A notoginseng anti-aging active polysaccharide, and a preparation method and application thereof

Abstract

This invention discloses an anti-aging active polysaccharide from *Codonopsis pilosula*, its preparation method, and its application, relating to the field of traditional Chinese medicine extraction technology. The polysaccharide is a crude polysaccharide from *Codonopsis pilosula*, with a weight-average molecular weight of (3-4) × 10⁻⁶. 4 Da, a triple helix structure, comprises the following monosaccharide components: glucose, xylose, galactose, mannose, and arabinose. The *Codonopsis pilosula* polysaccharide of this invention can delay D-galactose-induced aging of liver and brain tissue in mice, and can also activate T lymphocytes and B lymphocytes in the thymus and spleen, thus inhibiting aging through immune regulation. It has the potential to be used in the preparation of anti-aging or anti-aging pharmaceuticals, health products, and functional foods. This invention solves the problems of complex preparation, poor activity, and unclear composition of *Codonopsis pilosula* polysaccharides in the prior art.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extraction technology, specifically to an anti-aging active polysaccharide from *Codonopsis pilosula*, its preparation method, and its application. Background Technology

[0002] The root of *Anthriscus sylvestris* (L.) Hoffm is the root of *Anthriscus sylvestris*, a plant belonging to the genus *Anthriscus* in the family Apiaceae.

[0003] Polysaccharides are high-molecular-weight polymers widely found in organisms, composed of various monosaccharides linked by glycosidic bonds. They typically possess high biological activity. Numerous studies have found that natural polysaccharides from plants and fungi exhibit immunomodulatory, anti-aging, anti-tumor, antioxidant, and antibacterial activities. Polysaccharides extracted from plants and fungi such as Codonopsis pilosula, Astragalus membranaceus, Lycium barbarum, and Ganoderma lucidum possess anti-aging activity. They mainly delay aging by enhancing immunity, improving antioxidant capacity, controlling cell division and differentiation, regulating cell growth and aging, and maintaining normal metabolism in the organism. The activity of polysaccharides is influenced by their structural properties, including monosaccharide composition, functional groups, and spatial conformation. According to current research, polysaccharides with a triple-helix structure are the most active.

[0004] Current research on the active ingredients of *Codonopsis pilosula* both domestically and internationally mainly focuses on small molecules, including lignans, phenylpropanoids, and coumarins. However, research on *Codonopsis pilosula* polysaccharides has not been conducted abroad and is also limited domestically. Currently, there are no studies or technological applications of *Codonopsis pilosula* polysaccharides in anti-aging activities, and the structure of *Codonopsis pilosula* polysaccharides is still unclear. Furthermore, the traditional pretreatment process of *Codonopsis pilosula* roots involves peeling, which to some extent leads to the loss of active ingredients. Completely dried *Codonopsis pilosula* roots also have problems such as being too hard, difficult to crush, and difficult to dissolve, which are not conducive to the extraction of effective components such as polysaccharides. Therefore, there is an urgent need for a *Codonopsis pilosula* polysaccharide with anti-aging activity. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide an anti-aging active polysaccharide from *Codonopsis pilosula*, its preparation method, and its application, thereby resolving the issues of complex preparation, poor activity, and unclear composition of existing *Codonopsis pilosula* polysaccharides.

[0006] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A crude polysaccharide of *Codonopsis pilosula* with an anti-aging activity is provided, which has a weight-average molecular weight of (3-4)×10⁻⁶. 4 Da has a triple helix structure and includes the following monosaccharide components: glucose, xylose, galactose, mannose, and arabinose.

[0007] The beneficial effects of this invention are as follows: the monosaccharide composition of polysaccharides has an important influence on their biological activity. The monosaccharide composition of the Codonopsis pilosula polysaccharide of this invention contains glucose, xylose, galactose, mannose and arabinose, which have good activity. Therefore, the Codonopsis pilosula polysaccharide of this invention has good anti-aging activity.

[0008] Based on the above technical solution, the present invention can be further improved as follows:

[0009] Furthermore, the weight-average molecular weight is 3.42 × 10⁻⁶. 4 Da.

[0010] Furthermore, the molar ratio of the monosaccharide components glucose, xylose, galactose, mannose and arabinose is 80-90:3-7:2-6:1-5:0.1-2.

[0011] The beneficial effects of adopting the above-mentioned further technical solutions are as follows: the anti-aging activity of the monosaccharide composition of the present invention is very significant.

[0012] Furthermore, the molar ratio of the monosaccharide components glucose, xylose, galactose, mannose, and arabinose is 86.83:6.13:3.56:3.06:0.42.

[0013] This invention also provides a method for preparing the above-mentioned anti-aging active polysaccharide from *Codonopsis pilosula*, comprising the following steps:

[0014] (1) Dry the fresh medicinal material of Emei ginseng root with skin to half of its original moisture content, slice it, crush it for the first time, dry it completely, crush it for the second time, sieve it, and obtain Emei ginseng powder.

[0015] (2) Extract the ginseng powder obtained in step (1) by reflux with ethanol solution, filter the residue and dry it to obtain the residue powder.

[0016] (3) The residue powder obtained in step (2) is sonicated in NaOH solution and then centrifuged to obtain supernatant 1;

[0017] (4) Adjust the pH of the supernatant obtained in step (3) to 4-5 with glacial acetic acid, centrifuge, and obtain supernatant II.

[0018] (5) The supernatant obtained in step (4) is concentrated to obtain a concentrated solution. Ethanol solution is added for precipitation, centrifugation is performed, the precipitate is collected, and then washed and dried in sequence to obtain the anti-aging active polysaccharide of Ephedra sinica.

