CN117050204A

CN117050204A - Penthorn grass polysaccharide PCPP and application thereof

Info

Publication number: CN117050204A
Application number: CN202310951040.3A
Authority: CN
Inventors: 冯士令; 徐小艳; 邓官峰; 李逍; 李平进; 周莉君; 陈涛; 丁春邦
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2023-07-31
Filing date: 2023-07-31
Publication date: 2023-11-14
Anticipated expiration: 2043-07-31
Also published as: CN117050204B

Abstract

The invention discloses penthorum chinense pursh polysaccharide PCPP and application thereof, wherein the penthorum chinense pursh polysaccharide PCPP is separated from penthorum chinense pursh, and mainly comprises galactose, arabinose, glucose, mannose, xylose, galacturonic acid and glucuronic acid through glycosidic bonds. The invention adopts a method of hot water extraction and ethanol precipitation to obtain the penthorum chinense pursh crude polysaccharide, and the uniform Penthorum Chinense Pursh Polysaccharide (PCPP) is obtained through separation and purification by ion chromatography and agarose gel. The research shows that the PCPP has strong antioxidant activity in vivo and in vitro and has good application value.

Description

Penthorn grass polysaccharide PCPP and application thereof

Technical Field

The invention belongs to the technical field of foods and health-care products, and relates to penthorum chinense pursh polysaccharide PCPP and application thereof.

Background

Reactive oxygen species (Reactive Oxygen Species, ROS) are unstable intermediates produced by normal metabolism of cells or organisms. Under non-stress conditions, ROS are produced and cleared in a dynamic balance state, and when the organism is stressed, the balance state is destroyed to cause accumulation of a large amount of ROS, and normal functions of cells are destroyed, so that a series of diseases such as diabetes, neurodegenerative diseases, cancers and the like are caused. At present, most antioxidants used in the industries of foods and health care products are synthetic compounds, and have great toxic and side effects after long-term use. In recent years, natural antioxidants have been widely paid attention to because of their good antioxidant effect and small toxic and side effects, and development and utilization of natural antioxidants have become a trend in the technical field of foods and health products, so that natural antioxidants have a broad market application prospect.

Penthorum chinense pursh (Penthorum chinense Pursh), also known as penthorum chinense pursh, is a plant of the genus penthorum of the family Saxifragaceae. Modern pharmacological researches show that penthorum chinense pursh has various functions of resisting oxidation, protecting liver, promoting gallbladder, resisting virus, resisting tumor, resisting inflammation, reducing blood lipid and the like, and is rich in various active ingredients such as flavone, polyphenol, lignan, organic acid, polysaccharide and the like. Wherein the penthorum chinense pursh polysaccharide has the effects of resisting cancer, regulating immunity and resisting oxidization. At present, the research of active ingredients of penthorum chinense pursh is mainly focused on flavone, and the penthorum chinense pursh polysaccharide is used as one of important active ingredients of penthorum chinense pursh, and few reports are made. Therefore, in order to search for better natural antioxidants, the penthorum chinense pursh polysaccharide has important clinical significance.

Disclosure of Invention

In order to make up the defects of the prior art, the invention aims to provide penthorum chinense pursh polysaccharide and a preparation method and application thereof.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the first aspect of the invention provides penthorum chinense pursh polysaccharide PCPP, which mainly comprises 7 monosaccharides of galactose, arabinose, glucose, mannose, xylose, galacturonic acid and glucuronic acid.

Further, the penthorum chinense pursh polysaccharide PCPP is composed of galactose, arabinose, glucose, mannose, xylose, galacturonic acid and glucuronic acid through glycosidic bonds.

Further, the galactose content was 37.12%.

Further, the arabinose content was 35.21%.

Further, the glucose content was 9.14%.

Further, the mannose content was 9.98%.

Further, the xylose content was 3.75%.

Further, the content of galacturonic acid was 2.62%.

Further, the content of glucuronic acid is 2.18%.

Further, the penthorum chinense pursh polysaccharide PCPP comprises an arabinogalactan region (AG), a homogalactose region (HG), a mannose segment branched chain, a glucose segment branched chain, a homogalactose segment branched chain and a xylose segment branched chain.

