NL2036893A

NL2036893A - Polygonum multiflorum polysaccharide and extraction method and application thereof

Info

Publication number: NL2036893A
Application number: NL2036893A
Authority: NL
Inventors: Ma Shuangcheng; Yu Jiandong; Yang Jianbo; Wang Ying; Jin Hongyu; Cheng Xianlong; Wei Feng; Fan Jin
Original assignee: China Inst For Food And Drug Control
Priority date: 2023-06-09
Filing date: 2024-01-25
Publication date: 2024-03-05
Also published as: CN116693711A; CN116693711B

Abstract

The present invention provides a Polygonum mulliflorum polysaccharide and an extraction method and application thereof, and belongs to the technical field of medicine. The extraction method of the Polygonum mulliflorum polysaccharide of the present invention includes the following steps: (1) performing reflux extraction of Polygonum mulll'florum powder through petroleum ether, an ethanol solution and water sequentially to obtain a supernatant; (2) performing concentration, alcohol precipitation, purification and drying of the supernatant to obtain a crude polysaccharide of Polygonum multl'florum, and (3) sequentially using water and a sodium chloride solution as eluents, eluting the crude polysaccharide solution of Polygonum mulliflorum by using a Q-Sepharose Fast Flow column, removing impurities and drying to obtain a Polygonum mulll'florum polysaccharide RPMP-N and a Polygonum mulll'florum polysaccharide RPMP-A With different molecular weights. The Polygonum mulll'florum polysaccharides RPMP-N and RPMP-A extracted by the present invention can recover the activity of related antioxidant enzymes, protect visceral organ tissues, reduce the aging damage to visceral organs and delay the aging of organisms.

Description

POLYGONUM MULTIFLORUM POLYSACCHARIDE AND EXTRACTION

METHOD AND APPLICATION THEREOF

TECHNICAL FIELD

[01] The present invention relates to the technical field of medicine, and particularly relates to a Polygonum multiflorum polysaccharide and an extraction method and an application thereof.

BACKGROUND ART

[02] Tuber Fleeceflower Root (Chinese herb Heshouwu) is the dried root tuber of

Polygonum multiflorum Thunb., which has the medical effects of detoxifying, eliminating carbuncle, preventing malaria, relaxing bowels, etc. Modern pharmacological studies show that

Tuber Fleeceflower Root has the activity of resisting tumors, atherosclerosis, aging and inflammation, reducing blood lipid, enhancing immunity, etc. Therefore, Tuber Fleeceflower

Root has been widely applied to the fields of medicines, health care products, cosmetics and the like in recent years. Chemical component studies show that Tuber Fleeceflower Root tubers mainly contain active ingredients such as stilbene glycosides, polysaccharides, anthraquinones, flavonoids and phospholipids. The research on the activity of Tuber Fleeceflower Root mainly focuses on analyzing and studying the structure, activity and toxicity of stilbene glycosides and anthraquinones. It is found through research in recent years that polysaccharides in Tuber

Fleeceflower Root have the activity of resisting aging, fatigue, oxidation and tumors, reducing blood lipid, and achieving immunoregulation. In addition, polysaccharide is one of the main components of Tuber Fleeceflower Root, and further development of extraction methods of

Polygonum multiflorum polysaccharides and research on potential medicinal value of

Polygonum nmiltiflorum polysaccharides are of great significance.

SUMMARY

[03] An objective of the present invention is to provide a Polygonum multiflorum polysaccharide and an extraction method and an application thereof. The Polygonum mudtiflorum polysaccharides RPMP-N and RPMP-A extracted by the present invention can recover the activity of related antioxidant enzymes, protect visceral organ tissues, reduce the aging damage to visceral organs and delay the aging of organisms.