[0019] The beneficial effects of this invention are as follows: The raw material used in this invention is the root of *Gynostemma pentaphyllum* with its skin intact. *Gynostemma pentaphyllum* has a long history of use in traditional medicine and food, thus the raw material is abundant and safe to use. Furthermore, in this invention, the *Gynostemma pentaphyllum* is not peeled, allowing for full utilization of the medicinal material. This solves the problem of wasted active ingredients caused by peeling during the traditional pre-processing of *Gynostemma pentaphyllum* root, and addresses the issues of the root being too hard, difficult to pulverize, and difficult to dissolve components after complete drying. This invention uses fresh *Gynostemma pentaphyllum* for processing while it is still fresh. By controlling the moisture content during pulverization, pulverizing while it is semi-dry improves operability and pulverization effect, thereby promoting the dissolution of polysaccharide components, increasing the extraction rate and efficiency of *Gynostemma pentaphyllum* polysaccharides, and avoiding the loss of medicinal liquid during wet pulverization. It also facilitates further drying. Simultaneously, pulverizing the *Gynostemma pentaphyllum* root with its skin intact allows for the mixed pulverization of the softer skin and the harder root, which also helps solve problems such as high pulverization difficulty, poor effect, dust generation, and difficulties in subsequent extraction. This invention first uses ethanol extraction to remove lipid-soluble small molecule components, and then uses glacial acetic acid to adjust the pH and remove proteins. This process is beneficial for obtaining purer polysaccharides with well-defined structures, increasing the content of active ingredients in the extract and thus improving activity. It also improves the reproducibility of structural identification and activity test results. Since *Codonopsis pilosula* polysaccharides have better solubility in dilute alkaline solutions, using dilute NaOH solution for alkaline extraction can increase the extraction rate. Simultaneously, dilute alkali can reduce the dissolution of substances such as DNA and proteins. Ultrasonic-assisted extraction is employed, utilizing the cavitation, mechanical, and thermal effects of ultrasound to increase the frequency and speed of molecular motion, disrupt cell walls, enhance intracellular mass transfer, and increase solvent penetration, thereby improving the polysaccharide extraction rate.

[0020] Furthermore, in step (1), when the medicinal material is dried to half of its original moisture content, the moisture content is 5-30 wt%.

[0021] The beneficial effects of adopting the above-mentioned further technical solution are as follows: the water content of 5-30wt% is moderate, which avoids the problems of the dried ginseng being too hard, difficult to crush, and difficult to dissolve components, and also avoids the loss of medicinal liquid during wet crushing, and is also conducive to further drying.

[0022] Further, it is first crushed into coarse powder.

[0023] The beneficial effect of adopting the above-mentioned further technical solution is that it avoids the problem of the fine powder sticking together when the medicinal materials are directly crushed into fine powder when they are semi-dry.

[0024] Furthermore, the coarse powder has a particle size of 2-3 mm.

[0025] Furthermore, it is further pulverized into fine powder a second time.

[0026] The beneficial effects of adopting the above-mentioned further technical solutions are: increasing the specific surface area of ​​medicinal material particles, which is conducive to the leaching of effective components during subsequent extraction, improving the extraction rate and shortening the extraction time.

[0027] Furthermore, the fine powder has a particle size of 0.3-0.5 mm.

[0028] The beneficial effects of adopting the above-mentioned further technical solution are as follows: This is a moderate fine powder particle size, which is conducive to increasing the specific surface area of ​​medicinal material particles and promoting the leaching of effective ingredients, without being too fine and causing difficulties in filtering and separating the medicinal residue.

[0029] Furthermore, it is passed through a 50-70 mesh sieve.

[0030] The beneficial effects of adopting the above-mentioned further technical solutions are: sieving can obtain fine powder of medicinal materials with the most uniform particle size, which is conducive to the reproducibility of extraction effect.

[0031] Furthermore, in step (1), the fresh medicinal material of Emei ginseng root with skin was collected from Emei Mountain, Sichuan Province.

[0032] Furthermore, in step (2), the ratio of ginseng powder to ethanol solution is 1g:8-12mL.

[0033] The beneficial effect of adopting the above-mentioned further technical solution is that the above-mentioned material-liquid ratio is sufficient to fully dissolve and remove fat-soluble components.

[0034] Furthermore, in step (2), the sample is refluxed at 60-90℃ for 2-3 hours.

[0035] The beneficial effects of adopting the above-mentioned further technical solution are as follows: controlling the temperature at 60-90℃, appropriate heating improves the solubility and diffusion coefficient of fat-soluble components, thereby promoting the dissolution and removal of fat-soluble components in ethanol, and the temperature is not too high, so the effective component polysaccharide will not be degraded. Repeating the process 2-3 times in 2-3 hours is sufficient to fully dissolve and remove fat-soluble components.

[0036] Furthermore, in step (2), the volume fraction of the ethanol solution is 90-98%.

[0037] Furthermore, in step (2), the volume fraction of the ethanol solution is 95%.

[0038] Furthermore, the reflux extraction was repeated 2-3 times.

[0039] The beneficial effects of adopting the above-mentioned further technical solution are: repeated reflux extraction 2-3 times can fully remove fat-soluble components.

[0040] Furthermore, in step (3), the mass ratio of the residue powder to the NaOH solution is 1:8-12.

[0041] The beneficial effects of adopting the above-mentioned further technical solution are: a more suitable material-to-liquid ratio, which can fully extract polysaccharides without excessive solvent making subsequent concentration difficult.

[0042] Furthermore, the concentration of the NaOH solution is 0.01-0.03 mol / L.

[0043] The beneficial effects of adopting the above-mentioned further technical solutions are as follows: the solubility of Panax notoginseng polysaccharide in dilute alkaline solution is better, the concentration of dilute NaOH solution of 0.01-0.03 mol / L can improve the extraction rate, and at the same time, dilute alkali can reduce the dissolution of substances such as DNA and protein.