Further, the backbone of the arabinogalactan region is composed of 12 galactose residues and 12 arabinose residues which are sequentially cross-linked.

Further, the skeleton of the homogalactose region is formed by sequentially connecting 12 galactose residues.

Further, the mannose segment branches consist of 1 mannose residue, 1 galactose residue, and 1 galacturonic acid residue.

Further, the glucose segment branches consist of 2 glucose residues, 1 glucuronic acid residue and 1 galactose residue.

Further, the homogalactosylic segment branches consist of 4 galactose residues.

Further, the xylose segment branches consist of 1 xylose residue and 1 galactose residue.

Further, the penthorum chinense pursh polysaccharide PCPP is formed by sequentially connecting an arabinogalactan region and a homogalactosyl region.

Further, the skeleton of the homogalactose region is sequentially connected with a mannose segment branched chain, a glucose segment branched chain, a homogalactose segment branched chain and a xylose segment branched chain respectively.

Further, the penthorum chinense pursh polysaccharide PCPP is formed by sequentially connecting an arabinose segment, an arabinogalactan region and a galactose region framework.

Further, the penthorum chinense pursh polysaccharide PCPP comprises the following sugar residues: α -Araf- (1→β -Xylp- (1→α -Glcp- (1→α -Galp- (1→5) - α -Galp- (1→2) - β -Manp- (1→3, 4) - α -Galp- (1→4) - α -Glcp- (1→2) - α -GlcAp- (1→4, 6) - α -Galp- (1→4, 6) - β -Galp- (1→3, 4) - β -GalpA- (1→1).

Further, the Mw molecular weight of the penthorum chinense pursh polysaccharide PCPP is 14.96kDa.

Further, the Mn molecular weight of the penthorum chinense pursh polysaccharide PCPP is 10.97kDa.

Further, the method for extracting the penthorum chinense pursh polysaccharide PCPP comprises the following steps:

(1) Extracting penthorum chinense pursh crude polysaccharide;

(2) Separating the penthorum chinense pursh crude polysaccharide in the step (1) to obtain penthorum chinense pursh polysaccharide;

(3) Purifying the penthorum chinense pursh polysaccharide in the step (2) to obtain the penthorum chinense pursh polysaccharide PCPP.

Further, the specific steps of the step (1) include: extracting penthorum chinense pursh powder, concentrating, precipitating with ethanol, deproteinizing, and freeze-drying to obtain penthorum chinense pursh crude polysaccharide.

Further, the method for obtaining the penthorum chinense pursh powder comprises the steps of crushing penthorum chinense pursh, degreasing and drying.

Further, the degreasing includes petroleum ether, acetone, and/or ethanol degreasing.

Further, the extraction method comprises a hot water extraction method, an ultrasonic assisted extraction method, a microwave assisted extraction method, an enzymolysis method or an alkali liquor extraction method.

Further, the extraction method is a hot water extraction method.

Further, the extraction temperature of the hot water extraction method was 80 ℃.

Further, the extraction time of the hot water extraction method was 3 hours.

Further, the ratio of feed liquid in the hot water leaching method is 1:20.

Further, the concentration method includes precipitation method, adsorption method, ultrafiltration method, dialysis method, reduced pressure distillation method, and freeze drying method.

Further, the concentration method is a reduced pressure distillation method.

Further, the deproteinization method includes Sevag method, trifluorotrichloroethane method, trichloroacetic acid method, enzyme-Sevag method.

Further, the deproteinization method is a Sevag method.

Further, the drying method comprises freeze drying, vacuum drying, normal pressure drying or spray drying.

Further, the drying method is freeze drying.

Further, the specific steps of the step (2) include: and (3) separating the penthorum chinense pursh crude polysaccharide in the step (1) by adopting an ion exchange chromatographic column, performing gradient elution, collecting eluent, dialyzing and freeze-drying to obtain the penthorum chinense pursh polysaccharide.