[04] In order to achieve the objective described above, the present invention provides the following technical solution: 1

[05] the present invention provides an extraction method of Polygonum multiflorum polysaccharides, including the following steps:

[06] (1) performing reflux extraction of Polygonum multiflorum powder through petroleum ether, an ethanol solution and water sequentially to obtain a supernatant;

[07] (2) performing concentration, alcohol precipitation, purification and drying of the supernatant to obtain a crude polysaccharide of Polygonum multiflorum; and

[08] (3) sequentially using water and a sodium chloride solution as eluents, eluting the crude polysaccharide solution of Polygonum multiflorum by using a Q-Sepharose Fast Flow column, removing impurities and drying to obtain a Polygonum multiflorum polysaccharide RPMP-N and a Polvgonum multiflorum polysaccharide RPMP-A with different molecular weights.

[09] Preferably, the process of performing reflux extraction of Polygonum multiflorum powder through petroleum ether, an ethanol solution and water sequentially in the step (1) is specifically as follows: performing reflux extraction of the Polvgonum multiflorum powder with the petroleum ether to obtain a drug residue A; performing reflux extraction of the drug residue

A with the ethanol solution to obtain a drug residue B; and performing reflux extraction of the drug residue B with water to obtain a supernatant.

[10] Preferably, a mass-to-volume ratio of the Polygonum multiflorum powder to the petroleum ether is (40-60) g: (0.5-1.0) L; a boiling range of the petroleum ether is 60-90°C; the reflux extraction of the Polygonum multiflorum powder with the petroleum ether is performed at a temperature of 60-70°C for 2-4 h;

[11] a mass-volume ratio of the Polygonum maultiflorum powder to the ethanol solution is (40-60) g: (1.5-2.5) L; a volume fraction of the ethanol solution is 75-90%; the reflux extraction of the drug residue A with the ethanol solution is performed at a temperature of 80-90°C for 0.5-1.5 h; and

[12] a mass-to-volume ratio of the drug residue B to water is (40-60) g: (1.5-2.5) L; and the reflux extraction of the drug residue B with water is performed at a temperature of 95-100°C for 1.5-3 h.

[13] Preferably, a concentration of the concentrated supernatant 1s 0.4-0.6 kg/L; the alcohol precipitation refers to a process of mixing the concentrated supernatant and anhydrous ethanol at a volume ratio of 1: (3-5); the alcohol precipitation is performed at a temperature of 2-5°C for 10-15 h; the purification includes deproteinization and dialysis; the deproteinization method is a Sevag method, and the deproteinization frequency is 5-10 times; a molecular weight cutoff of a dialysis bag used for the dialysis is 10 kDa; and time of the dialysis is 20-30 h.

[14] Preferably, a concentration of the crude polysaccharide solution of Polygonum 2 multiflorum is 18-22 mg/mL.

[15] Preferably, a flow rate of the elution is 0.5-2 ml/min; and a process of the elution is as follows: sequentially using water and a sodium chloride solution as eluents, where a use amount of the water is 350-500 ml, a use amount of the sodium chloride solution is 350-500 ml, and a concentration of the sodium chloride solution is 0.4-0.6 mol/L; collecting the eluent through tubes with a unit volume of 10 mL, removing impurities and drying the eluent in the 111-23 tubes to obtain the Polygomun multiflorum polysaccharide RPMP-N; and removing impurities and drying the eluent in the 42™-46™ tubes to obtain the Polygonum multiflorum polysaccharide

RPMP-A.

[16] Preferably, the impurity removal is performed by dialysis, a molecular weight cutoff of a dialysis bag used for the impurity removal is 10 kDa, and the impurity removal lasts for 40- 60 h at a temperature of 2-5°C.

[17] The present invention further provides a Polygonum multiflorum polysaccharide

RPMP-N extracted through the above extraction method.

[18] The present invention further provides a Polygonum multiflorum polysaccharide

RPMP-A extracted through the above extraction method.

[19] The present invention further provides an application of the Polygonum multiflorum polysaccharide RPMP-N and/or the Polygomum multiflorum polysaccharide RPMP-A in preparing anti-aging drugs.

