while decreasing the proportion of effector T cells. In conclusion, Polygonatum saponins have a regulatory effect on the peripheral export bias of lymphocytes and myeloid cells, and this effect is significantly superior to that of Polygonatum polysaccharides.

[0079] The foregoing description of embodiments of the present invention, through which those skilled in the art are able to implement or use the present invention, will be readily apparent to those skilled in the art. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novelty disclosed herein.

Claims

1. A composition for regulating the myeloid differentiation bias of senescent bone marrow hematopoietic stem cells and achieving the purpose of anti-immune aging, characterized in that, The composition is composed of the following effective components: Polygonatum sibiricum extract; Peach gum polysaccharide extract; Wherein: 1) The mass ratio of the two is (1-3):(1-2) based on the dry weight of the Polygonatum sibiricum extract and the peach gum polysaccharide extract; 2) The total saponin content in the Polygonatum sibiricum extract is 20-35wt%; 3) The polysaccharide purity of the peach gum polysaccharide extract is 80-95wt%.

2. The composition of claim 1, wherein, The mass ratio of the Polygonatum sibiricum extract and the peach gum polysaccharide extract is 2:

1.

3. The composition according to claim 1 or 2, characterized in that, The extract of saponins of polygonatum is derived from the dried rhizome of Polygonatum kingianum Coll. et Hemsl., Polygonatum sibiricum Red. or Polygonatum cyathopetalum Hua. Polygonatum kingianum The extract of saponins of polygonatum is derived from the dried rhizome of Polygonatum kingianum Coll. et Hemsl., Polygonatum sibiricum Red. or Polygonatum cyathopetalum Hua. Polygonatum sibiricum The extract of saponins of polygonatum is derived from the dried rhizome of Polygonatum kingianum Coll. et Hemsl., Polygonatum sibiricum Red. or Polygonatum cyathopetalum Hua. Polygonatum cyrtonema The extract of saponins of polygonatum is derived from the dried rhizome of Polygonatum kingianum Coll. et Hemsl., Polygonatum sibiricum Red. or Polygonatum cyathop 4. Oral formulation comprising the composition according to any one of claims 1 to 3, characterized in that, The oral preparation is selected from one or more of tablets, capsules, granules, oral liquids, oral solutions or oral emulsions, and can further contain pharmaceutically acceptable fillers, disintegrants, binders, wetting agents, sweeteners and / or flavoring agents.

5. The oral formulation according to claim 4, characterized in that, The mass percentage of the Polygonatum sibiricum extract and the peach gum polysaccharide extract in the composition is 1-60wt% based on the total mass of the preparation.

6. A process for the preparation of a composition according to any one of claims 1 to 3, characterised in that, The method comprises the following steps: 1) Preparation of Polygonatum sibiricum extract: ① Take Polygonatum sibiricum medicinal materials, add 8-12 times the volume of 75-95% ethanol, and soak at 65-75°C for 2-3 times of reflux extraction, each for 1.5-2h, and combine the ethanol extract; ② After the extraction residue is left to stand to volatilize the residual ethanol, add 8-12 times the volume of water, and extract with water at 65-75°C for 2-3 times, each for 1.5-2h, combine the water extract and concentrate; ③ The concentrated water extract is subjected to water extraction and alcohol precipitation, 75-95% ethanol is added to adjust the pH of the solution to 5.5-6.0, stand for 5-6h, then filter, take the supernatant, combine with the ethanol extract of step ①, recover ethanol by rotary evaporation, and then add pure water to suspend; ④ Add dichloromethane, water-saturated ethyl acetate, water-saturated n-butanol and pure water to the suspension in a volume ratio of 1:1 in sequence for liquid-liquid extraction, each for 2-3 times, collect the n-butanol phase, and concentrate and dry under reduced pressure to obtain Polygonatum sibiricum n-butanol extract as the Polygonatum sibiricum saponin extract; 2) Preparation of peach gum polysaccharide extract: ① Take peach gum raw materials, add 12-15 times the volume of water, heat and reflux for 1-2h, repeat 2 times, filter and combine the filtrate, and concentrate under reduced pressure; ② Add 1 / 4-1 / 3 times the volume of Sevage reagent to the concentrated solution, shake at room temperature for 20-25min, then centrifuge at 2000r / min for 5min, collect the supernatant, and repeat the deproteinization step 3-4 times; ③ Add 3-4 times the volume of 80-90% ethanol to the combined supernatant, stand overnight at 4°C, and collect the precipitate by filtration to obtain crude peach gum polysaccharide; ④ Dissolve the crude peach gum polysaccharide and load it onto a DEAE-52 cellulose column, elute with 0, 0.1 and 0.2mol / L NaCl solution in sequence, detect the absorbance of the eluate online by anthrone-sulfuric acid method, collect the elution fractions with absorbance>0.3, concentrate at 50°C and freeze-dry to obtain peach gum polysaccharide extract; 3) Preparation of the composition: The obtained polygonatum sibiricum redoute saponin extract and the obtained peach gum polysaccharide extract are mixed according to the mass ratio, pharmaceutically acceptable adjuvants are added, and the mixture is mixed, dried and shaped to obtain the composition.

7. The production method according to claim 6, wherein In step 1), the volume ratio of polygonatum sibiricum redoute medicinal materials to 95% ethanol is 1:10-1:12, and the number of ethanol extraction is 3 times and the number of water extraction is 3 times; in step 2), the volume ratio of dichloromethane to n-butanol in Sevage reagent is 4:1, and the deproteinization step is repeated 4 times.

8. Use of a composition according to any one of claims 1 to 3 for the manufacture of a medicament for modulating the myeloid differentiation bias of senescent bone marrow hematopoietic stem cells, characterized in that, The drug is used for: reducing the proportion of myeloid common progenitor cells CMP, granulocyte-monocyte progenitor cells GMP and megakaryocyte-erythroid progenitor cells MEP in the aging bone marrow; increasing the proportion of lymphoid common progenitor cells CLP; reducing the proportion of bone marrow myeloid cells, including inflammatory monocytes, granulocytes and macrophages; increasing the proportion of B cells, T cells and NK cells; to improve the imbalance of bone marrow hematopoiesis related to aging.

9. Use according to claim 8, characterized in that, The drug is an oral preparation suitable for middle-aged and elderly individuals with signs of immune aging, and is used to improve the problems of decreased immune function, increased susceptibility to infection and weakened vaccine response caused by aging.

10. Use of the composition according to any one of claims 1-3 in the preparation of health products for increasing immunity.

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