[0044] Furthermore, the ultrasound was performed at room temperature for 1.5-2.5 hours.

[0045] The advantages of adopting the above-mentioned further technical solution are: mild room temperature conditions, simplicity, and ease of implementation. Ultrasonic-assisted extraction utilizes the cavitation, mechanical, and thermal effects of ultrasound to increase the frequency and speed of molecular motion, disrupt cell walls, enhance intracellular mass transfer, and increase solvent penetration, thereby improving the polysaccharide extraction rate. Thanks to ultrasound assistance, polysaccharides can be fully extracted in 1.5-2.5 hours.

[0046] Furthermore, ultrasound was performed at 35-45 kHz and 700-800 W.

[0047] The beneficial effects of adopting the above-mentioned further technical solution are as follows: it is easy to achieve the working frequency and power of commonly available ultrasonic instruments. Under this ultrasonic condition, the cell wall can be effectively destroyed, mass transfer can be enhanced, and the extraction rate and extraction efficiency can be improved.

[0048] Furthermore, in step (3), centrifuge at 4500-5500 rpm for 8-12 min.

[0049] The beneficial effects of adopting the above-mentioned further technical solution are as follows: the extracted polysaccharide-containing solution has a high viscosity, and centrifugation instead of filtration is used to separate the polysaccharide solution and the residue, which makes the solid-liquid separation more efficient and can effectively solve the problem of slow filtration speed of high viscosity solutions. The conditions of 4500-5500 rpm and 8-12 min are easy to achieve and have good results, and the solid and liquid can be fully separated.

[0050] Furthermore, in step (4), the pH value is 4.5.

[0051] Furthermore, in step (5), the volume is concentrated to 1 / 4 to 1 / 3 of the original volume.

[0052] The beneficial effects of adopting the above-mentioned further technical solution are as follows: appropriate concentration of polysaccharide solution can reduce the amount of ethanol used in the subsequent alcohol precipitation step, and concentration to 1 / 4-1 / 3 of the original volume will not be too concentrated and will cause the large polysaccharide molecules to swell, thus affecting the alcohol precipitation effect.

[0053] Furthermore, in step (5), the volume ratio of the concentrate to the ethanol solution is 1:3-5.

[0054] The beneficial effect of adopting the above-mentioned further technical solution is that after the polysaccharide solution is added to 3-5 times the volume of ethanol, the final concentration of ethanol is high enough to allow the polysaccharide to fully precipitate and obtain Ephedra polysaccharide solid powder.

[0055] Furthermore, in step (5), precipitation is carried out at 3-6℃ for 20-25 hours.

[0056] The beneficial effects of adopting the above-mentioned further technical solution are as follows: the low temperature of 3-6℃ can further reduce the solubility of polysaccharides in ethanol-rich solutions, which is conducive to precipitation. Letting it stand for 20-25 hours allows the polysaccharides to fully precipitate.

[0057] Furthermore, in step (5), the volume fraction of the ethanol solution is 90-98%.

[0058] Furthermore, in step (5), the volume fraction of the ethanol solution is 95%.

[0059] The beneficial effects of adopting the above-mentioned further technical solution are: 95% ethanol has high purity, which can meet the requirements for fully analyzing polysaccharides, and the 95% ethanol used is common and inexpensive.

[0060] Furthermore, in step (5), anhydrous ethanol is used for washing.

[0061] The beneficial effect of adopting the above-mentioned further technical solution is that using anhydrous ethanol when washing polysaccharide precipitate can prevent the polysaccharide precipitate from being partially redissolved in water and lost.

[0062] Furthermore, in step (5), the drying process is completed by vacuum drying and freeze drying in sequence.

[0063] Furthermore, vacuum drying was carried out for 20-30 minutes at a vacuum level of 0.09-0.1 MPa and room temperature.

[0064] The beneficial effects of adopting the above-mentioned further technical solution are as follows: under the vacuum drying conditions, most of the residual ethanol in the Codonopsis pilosula polysaccharide powder can be removed, and subsequent freeze drying can completely remove the residual ethanol and any water that may be present, thus obtaining dry Codonopsis pilosula polysaccharide powder. Vacuum drying followed by freeze drying can greatly shorten the freeze drying time, and the vacuum drying time is also not long, so the overall drying time is shorter.

[0065] This invention also provides the application of the above-mentioned ginseng anti-aging active polysaccharide in the preparation of anti-aging or anti-aging pharmaceutical preparations, health products or functional foods.

[0066] The present invention also provides a pharmaceutical preparation, health product or functional food, including the above-mentioned anti-aging active polysaccharide of Codonopsis pilosula.

[0067] The present invention has the following beneficial effects:

[0068] 1. The anti-aging active polysaccharide of the present invention can improve the depressive mood and cognitive ability of D-galactose-aged mice, improve liver function indicators, promote liver lipid metabolism, maintain the balance of the cholinergic system, increase antioxidant enzyme activity, reduce the level of pro-inflammatory factors, downregulate the expression of aging genes p53 and p21, exert anti-aging activity by activating the Nrf2 / HO-1 / NQO1 signaling pathway, thereby delaying the aging of liver and brain tissue in D-galactose-induced aging mice, and can also activate T lymphocytes and B lymphocytes in the thymus and spleen, thus anti-aging through immune regulation.

[0069] 2. The anti-aging active polysaccharide prepared by this invention has a clear structure, and the structural characteristics of the polysaccharide are characterized for the first time. The weight-average molecular weight is 3.42 × 10⁻⁶. 4 The molar ratio of glucose, xylose, galactose, mannose and arabinose is 86.83:6.13:3.56:3.06:0.42, and the polysaccharide chain has a stable triple helix structure.