[20] The present invention provides a Polygonum multiflorum polysaccharide and an extraction method and application thereof. In the present invention, taking petroleum ether, ethanol and water as extracts respectively, and water and sodium chloride as eluents,

Polygonum multiflorum polysaccharide components are separated to finally obtain the

Polygonum multiflorum polysaccharides RPMP-N and RPMP-A with anti-aging activity, through a reflux extraction method and use of the Q-Sepharose Fast Flow column. The

Polygonum multiflorum polysaccharides RPMP-N and RPMP-A of the present invention are capable to recover the activity of related antioxidant enzymes, resist the accumulation of ROS in organisms, protect visceral organ tissues, reduce the aging damage to visceral organs, and inhibit an expression of a key protein P16 in expressions of P53-P21 and CDKs, thus delaying the aging of organisms.

BRIEF DESCRIPTION OF THE DRAWINGS

[21] FIG. 1 is an elution curve of a Polygonum multiflorum polysaccharide in Example 1.

[22] FIG. 2is a diagram of hematoxylin and eosin (H&E) stained sections of brain tissues 3 of mice in each group in Example 2.

[23] FIG. 3 is a diagram of H&E stained sections of hepatic tissues of mice in each group in Example 2.

[24] FIG. 4 is a diagram of detection results of malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity, and catalase (CAT) enzyme activity of brain tissues of mice in each group in Example 2.

[25] FIG. 5 is a diagram of detection results of MDA content, SOD activity, GSH-

Px activity, and CAT enzyme activity of hepatic tissues of mice in each group in Example 2.

[26] FIG. 6 is a diagram of determination results of expression levels of P16, P21 and P53

IO proteins in hepatic tissues of mice in each group in Example 2.

[27] FIG. 7 is a diagram of determination results of expression levels of P16, P21 and P53 proteins in brain tissues of mice in each group in Example 2.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[28] The technical solution provided in the present invention is described in detail below in combination with the embodiments, but such embodiments are not to be construed as limiting the scope of protection of the present invention.

[29] Example 1

[30] In this example, there is provided an extraction method of Polvgonum multiflorum polysaccharides, including the following steps:

[31] Polvgonum multiflorum decoction pieces were crushed and then sieved through a No. 3 sieve, Polygonum multiflorum powder sieved out was mixed with petroleum ether (a boiling range of 60-90°C) at a ratio of 50 g:0.75 L, and reflux extraction was performed at 65°C for 3 h to remove lipids; after the reflux extraction was completed, filtering was performed to remove a solvent, and evaporation drying was performed to obtain a drug residue A; at a ratio of 50 g: 2L between the Polygonum multiflorum powder and an ethanol solution (a volume fraction of 80%), the drug residue A was mixed with the ethanol solution, and reflux extraction was performed at 85°C for 1 h to remove small molecular substances and pigments; after the reflux extraction was completed, filtering was performed to remove a solvent, and evaporation drying was performed to obtain a drug residue B; the drug residue B was mixed with deionized water at a ratio of 50 g:2 L, reflux extraction was performed at 100°C for 2 h, and after the reflux extraction was completed, centrifuging at 5000 r/min was performed for 5 min to obtain a supernatant; the supernatant was concentrated under reduced pressure to obtain a concentrated solution with a concentration of 0.5 kg/L (that is, each liter of the concentrated solution 4 corresponds to 0.5 kg of Polygonum multiflorum decoction pieces), and the concentrated solution was mixed with anhydrous ethanol until an ethanol volume fraction reached 80%, a mixture obtained was placed in a refrigerator and kept static at 4°C for 12 h, the mixture was taken out and centrifuged at 5000 r/min for 5 min to obtain a precipitate, and the precipitate was washed with the anhydrous ethanol twice to obtain a precipitate of Polvgonum multiflorum polysaccharides.

[32] deionized water was used to dissolve the obtained precipitate of Polygonum mudtiflorum polysaccharides into a polysaccharide precipitate solution with a concentration of 5 mg/mL, and a Sevag method was used to remove proteins, with specific operation steps as follows: the polysaccharide precipitate solution was mixed with a Sevag reagent (obtained by mixing trichloromethane and n-butyl alcohol at a volume ratio of 4:1) at a volume ratio of 5:1, and a mixture obtained was mixed well on a shaker at 200 r/min for 60 min, and then centrifuged at 5000 r/min for 5 min to obtain a supernatant; the supernatant was further mixed with Sevag reagent, and the above operation steps were repeated; a total of 8 treatments were performed, and during the last treatment, the obtained supernatant was dialyzed in deionized water for 24 h by using a dialysis bag (a molecular weight cutoff of 10 kDa), and then freeze-dried at -90°C and 0.3 mbar for 48 h to obtain a crude polysaccharide of Polygonum multiflorum.