Further, the ion exchange chromatography column comprises an anion exchange chromatography column and a cation exchange chromatography column; the anion exchange chromatographic column comprises a strong anion exchange chromatographic column and a weak anion exchange chromatographic column; the strong anion exchange chromatographic column comprises Q-Sephadex A-25, Q-Sephadex A-50, Q-Sephadex C-25 and Q-Sephadex C-50; the weak anion exchange chromatographic column comprises DEAE-Cellulose DE-22, DEAE-Cellulose DE-23, DEAE-Cellulose DE-51, DEAE-Cellulose DE-52, DEAE-Cellulose DE-53 and DEAE-Sepharose Fast Flow.

Further, the ion exchange chromatography column is DEAE-Sepharose Fast Flow.

Further, the gradient elution method comprises the step of sequentially adopting deionized water and/or salt solution for gradient elution.

Further, the salt solution includes a sodium chloride solution, a sodium sulfate solution, a magnesium sulfate solution, or an ammonium sulfate solution.

Further, the flow rate of deionized water was 2mL/min.

Further, the concentration of the sodium chloride solution was 0.1M.

Further, the drying method is freeze drying.

Further, the specific steps of the step (3) include: and (3) purifying the penthorum chinense pursh polysaccharide in the step (2) by adopting a hydrophobic chromatography column, performing gradient elution by adopting deionized water, collecting eluent, filtering and freeze-drying to obtain the penthorum chinense pursh polysaccharide PCPP.

Further, the hydrophobic chromatography column comprises SephacrylS-100, sephacrylS-200, sephacrylS-300, sephacrylS-400, superose 12, superose 6, superdex 12 or Superdex 6; the Superose 6 includes Sepharose 6Fast Flow.

Further, the hydrophobic chromatography column is a Sepharose 6Fast Flow.

Further, the flow rate of deionized water was 1mL/min.

In a second aspect, the invention provides a composition, which comprises the penthorum chinense pursh polysaccharide PCPP in the first aspect.

Further, the composition may further comprise a pharmaceutically acceptable carrier, adjuvant or vehicle.

A third aspect of the invention provides the use of any one of:

(1) The application of the penthorum chinense pursh polysaccharide PCPP in the first aspect or the composition in the second aspect in preparing antioxidant products.

(2) The application of the penthorum chinense pursh polysaccharide PCPP in the first aspect or the composition in the second aspect in preparation of products for delaying aging and preventing or treating aging-related diseases.

(3) The application of the penthorum chinense pursh polysaccharide PCPP in the first aspect or the composition in the second aspect in the preparation of products for scavenging free radicals is provided.

(4) Use of the penthorum chinense pursh polysaccharide PCPP of the first aspect of the invention or the composition of the second aspect of the invention in the preparation of a product for reducing ROS levels.

(5) The use of the penthorum chinense pursh polysaccharide PCPP according to the first aspect of the invention or the composition according to the second aspect of the invention in the preparation of products for enhancing SODs or CATs activity.

(6) The use of the penthorum chinense pursh polysaccharide PCPP according to the first aspect of the invention or the composition according to the second aspect of the invention in the preparation of products for reducing MDA levels.

(7) The application of the penthorum chinense pursh polysaccharide PCPP in the first aspect or the composition in the second aspect in preparation of products for promoting expression of DAF-16 or HSP-16.2.

Further, the free radicals include superoxide anion free radicals, H ₂ O ₂ Singlet oxygen, HO, alkylperoxide radical, lipoperoxide radical, ABTS+, DPPH.

Further, the free radical is DPPH.

Further, the aging-related diseases include atherosclerosis, cardiovascular diseases, arthritis, cataract, osteoporosis, type 2 diabetes, hypertension, neurodegeneration, stroke, atrophic gastritis, NASH, trunk prodromal disease, chronic obstructive pulmonary disease, coronary artery disease, dopamine imbalance syndrome, metabolic syndrome, fatigue urinary incontinence, hashimoto thyroiditis, heart failure, senile depression, immune aging, myocardial infarction, acute coronary syndrome, sarcopenia obesity, senile osteoporosis, and urinary incontinence.

Further, the products include pharmaceuticals, solid beverages, liquid beverages, dairy products, flavoured powders, and nutraceuticals.

Further, the dosage forms of the medicament include solutions, suspensions, emulsions, extracts, elixirs, powders, granules, tablets, capsules.