[0070] 3. The anti-aging activity of the ginseng root anti-aging active polysaccharide of the present invention is related to the use of fresh ginseng root with peel as raw material, the specific extraction method, and the specific structure of the polysaccharide obtained therefrom. The ginseng root anti-aging active polysaccharide has the potential to be used to prepare anti-aging or anti-aging pharmaceutical preparations, health products, and functional foods. Attached Figure Description

[0071] Figure 1 The infrared spectrum of the anti-aging active polysaccharide of *Codonopsis pilosula* in Example 1 is shown below.

[0072] Figure 2 This is a diagram showing the Congo red test results of the anti-aging active polysaccharides from *Codonopsis pilosula* in Example 1.

[0073] Figure 3 This is a graph showing the results of monosaccharide composition determination of the anti-aging active polysaccharides of *Codonopsis pilosula* in Example 1;

[0074] Figure 4 Example 1: Brain tissue T-NOS activity of anti-aging active polysaccharides from *Codonopsis pilosula*.

[0075] Figure 5 Example 1: CAT activity of anti-aging polysaccharides from *Codonopsis pilosula* in brain tissue;

[0076] Figure 6 Example 1: Brain tissue T-AOC activity of anti-aging active polysaccharides from *Codonopsis pilosula*.

[0077] Figure 7 Example 1: Brain tissue SOD activity of anti-aging active polysaccharides from *Codonopsis pilosula*.

[0078] Figure 8Example 1: Brain tissue GSH-Px activity of anti-aging polysaccharides from *Codonopsis pilosula*.

[0079] Figure 9 Example 1: Brain tissue MDA activity of anti-aging active polysaccharides from *Codonopsis pilosula*.

[0080] Figure 10 Example 1: Liver T-NOS activity of ginseng anti-aging active polysaccharide;

[0081] Figure 11 Example 1: Liver CAT activity of the anti-aging polysaccharide from *Codonopsis pilosula*.

[0082] Figure 12 Example 1: Liver T-AOC activity of Panax notoginseng anti-aging polysaccharide;

[0083] Figure 13 Example 1: Liver SOD activity of the anti-aging polysaccharide from *Codonopsis pilosula*.

[0084] Figure 14 Example 1: Liver GSH-Px activity of the anti-aging polysaccharide from *Codonopsis pilosula*.

[0085] Figure 15 Example 1: Liver MDA activity of the anti-aging polysaccharide from *Codonopsis pilosula*.

[0086] Figure 16 Example 1: Brain tissue TNF-α levels of anti-aging active polysaccharides from *Codonopsis pilosula*.

[0087] Figure 17 Example 1: Brain tissue IL-1β levels of anti-aging active polysaccharides from *Codonopsis pilosula*.

[0088] Figure 18 Example 1: Brain tissue IL-6 levels of anti-aging active polysaccharides from *Codonopsis pilosula*.

[0089] Figure 19 Example 1: Liver TNF-α levels of anti-aging active polysaccharides from *Codonopsis pilosula*.

[0090] Figure 20 The liver IL-1β level of the anti-aging active polysaccharide of Codonopsis pilosula in Example 1;

[0091] Figure 21 The liver IL-6 level of the anti-aging active polysaccharide of Codonopsis pilosula in Example 1;

[0092] Figure 22 Example 1: Brain tissue p53 mRNA expression level of anti-aging active polysaccharides from *Codonopsis pilosula*.

[0093] Figure 23 Example 1: Brain tissue p21 mRNA expression level of anti-aging active polysaccharide from *Codonopsis pilosula*.

[0094] Figure 24 The expression level of liver p53 mRNA of the anti-aging active polysaccharide of *Codonopsis pilosula* in Example 1;

[0095] Figure 25 The expression level of liver p21 mRNA of the anti-aging active polysaccharide of Codonopsis pilosula in Example 1;

[0096] Figure 26 This is a brain tissue-related protein band diagram of the anti-aging active polysaccharide of *Codonopsis pilosula* from Example 1;

[0097] Figure 27 The relative expression level of Nrf2 protein in brain tissue of the anti-aging active polysaccharide of Codonopsis pilosula in Example 1;

[0098] Figure 28 The relative expression level of NQO1 protein in brain tissue of the anti-aging active polysaccharide of Codonopsis pilosula in Example 1;

[0099] Figure 29 The relative expression level of HO-1 protein in brain tissue of the anti-aging active polysaccharide of Codonopsis pilosula in Example 1;

[0100] Figure 30 This is a liver-related protein band diagram of the anti-aging active polysaccharide of *Codonopsis pilosula* in Example 1;

[0101] Figure 31 The relative expression level of Nrf2 protein in the liver of the anti-aging active polysaccharide of Codonopsis pilosula in Example 1;

[0102] Figure 32 The relative expression level of NQO1 protein in the liver of the anti-aging active polysaccharide of Codonopsis pilosula in Example 1;

[0103] Figure 33 The relative expression level of liver HO-1 protein in the anti-aging active polysaccharide of Codonopsis pilosula in Example 1. Detailed Implementation

[0104] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0105] Example 1:

[0106] A crude polysaccharide from *Codonopsis pilosula* with anti-aging activity was discovered, having a weight-average molecular weight of 3.42 × 10⁻⁶. 4 Da has a triple helix structure, and its monosaccharide components are glucose, xylose, galactose, mannose and arabinose in a molar ratio of 86.83:6.13:3.56:3.06:0.42.

[0107] A polysaccharide with anti-aging activity from *Codonopsis pilosula* is prepared by the following steps:

[0108] (1) 500g of fresh Emeishan root with skin was collected from Emeishan, Sichuan Province and dried to a moisture content of 20wt% (a small sample was taken and the moisture content of the medicinal material was determined to be 20wt% by drying method). The root was sliced, crushed into coarse powder (particle size of 2-3mm) for the first time, then dried completely, crushed into fine powder (particle size of 0.3-0.5mm) for the second time, and passed through a 60-mesh sieve to obtain Emeishan powder.