[33] Deionized water was used to dissolve the obtained crude polysaccharide of Polygonum multiflorum into a polysaccharide solution of 20 mg/mL, and a Q-Sepharose Fast Flow column was used for separation, with specific steps as follows: 400 ml of deionized water and 400 ml of an aqueous solution of sodium chloride (0.5 mol/L) were used sequentially to perform elution at a flow rate of 1 ml/min, and an eluent was collected by tube with a unit volume of 10 mL; 1 mL of the eluent from each tube was taken as a sample solution, 1 mL of deionized water, 1 mL of an aqueous solution of phenol with a concentration of 0.05 g/mL and 5 mL of concentrated sulfuric acid (a concentration of 98%) were added to the 1 mL sample solution, after mixing well, a mixture obtained was heated at 70°C in a water bath for 15 min, the mixture was taken out and kept in an ice bath for 10 min, an absorbance value (value A) was measured at 490 nm according to Ultraviolet—visible spectrophotometry, where the number of elution tubes was taken as an X-coordinate and the value A was taken as a Y-coordinate, and an elution curve was drawn, as shown in FIG. 1; according to the elution curve shown in FIG. 1, the eluents in the 11-23" tubes of the same elution peak were combined, a mixture of the eluents was dialyzed in deionized water at 4°C for 48 h by using a dialysis bag with a molecular weight cutoff of 10 kDa, and then freeze-dried at -90°C and 0.3 mbar for 48 h to obtain a Polygonum multiftorum polysaccharide RPMP-N; and the eluents in the 424-46" tubes of the same elution 5 peak were combined, a mixture of the eluents was dialyzed in deionized water at 4°C for 48 h by using a dialysis bag with a molecular weight cutoff of 10 kDa, and then freeze-dried at - 90°C and 0.3 mbar for 48 h to obtain a Polvgonum multiflorum polysaccharide RPMP-A.

[34] Structural analysis of the Polygonum multiflorum polysaccharides RPMP-N and

RPMP-A was performed respectively, with structural characteristics as follows: with a total sugar content of greater than 97%, and a relative molecular weight of 167 kDa, the Polygonum multiflorum polysaccharide RPMP-N was mainly composed of glucose, mannose and arabinose; and with a total sugar content of greater than 77%, and a relative molecular weight of 47 kDa, the Polvgonum mudtiflorum polysaccharide RPMP-A was mainly composed of glucose, galacturonic acid, mannose and arabinose.

[35] Example 2

[36] In this example, D-galactose (D-Gal)-induced mouse aging model was used to verify the anti-aging efficacy of the Polygomum multiflorum polysaccharides RPMP-N and RPMP-A prepared in Example 1, with a specific process as follows:

[37] 1. Constructing a mouse model

[38] A total of 70 male Institute of Cancer Research (ICR) mice (a Specific Pathogen Free (SPF) grade, an average weight of 18-22g, an average age of 3-4 weeks, purchased from SiPeiFu (Beijing) Biotechnology Co., Ltd.) were randomly divided into several groups including a normal control (NC) group, a model (Model) group, a positive control (PCA) group, alow-dose RPMP-N (LRPMP-N) group, a high-dose RPMP-N (HRPMP-N) group, a low-dose

RPMP-A polysaccharide (LRPMP-A) group, and a high-dose RPMP-A polysaccharide (HRPMP-A) group, with 10 mice per group. Except for the mice in the NC group, each mouse in other groups was intraperitoneally injected 100 mg/kg D-Gal every day for 6 weeks. Each mouse in the NC group was injected with the same dose of normal saline for 6 weeks. The content of malondialdehyde (MDA) in blood of the mice in other groups was measured and compared with that of the NC group. An increase in the MDA content was statistically significant and could be used as an indicator for measuring effectiveness of the model. From the seventh week after the aging model was verified to be effective, each mouse in the PCA group was intraperitoneally injected with 100 mg/kg of vitamin C (in addition to the injection of D-Gal) every day. For the four dose groups of RPMP-N and RPMP-A, samples with doses of 50 mg/kg and 100 mg/kg were intragastrically administered for 4 weeks respectively.