The invention has the advantages and beneficial effects that:

the method is used for separating and purifying penthorum chinense pursh polysaccharide PCPP from penthorum chinense pursh for the first time, has strong in-vivo and in-vitro antioxidant activity, and can be developed and utilized as a novel antioxidant.

Drawings

FIG. 1 is an ultraviolet spectrum of PCPP;

FIG. 2 is a graph showing the results of measurement of the molecular weight of PCPP by gel permeation chromatography, wherein 2A is a standard chart of weight average molecular weight (Mw), and 2B is a chromatogram;

FIG. 3 is a diagram showing the monosaccharide composition of an electrochemical detector for PCPP;

FIG. 4 is a GC diagram of a methylated derivative of PCPP;

FIG. 5 is an NMR spectrum of PCPP, wherein 5A is ¹ H NMR spectrum, 5B is ¹³ C NMR spectrum, 5C is COSY NMR spectrum, 5D is TOCOSY NMR spectrum, 5E is HSQC NMR spectrum, 5F is HMBC NMR spectrum;

FIG. 6 is a primary structural diagram of PCPP;

FIG. 7 is an infrared spectrum of PCPP;

FIG. 8 is a chart of Congo red experimental results for PCPP;

FIG. 9 is a different magnification scanning electron microscope image of PCPP, wherein 9A is a 50 Xscanning electron microscope image, 9B is a 300 Xscanning electron microscope image, 9C is a 500 Xscanning electron microscope image, and 9D is a 1000 Xscanning electron microscope image;

FIG. 10 is a DSC crystallization profile of PCPP;

FIG. 11 is a graph showing the results of an in vitro antioxidant activity test of PCPP, wherein 11A is a graph showing the results of a DPPH radical scavenging force test, 11B is a graph showing the results of an iron ion reducing force test, and 11C is a graph showing the results of a total antioxidant force test;

FIG. 12 is a graph showing the results of the effect of PCPP on HHT-5 cell viability, wherein 12A is a graph showing the results of the effect of different concentrations of PCPP on HHT-5 cell viability under non-stress conditions, and 12B is a graph showing the results of the effect of different concentrations of PCPP on HHT-5 cell viability under non-stress conditions;

FIG. 13 is a graph of the results of the effect of PCPP on ROS in HIL-5 cells;

FIG. 14 is a graph showing the results of the effect of PCPP on ROS in HHT-5 cells, wherein 14A is a graph showing the results of JC-1 detection of the effect of PCPP on mitochondrial membrane potential in HHT-5 cells, and 14B is a graph showing the results of PCPP maintaining mitochondrial membrane potential in HHT-5 cells;

FIG. 15 is a graph showing the effect of PCPP on HHT-5 apoptosis, wherein 15A is a graph showing the results of flow cytometry analysis of apoptotic cells in the control group, 15B is a graph showing the results of flow cytometry analysis of apoptotic cells in the PCPP-treated group, 15C is a graph showing the results of flow cytometry analysis of apoptotic cells in the damaged group, and 15D is a graph showing the results of reduction of the proportion of apoptosis of HHT-5 cells by PCPP;

FIG. 16 is a graph of the results of the effect of PCPP on caenorhabditis elegans longevity under heat stress;

FIG. 17 is a graph of the results of the effect of PCPP on caenorhabditis elegans longevity under oxidative stress;

FIG. 18 is a graph showing the effect of PCPP on the variation of ROS content in an insect, wherein 18A is H ₂ Results of DCF-DA detection of ROS levels in caenorhabditis elegans, 18B is a graph of results of PCPP inhibiting ROS production in caenorhabditis elegans;

FIG. 19 is a graph showing the effect of PCPP on the localization of C.elegans DAF-16, wherein 19A is a graph showing the effect of DAF-16 on the subcellular localization of GFP and 19B is a graph showing the effect of PCPP on the promotion of nuclear entry of DAF-16;

FIG. 20 is a graph showing the effect of PCPP on the expression of nematode HSP-16.2 and SOD-3, wherein 20A is a graph showing the effect of PCPP on the expression of nematode HSP-16.2:: GFP and SOD-3:: GFP expression fluorescence, and 20B is a graph showing the effect of PCPP on the expression of HSP-16.2 and SOD-3;