[0109] (2) With a material-to-liquid ratio of 1:10 g / mL, the ginseng powder obtained in step (1) was extracted by reflux extraction with ethanol solution (95 wt%) at 80°C for 2.5 h to remove fat-soluble small molecule components. The extraction was repeated twice, the residue was filtered and dried to obtain the residue powder.

[0110] (3) Add the residue powder obtained in step (2) to NaOH solution (0.01mol / L) at a mass ratio of 1:10, sonicate at room temperature, 40kHz and 760W for 2h, and then centrifuge at 5000rpm for 10min to obtain supernatant.

[0111] (4) Adjust the pH of the supernatant obtained in step (3) to 4.5 with glacial acetic acid, centrifuge to remove the precipitated protein, and obtain supernatant II.

[0112] (5) The supernatant obtained in step (4) was concentrated to 1 / 5 of its original volume using a rotary evaporator to obtain a concentrated solution. An ethanol solution (95 wt%) was added at a volume ratio of 1:4, and the solution was precipitated at 4°C for 24 h. The precipitate was collected by centrifugation, washed with anhydrous ethanol, and vacuum dried at 0.095 MPa and room temperature for 25 min. The solution was then freeze-dried to obtain the anti-aging active polysaccharide of Codonopsis pilosula.

[0113] Example 2:

[0114] A type of anti-aging active polysaccharide from *Codonopsis pilosula*, specifically crude *Codonopsis pilosula* polysaccharide, has a weight-average molecular weight of 3 × 10⁻⁶. 4 Da has a triple helix structure, and its monosaccharide components are glucose, xylose, galactose, mannose and arabinose in a molar ratio of 80:3:2:1:0.1.

[0115] A polysaccharide with anti-aging activity from *Codonopsis pilosula* is prepared by the following steps:

[0116] (1) 500g of fresh Emeishan root with skin was collected from Emeishan, Sichuan Province and dried to a moisture content of 5wt% (a small sample was taken and the moisture content of the medicinal material was determined to be 5wt% by drying method). The root was sliced, crushed into coarse powder (particle size of 2-3mm) for the first time, then dried completely, crushed into fine powder (particle size of 0.3-0.5mm) for the second time, and passed through a 50-mesh sieve to obtain Emeishan powder.

[0117] (2) With a material-to-liquid ratio of 1:8 g / mL, the ginseng powder obtained in step (1) was extracted by reflux at 60°C for 3 hours using an ethanol solution (90 vt%) to remove fat-soluble small molecule components. The extraction was repeated twice, the residue was filtered and dried to obtain the residue powder.

[0118] (3) Add the residue powder obtained in step (2) to NaOH solution (0.01mol / L) at a mass ratio of 1:8, sonicate at room temperature, 35kHz and 800W for 2.5h, and then centrifuge at 4500rpm for 8min to obtain supernatant.

[0119] (4) Adjust the pH of the supernatant obtained in step (3) to 4 using glacial acetic acid, centrifuge to remove the precipitated protein, and obtain supernatant II.

[0120] (5) The supernatant obtained in step (4) is concentrated to 1 / 3 of its original volume using a rotary evaporator.

[0121] The concentrated solution was added to an ethanol solution (90 wt%) at a volume ratio of 1:3, and precipitated at 3°C ​​for 25 h. After centrifugation, the precipitate was collected, washed with anhydrous ethanol, vacuum dried at 0.09 MPa and room temperature for 30 min, and then freeze-dried to obtain the anti-aging active polysaccharide of Codonopsis pilosula.

[0122] Example 3:

[0123] A type of anti-aging active polysaccharide from *Codonopsis pilosula*, specifically crude *Codonopsis pilosula* polysaccharide, has a weight-average molecular weight of 4 × 10⁻⁶. 4 Da has a triple helix structure, and its monosaccharide components are glucose, xylose, galactose, mannose and arabinose in a molar ratio of 90:7:6:5:2.

[0124] A polysaccharide with anti-aging activity from *Codonopsis pilosula* is prepared by the following steps:

[0125] (1) 500g of fresh Emeishan root with skin was collected from Emeishan, Sichuan Province and dried to a moisture content of 30wt% (a small sample was taken and the moisture content of the medicinal material was determined to be 30wt% by drying method). The root was sliced, crushed into coarse powder (particle size of 2-3mm) for the first time, then dried completely, crushed into fine powder (particle size of 0.3-0.5mm) for the second time, and passed through a 70-mesh sieve to obtain Emeishan powder.

[0126] (2) With a material-to-liquid ratio of 1:12 g / mL, the ginseng powder obtained in step (1) was extracted by reflux at 90°C for 2 hours using an ethanol solution (98 wt%) to remove fat-soluble small molecule components. This process was repeated 3 times. The residue was filtered and dried to obtain the residue powder.

[0127] (3) Add the residue powder obtained in step (2) to NaOH solution (0.03mol / L) at a mass ratio of 1:12, sonicate at room temperature, 45kHz and 800W for 1.5h, and then centrifuge at 5500rpm for 8min to obtain supernatant.

[0128] (4) Adjust the pH of the supernatant obtained in step (3) to 5 with glacial acetic acid, centrifuge to remove the precipitated protein, and obtain supernatant II.

[0129] (5) The supernatant obtained in step (4) was concentrated to 1 / 4 of its original volume using a rotary evaporator to obtain a concentrated solution. An ethanol solution (98 wt%) was added at a volume ratio of 1:5. The solution was precipitated at 6°C for 20 h. After centrifugation, the precipitate was collected and washed with anhydrous ethanol. The solution was then vacuum dried at 0.1 MPa and room temperature for 20 min and freeze-dried to obtain the anti-aging active polysaccharide of Codonopsis pilosula.