[39] 2. Performing pathological examination of model mice

[40] The mice were fasted overnight, the next day, their eyeballs were enucleated to collect blood samples, and the blood samples were centrifuged at 4000 rpm/min and 4°C for 10 min to 6 obtain a supernatant (namely serum). After a mouse was killed by cervical dislocation, its brain tissues and hepatic tissues were peeled off, immediately fixed in paratormaldehyde with a concentration of 4%, conventionally dehydrated, transparentized, immersed in wax, embedded, and then serially sectioned (5 um thick per section, with a section interval of 30 um), and after routine dewaxing, the brain tissues and liver were stained with hematoxylin-eosin, and panoramically scanned under a high-power microscope. Pathological changes of brain tissues and hepatic tissues of mice in each experimental group were observed, as shown in FIGs. 2-3.

FIG. 2 showed the pathological changes in brain tissues of the mice, and FIG. 3 showed the pathological changes in hepatic tissues of the mice.

[41] It can be seen from FIGs. 2 and 3 that the polysaccharides RPMP-N and RPMP-A can play a certain role in repairing and protecting the liver issues and brain tissues damaged by D- gal, and alleviate arrangement disorders of hepatic cords and hepatic sinusoids, hepatocellular edema and inflammatory cell infiltration, as well as neuropil edema, inflammatory cell infiltration and necrosis in the hippocampal tissues of brain.

[42] 3. Detecting malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity, and catalase (CAT) enzyme activity of model mice

[43] Reagent kits (purchased from Jiangsu Meimian Industrial Co., Ltd.) were used to detect

GSH-Px activity, SOD activity, MDA content and CAT enzyme activity of hepatic tissues and brain tissues, so as to evaluate the degrees of oxidative stress in the hepatic tissues and brain tissues. The MDA content, SOD activity, GSH-Px activity and CAT enzyme activity of brain tissues of the mice were shown in FIG. 4, and the MDA content, SOD activity, GSH-Px activity and CAT enzyme activity of hepatic tissues of the mice were shown in FIG. 5.

[44] It can be seen from FIGs. 4 and 5 that, compared with the mice in the NC group, the mice in the model group showed a significantly increased MDA content of brain tissues and hepatic tissues, indicating that intraperitoneal injection of D-gal could cause accumulation of a large number of MDA products, and an obvious peroxidation trend; while the SOD activity,

GSH-Px activity and CAT enzyme activity declined with significant differences, indicating that intraperitoneal injection of D-gal could affect the antioxidant enzyme activity and a balance of

ROS in the organisms, and cause damage to related organs. Compared with the mice in the model group, the mice in the PCA group and different dose groups of the polysaccharides

RPMP-N and RPMP-A showed a significant decrease in the MDA content, and a significant increase in the SOD activity, GSH-Px activity and CAT enzyme activity with significant differences, indicating that peroxidation damage to the hepatic tissues and brain tissues of the mice was repaired somewhat, and the polysaccharides RPMP-N and RPMP-A fought against 7 the excessive accumulation of ROS by restoring the activity of related antioxidant enzymes, thereby protecting related organs and tissues and resisting D-gal-induced aging of the hepatic tissues and brain tissues.

[45] 4. Detecting expression levels of aging-related proteins in model mice

[46] Western Blot was used to detect the expression levels of aging-related proteins P16,

P21 and P53 in the hepatic tissues and brain tissues of aging mice, and to explore a possible mechanism by which the polysaccharides RPMP-N and RPMP-A can delay aging of D-gal model mice.

[47] Some fresh hepatic tissues and brain tissues were taken and weighed, added to a protein lysis solution prepared at a ratio of W: V = 1:6, ground into a pasty state by using a grinder, placed on ice for 30 min, and shaken once every 10 min. The pasty tissues were centrifuged at 12,000 r/min for 30 min by using a centrifuge that had been pre-cooled to 4°C, to obtain a supernatant, and the proteins were quantified by using Bradford protein assay kits (purchased from Wuhan Boster Biological Technology., LTD). 10% sodium dodecyl sulfate (SDS)- denatured polyacrylamide gel (lower separation gel, single- sided) was prepared and quickly filled until 1ts height reached about 2/3 of a total height of a glass plate, then 1 mL of water- saturated n-butyl alcohol was added above the gel to ensure that flatness of the gel at an upper layer, and the gel was kept static until solidification. 5% SDS-denatured polyacrylamide gel (upper laminating gel, single-sided) was prepared and quickly filled into a glass plate until it was stuffed up, and after a comb was inserted, the gel was kept static until solidification. Before electrophoresis, the comb was removed, the gel was placed in a 1 xTris-glycine electrophoresis buffer, and a syringe needle was used to blow and clean sample loading holes. After protein samples were mixed with a 5x sample loading buffer (containing B-mercaptoethanol), a mixture obtained was boiled and denaturated for 5 min, and kept in an ice bath for 5 min. An appropriate number of protein samples were loaded, and SDS-denatured polyacrylamide gel electrophoresis (SDS-PAGE) was performed until target proteins were effectively separated to stop electrophoresis. After the electrophoresis, the gel was taken out and placed in a special sandwich clamp for membrane transfer, the gel was placed on a negative electrode, the polyvinylidene fluoride (PVDF) membrane was placed on a positive electrode, and membrane transfer in a transfer buffer was performed at 4°C and a constant current of 350 mA for 2 h, so that the proteins in the gel were transferred to the PVDF membrane to form a blot. The membrane was placed in a 1xBlotto solution, and shaken and sealed at room temperature for 2 h. After being taken out, the membrane was cut open according to a blotting position of the protein, then placed in the Blotto solution containing primary antibodies P16, P21 and P53 8

(1:1000), and shaken at 4°C overnight. The membrane was taken out the day after tomorrow, and placed in a 1xTris Buffered Saline with Tween 20 (TBST) solution, and a mixture obtained was shaken and rinsed for 5 min, with a total of 4 times. The mixture was put into a Blotto solution containing corresponding secondary antibodies (1:8000), incubated at room temperature for 1.5 h, and then shaken and rinsed with the TBST solution for 5 min, with a total of 4 times. The membrane was placed in an electrochemiluminescence (ECL) chromogenic agent for 30 sec, and photos were taken through a chemiluminescence imaging and analysis system to analyze a brightness values of each group of protein bands and calculate a ratio between the brightness value of the protein band of each sample and the brightness value of a corresponding Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) band (internal reference), so as to obtain the corrected brightness values of protein bands. Taking the brightness value of the protein band as a standard value 1, histograms were drawn, as shown in

FIGs. 6 and 7. FIG. 6 showed determination results of expression levels of P16, P21 and P53 proteins in hepatic tissues of mice, and FIG. 7 showed determination results of expression levels of P16, P21 and P53 proteins in brain tissues of mice.

[48] It can be seen from FIGs. 6 and 7 that, compared with the mice in the NC group, the mice in the model group show a significant increase in expression levels of P16, P21 and P53 proteins in the hepatic and brain tissues. Different doses of polysaccharides RPMP-N and

RPMP-A have an obvious effect on down-regulating the expressions of related proteins, showing a certain concentration dependence, indicating that the polysaccharides RPMP-N and

RPMP-A, by inhibiting an expression of a key protein P16 in expressions of P53-P21 and

CDK, are capable to delay the aging of D-gal model mice.