[0130] Test case

[0131] I. Structural Inspection

[0132] The anti-aging active polysaccharide of *Codonopsis pilosula* prepared in Example 1 was subjected to infrared spectroscopy, molecular weight, Congo red test and monosaccharide composition detection.

[0133] The specific method for infrared spectroscopy determination is as follows: The *Codonopsis pilosula* polysaccharide sample is dried at low temperature in a vacuum drying oven. 5-10 mg of sample powder is taken, pressed into a pellet using KBr, and incubated at 4000-500 cm⁻¹. -1 Fourier transform infrared spectroscopy was performed, and the results are shown below. Figure 1 ;

[0134] The molecular weight determination method was as follows: HPGPC (High Performance Gel Permeation Chromatography) was used, with a gel column (8.0 mm × 300 mm, 35℃) and a refractive index detector (30℃) to determine the relative molecular mass of the polysaccharide. The sample solution (20 μL, 5 mg / mL) was injected into the instrument. The mobile phase was deionized water, and the flow rate was 0.8 mL / min. A standard curve was established using T-series dextran. Empower software was used to establish a standard curve for the logarithm of the weight-average molecular weight (Mw) related to retention time (t) using a series of dextran solutions. The weight-average molecular weight of the sample was calculated based on the standard curve. The calculation results showed that the weight-average molecular weight was 3.42 × 10⁻⁶. 4 Da.

[0135] The specific method for detecting Congo red is as follows: 1.5 mL of 0.2 mmol / L Congo red solution and 1 mL of 2 mg / mL Panax notoginseng anti-aging active polysaccharide solution are mixed, and the final NaOH concentration is adjusted to 0, 0.05 mol / L, 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.4 mol / L, respectively. The maximum absorption wavelength λmax of the Congo red control solution and the Panax notoginseng polysaccharide mixed solution under different NaOH concentrations is then measured and plotted. The results are shown in the figure below. Figure 2 .

[0136] The specific method for detecting monosaccharide composition is as follows: Monosaccharide reference standards mannose (Man), rhamnose (Rha), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara), glucuronic acid (GlcA), and galacturonic acid (GalA) were dissolved in ultrapure water to prepare a mixed standard solution with a total molar concentration of 40 mmol / L. 5 mg of *Codonopsis pilosula* polysaccharide sample was weighed and hydrolyzed with 2 mol / L trifluoroacetic acid (TFA). Both the monosaccharide mixed standard solution and the hydrolyzed polysaccharide solution were subjected to PMP derivatization. After filtration through a 0.22 μm filter membrane, the samples were analyzed by HPLC. The content of each monosaccharide in the hydrolyzed polysaccharide sample was calculated based on the peak area and regression equation. The HPLC detection conditions were Megres. C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: A: acetonitrile, B: 0.1 mol / L phosphate buffer, pH = 6.8; detection wavelength: 254 nm; column temperature: 30 ℃; flow rate: 1 mL / min; injection volume: 10 μL; gradient elution (0-40 min: 19.5-16A%, 40-43 min: 16-15A%, 43-80 min: 15-15A%, 80-90 min: 15-19.5A%). Results are shown below. Figure 3 .

[0137] Depend on Figure 1 It can be known that 3427cm -1 The stronger bands are due to the stretching vibration of the polysaccharide -OH group; 2927 cm⁻¹ -1 This is a CH stretching vibration; 1700-1780cm -1 No absorption indicates the absence of uronic acid; 1631cm -1 It is a bending vibration of -OH; 1152 cm -1 and 1026cm -1 1081cm -1 Together, they indicate that the sugar ring of the *Panax notoginseng* polysaccharide is a pyranose ring; 890cm -1 The small peaks nearby indicate the presence of β-glycosidic bonds; 850 cm⁻¹ -1 The nearby peak indicates the presence of an α-glycosidic bond; 810 cm⁻¹ -1 The nearby peaks indicate the presence of mannose.

[0138] Depend on Figure 2 It is known that Congo red can form a complex with polysaccharides with a triple helix structure, thereby increasing the maximum absorption wavelength (red shift) within a certain range of NaOH concentration, indicating that the anti-aging active polysaccharides of Codonopsis pilosula have a triple helix structure.

[0139] Depend on Figure 3 It is known that the anti-aging active polysaccharides of Emei ginseng are composed of five monosaccharides: glucose, xylose, galactose, mannose, and arabinose. Figure 3 Peak 1 is PMP residue from the derivatization reaction, peak 2 is mannose, peak 3 is glucose, peak 4 is galactose, peak 5 is xylose, and peak 6 is arabinose. Based on the peak area of ​​each monosaccharide and the regression equation, the molar ratio of each monosaccharide in the anti-aging active polysaccharide of Emei ginseng is calculated to be 86.83:6.13:3.56:3.06:0.42.

[0140] II. Performance Testing

[0141] The following pharmacological tests are recognized detection methods in this field.

[0142] The D-galactose aging model mouse is created by long-term injection of D-galactose into mice. Excess D-galactose is converted into excess reactive oxygen species, ultimately producing advanced glycation end products (AGEs) that further accelerate the aging process, resulting in characteristics similar to natural aging. Numerous studies have shown that D-galactose injection for 6-10 weeks can induce oxidative stress, neuroinflammation, and apoptosis in mice, making this a commonly used mouse model for aging.

[0143] The effective dose of *Codonopsis pilosula* anti-aging polysaccharides for the anti-aging effect in D-galactose-induced aging model mice is 100-400 mg / kg / day, with low doses at 100 mg / kg / day, medium doses at 200 mg / kg / day, and high doses at 400 mg / kg / day. The optimal effective dose of *Codonopsis pilosula* anti-aging polysaccharides for the anti-aging effect in D-galactose-induced aging model mice is 400 mg / kg / day.