[49] The above examples are merely the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, several improvements and embellishments can be made within the protection range of the disclosure without departing the principle of the present invention. 9

Claims

Conclusions

1. An extraction method of Polygonum multiflorum polysaccharides, comprising the following steps: (1) performing reflux extraction of Polygonum multiflorum powder by means of petroleum ether, an ethanol solution and water successively to obtain a supernatant; (2) carrying out concentration, alcohol precipitation, purification and drying of the supernatant to obtain a crude polysaccharide from Polygonum multiflorum; and (3) successively using water and sodium chloride solution as eluents, eluting the crude polysaccharide solution from Polygonum multiflorum using a Q Sepharose Fast Flow column, removing impurities and drying to obtain a Polygonum multiflorum polysaccharide RPMP N and a Polygonum multiflorum to obtain polysaccharide RPMP A with different molecular weights; an elution flow rate is 0.5-2 ml/min; and a process of the elution is as follows: successively using water and sodium chloride solution as eluents, where a usage amount of the water is 350-500 ml, a usage amount of the sodium chloride solution is 350-500 ml, and a concentration of the sodium chloride solution is 0.4-0.6 mol/L; collecting the eluent through tubes with a unit volume of 10 mL, removing impurities and drying the eluent in the 11th-23rd tubes to obtain the Polygonum multiflorum polysaccharide RPMP N; and removing impurities and drying the eluent in the 42nd-46th tubes to obtain the Polygonum multiflorum polysaccharide RPMP A.

The extraction method according to claim 1, wherein the process of carrying out reflux extraction of Polygonum multiflorum powder by means of petroleum ether, an ethanol solution and water successively in step (1) is specifically as follows: carrying out reflux extraction of the Polygonum multflorum powder with the petroleum ether to obtain a drug residue A; performing reflux extraction of the drug residue A with the ethanol solution to obtain a drug residue B; and performing reflux extraction of the drug residue B with water to obtain a supernatant.

The extraction method according to claim 2, wherein a mass-volume ratio of the Polygonum multiflorum powder to the petroleum ether is (40-60) g: (0.5-1.0) L; a boiling range 10 of the petroleum ether is 60-90°C; the reflux extraction of the Polygonum multiflorum powder with the petroleum ether is carried out at a temperature of 60-70°C for 2-4 hours; a mass-volume ratio of the Polygonum multiflorum powder to the ethanol solution is (40-60) g: (1.5-2.5) L; a volume fraction of the ethanol solution is 75-90%; the reflux extraction of the drug residue A with the ethanol solution is carried out at a temperature of 80-90°C for 0.5-1.5 hours; and a mass-volume ratio of the drug residue B to water is (40-60) g: (1.5-2.5) L; and the reflux extraction of the drug residue B with water is carried out at a temperature of 95-100°C for 1.5-3 hours.

The extraction method according to claim 3, wherein a concentration of the concentrated supernatant is 0.4-0.6 kg/L; the alcohol precipitation refers to a process of mixing the concentrated supernatant and anhydrous ethanol at a volume ratio of 1:(3-5); and the alcohol precipitation is carried out at a temperature of 2-5°C for 10-15 hours; the purification includes deproteinization and dialysis; the deproteinization method is a Sevag method, and the deproteinization frequency is 5-10 times; a molecular weight cutoff of a dialysis bag used for dialysis is 10 kDa; and the time of dialysis is 20-30 hours.

The extraction method according to claim 4, wherein a concentration of the crude polysaccharide solution of Polygonum multiflorum is 18-22 mg/mL.

The extraction method according to claim 5, wherein the removal of impurities is carried out by dialysis, a molecular weight cutoff of a dialysis bag used for the removal of impurities is 10 kDa, and the removal of impurities takes 40-60 hours at a temperature of 2 -5°C.

A Polygonum multiflorum polysaccharide RPMP N extracted by the extraction method according to any one of claims 1-6.

A Polygonum multiflorum polysaccharide RPMP A extracted by the extraction method according to any one of claims 1-6. 11

An application of the Polygonum multiflorum polysaccharide RPMP N according to claim 7 and/or the Polygonum multiflorum polysaccharide RPMP A according to claim 8 in the preparation of anti-aging drugs.

12