[0144] 1. Effects on antioxidant enzyme activity

[0145] The effects of the anti-aging active polysaccharide from *Codonopsis pilosula* prepared in Example 1 on the activity of antioxidant enzymes in the brain tissue and liver of D-galactose-induced aging model mice were detected. The specific detection method was as follows: Seventy-two male KM mice were acclimatized for one week and then randomly divided into six groups: blank control group (NC), model control group (MC), positive control group (VE), low-dose polysaccharide group (AP-L), medium-dose polysaccharide group (AP-M), and high-dose polysaccharide group (AP-H), with 12 mice in each group. The mice were weighed every three days, and the dosage volume (0.1 mL / 10 g) was calculated based on their weight. The NC group received a subcutaneous injection of an equal volume of physiological saline in the neck and back. The other groups received D-galactose solution (200 mg / kg / d). The NC and MC groups were administered 0.05% sodium carboxymethyl cellulose solution by gavage, the VE group was administered vitamin E suspension (200 mg / kg / d) by gavage, and the low, medium, and high dose polysaccharide groups were administered Ephedra polysaccharide solution (100, 200, and 400 mg / kg / d), respectively. Injections were administered continuously for 49 days, with gavage starting on day 15. The levels of oxidative stress factors SOD, CAT, GSH-Px, T-NOS, T-AOC, and MDA in the liver and brain tissues of mice in each group were measured according to the instructions of the biochemical reagent kits from Nanjing Jiancheng Biotechnology Co., Ltd. Results are shown below. Figure 4-15 (# indicates a significant difference from the control group (P < 0.05), ## indicates a highly significant difference from the control group (P < 0.01), * indicates a significant difference from the model group (P < 0.05), ** indicates a highly significant difference from the model group (P < 0.01), and ns indicates no statistical significance).

[0146] Depend on Figure 4-15 Compared with the NC group, the MC group showed decreased levels of SOD, CAT, T-AOC, and GSH-Px (p<0.01), and significantly increased levels of MDA and T-NOS (p<0.01), indicating that D-galactose treatment increased oxidative stress levels in the liver and brain tissues of mice. Compared with the MC group, all treatment groups showed increased levels of SOD, CAT, and T-AOC (p<0.05, p<0.01, or increased but not significantly), and decreased levels of MDA and T-NOS (p<0.01). These results indicate that Panax notoginseng polysaccharide can enhance the antioxidant capacity of mouse liver and brain tissues.

[0147] 2. Effects on inflammatory factors

[0148] The effects of the anti-aging active polysaccharide from *Gynostemma pentaphyllum* (Example 1) on inflammatory factors in the brain and liver of D-galactose-induced aging mice were investigated. The specific detection methods were as follows: animal grouping, aging model establishment, and drug administration were the same as described above. The levels of IL-6, IL-1β, and TNF-α in the liver and brain tissue of mice were detected using an ELISA kit from Jiangsu Jingmei Biotechnology Co., Ltd. The results are shown below. Figure 16-21(# indicates a significant difference from the control group (P < 0.05), ## indicates a highly significant difference from the control group (P < 0.01), * indicates a significant difference from the model group (P < 0.05), ** indicates a highly significant difference from the model group (P < 0.01), and ns indicates no statistical significance).

[0149] Depend on Figure 16-21 It was found that, compared with the NC group, the MC group showed a significant increase in pro-inflammatory cytokines in liver and brain tissue (p<0.01). Compared with the MC group, the levels of pro-inflammatory cytokines in each polysaccharide dosage group were decreased (p<0.05, p<0.01). These results indicate that Panax notoginseng polysaccharide can resist D-galactose-induced inflammatory responses in liver and brain tissue.

[0150] 3. Effects on the expression of aging gene mRNA

[0151] The effect of the anti-aging active polysaccharide of *Codonopsis pilosula* prepared in Example 1 on the expression of aging gene mRNA in the brain and liver of D-galactose-induced aging model mice was detected by real-time quantitative PCR (RT-qPCR). The specific detection method was as follows: animal grouping, aging model establishment, and drug administration were the same as above. RNA was extracted from mouse liver and brain tissue according to the TRIZOL kit instructions; RNA was reverse transcribed into cDNA according to the reverse transcription kit instructions; PCR amplification was performed using a real-time quantitative PCR instrument (95℃ for 3 min; 95℃ for 15 s; 60℃ for 60 s; temperature increase of 0.3℃ every 15 s from 60 to 95℃); data processing was performed using the ΔΔCT method. The complete gene sequences were searched from the National Center for Biotechnology Information (NCBI) database, and gene-specific primers were designed and screened using Primer Premier primer design software. Results are shown below. Figure 22-25 (# indicates a significant difference from the control group (P < 0.05), ## indicates a highly significant difference from the control group (P < 0.01), * indicates a significant difference from the model group (P < 0.05), ** indicates a highly significant difference from the model group (P < 0.01), and ns indicates no statistical significance).

[0152] Depend on Figure 22-25 The results showed that, compared with the NC group mice, the mRNA expression levels of p53 and p21 in the liver and brain tissues of mice induced by long-term D-galactose were significantly increased (p<0.01). Compared with the MC group mice, the mRNA expression levels of p21 in the liver and brain tissues of mice in each polysaccharide dosage group were significantly decreased (p<0.01), and the mRNA expression levels of p53 in the liver and brain tissues of mice in the AP-M and AP-H groups were significantly decreased (p<0.01). These results indicate that Panax notoginseng polysaccharide has an inhibitory effect on the aging proteins p53 and p21.

[0153] 4. Impact on the Nrf2 / HO-1 / NQO1 signaling pathway

[0154] The effects of the anti-aging active polysaccharide from *Codonopsis pilosula* prepared in Example 1 on the Nrf2 / HO-1 / NQO1 signaling pathway in the brain and liver of D-galactose-induced aging mice were detected by Western blotting. The specific detection method was as follows: animal grouping, aging model establishment, and drug administration were the same as above. Tissues were rinsed 2-3 times with pre-cooled PBS buffer to remove blood contamination, cut into small pieces, and placed in a homogenizer. Proteins were extracted from the tissues, and protein concentration was detected using a BCA kit. Gel was prepared, and the protein solution was denatured before loading. The membrane was transferred, blocked with skim milk powder, and incubated with primary and secondary antibodies. The membrane was exposed, developed, and fixed in a darkroom, and the films were scanned and archived. Results are shown below. Figure 26-33 (# indicates a significant difference from the control group (P < 0.05), ## indicates a highly significant difference from the control group (P < 0.01), * indicates a significant difference from the model group (P < 0.05), ** indicates a highly significant difference from the model group (P < 0.01), and ns indicates no statistical significance).

[0155] Depend on Figure 26-33 It was found that long-term administration of D-galactose significantly downregulated the expression of NQO1, HO-1, and Nrf2 in the liver and brain tissues of mice. Using ImageJ software to obtain the optical density values ​​of the protein bands, treatment with *Codonopsis pilosula* polysaccharide significantly upregulated the expression of NQO1, HO-1, and Nrf2 (p<0.01). *Codonopsis pilosula* polysaccharide can cause significant differences in the expression of these three proteins, therefore it exerts its anti-aging effect through the Nrf2 / HO-1 / NQO1 signaling pathway.

[0156] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polysaccharide with anti-aging activity from *Panax notoginseng*, characterized in that, It is a crude polysaccharide from *Codonopsis pilosula*, with a weight-average molecular weight of 3.42 × 10⁻⁶. 4 Da has a triple helix structure, and its monosaccharide components are glucose, xylose, galactose, mannose and arabinose in a molar ratio of 86.83:6.13:3.56:3.06:0.

42. The method for preparing the anti-aging active polysaccharide from Codonopsis pilosula includes the following steps: (1) 500g of fresh Emeishan root with skin was dried to a moisture content of 20wt%, sliced, and first crushed into coarse powder with a particle size of 2-3mm. Then it was completely dried and crushed into fine powder with a particle size of 0.3-0.5mm. It was then passed through a 60-mesh sieve to obtain Emeishan root powder. (2) With a material-to-liquid ratio of 1:10 g / mL, the ginseng powder obtained in step (1) was extracted by reflux at 80°C for 2.5 h using a 95% ethanol solution to remove fat-soluble small molecule components. The extraction was repeated twice, the residue was filtered and dried to obtain the residue powder. (3) Add the residue powder obtained in step (2) to a 0.01 mol / L NaOH solution at a mass ratio of 1:10, sonicate at room temperature, 40 kHz and 760 W for 2 h, and then centrifuge at 5000 rpm for 10 min to obtain supernatant. (4) Adjust the pH of the supernatant obtained in step (3) to 4.5 with glacial acetic acid, centrifuge to remove the precipitated protein, and obtain supernatant II. (5) The supernatant obtained in step (4) is concentrated to 1 / 5 of its original volume using a rotary evaporator to obtain a concentrated solution. A 95% ethanol solution is added at a volume ratio of 1:

4. The solution is precipitated at 4°C for 24 hours. After centrifugation, the precipitate is collected and washed with anhydrous ethanol. The solution is then vacuum dried at 0.095 MPa and room temperature for 25 minutes and freeze-dried to obtain the anti-aging active polysaccharide of Codonopsis pilosula.

2. The method for preparing the anti-aging active polysaccharide of *Codonopsis pilosula* according to claim 1, characterized in that, Includes the following steps: (1) 500g of fresh Emeishan root with skin was dried to a moisture content of 20wt%, sliced, and first crushed into coarse powder with a particle size of 2-3mm. Then it was completely dried and crushed into fine powder with a particle size of 0.3-0.5mm. It was then passed through a 60-mesh sieve to obtain Emeishan root powder. (2) With a material-to-liquid ratio of 1:10 g / mL, the ginseng powder obtained in step (1) was extracted by reflux at 80°C for 2.5 h using a 95% ethanol solution to remove fat-soluble small molecule components. The extraction was repeated twice, the residue was filtered and dried to obtain the residue powder. (3) Add the residue powder obtained in step (2) to a 0.01 mol / L NaOH solution at a mass ratio of 1:10, sonicate at room temperature, 40 kHz and 760 W for 2 h, and then centrifuge at 5000 rpm for 10 min to obtain supernatant. (4) Adjust the pH of the supernatant obtained in step (3) to 4.5 with glacial acetic acid, centrifuge to remove the precipitated protein, and obtain supernatant II. (5) The supernatant obtained in step (4) is concentrated to 1 / 5 of its original volume using a rotary evaporator to obtain a concentrated solution. A 95% ethanol solution is added at a volume ratio of 1:

4. The solution is precipitated at 4°C for 24 hours. After centrifugation, the precipitate is collected and washed with anhydrous ethanol. The solution is then vacuum dried at 0.095 MPa and room temperature for 25 minutes and freeze-dried to obtain the anti-aging active polysaccharide of Codonopsis pilosula.

3. The use of the ginseng anti-aging active polysaccharide according to claim 1 in the preparation of anti-aging or anti-aging pharmaceutical preparations.

4. A pharmaceutical preparation, characterized in that, Including the anti-aging active polysaccharide of Codonopsis pilosula as described in claim 1.

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