FIG. 21 is a graph showing the results of PCPP inducing the expression of antioxidant enzyme and inhibiting the accumulation of MDA in caenorhabditis elegans;

FIG. 22 is a graph of the results of the effect of PCPP on caenorhabditis elegans longevity;

FIG. 23 is a graph of the results of the effect of PCPP on caenorhabditis elegans longevity;

FIG. 24 is a graph of the results of the effect of PCPP on caenorhabditis elegans locomotor ability;

FIG. 25 is a graph showing the effect of PCPP on the accumulation of Xylodes albopictus lipofuscin, wherein 25A is a graph showing the effect of PCPP on the accumulation of Xylodes albopictus lipofuscin, and 25B is a graph showing the effect of PCPP on the inhibition of the accumulation of Xylodes albopictus lipofuscin on day 5.

And (3) injection: * p <0.05, < p <0.01, < p <0.001, < p <0.0001, < ns means no significant difference.

Detailed Description

The following provides definitions of some of the terms used in this specification. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

According to the invention, through extensive and intensive research, the uniform penthorum chinense pursh polysaccharide PCPP is separated and purified from penthorum chinense pursh. In-vivo and in-vitro experiments show that the PCPP has good free radical scavenging capability and in-vitro antioxidation potential, and can inhibit the generation of ROS in HIL-5 cells, maintain mitochondrial membrane potential and relieve apoptosis; PCPP can promote the penetration of caenorhabditis elegans DAF-16 into the nucleus, regulate the expression of nematode downstream genes sod-3 and hsp-16.2, promote the production of antioxidant enzyme and prolong the service life of caenorhabditis elegans under the conditions of stress and non-stress.

In the present invention, the term "monosaccharide" means a simple sugar. Such simple sugars may contain 3 to 10 carbon atoms, preferably 5 to 7 carbon atoms, and may be aldoses or ketoses, which may be in the D-or L-configuration compared to D-or L-glyceraldehyde. The monosaccharides are, for example, threose, erythrose or erythrulose (4 carbon atoms), or arabinose, xylose, ribose, lyxose, ribulose or xylulose (5 carbon atoms), or allose, glucose, fructose, maltose, mannose, galactose, gulose, idose, altrose, talose, allose, sorbose or tagatose (6 carbon atoms), mannoheptulose or sedoheptulose (7 carbon atoms) or salivary sugar (sialose, 9 carbon atoms), or galacturonic acid or glucuronic acid. Further, the term "monosaccharide" means galactose, arabinose, glucose, mannose, xylose, galacturonic acid, and glucuronic acid.

In the present invention, the term "polysaccharide" means a molecule composed of chains of monosaccharide units linked by glycosidic bonds. The distinction between "polysaccharides" and "oligosaccharides" is based on the number of monosaccharide units present in the chain. Oligosaccharides generally contain 2 to 9 monosaccharide units, whereas polysaccharides contain 10 or more monosaccharide units. In the present invention, the term "polysaccharide" includes molecules consisting of two or more monosaccharide units, in particular molecules in which the longest monosaccharide chain is 3 to 9 monosaccharide units. The term "polysaccharide" includes both linear and branched molecules, isolated and bound to polypeptides, sialylated and non-sialylated molecules. In a specific embodiment of the invention, the polysaccharide consists of 7 monosaccharides galactose, arabinose, glucose, mannose, xylose, galacturonic acid and glucuronic acid.

In the present invention, the method for extracting the penthorum chinense pursh crude polysaccharide includes but is not limited to: hot water extraction, ultrasonic assisted extraction, microwave assisted extraction, enzymatic hydrolysis or alkaline extraction. The term hot water extraction method is a classical method for extracting polysaccharide, and the method utilizes the principle of similar compatibility to dissolve polar macromolecular compound polysaccharide in polar solvents such as water for extraction, and has the characteristics of low equipment requirement, simple operation and the like. The term ultrasonic assisted extraction method is to utilize ultrasonic waves to generate high-speed and strong cavitation effect and stirring effect, so that the cell walls of the traditional Chinese medicine plants are easy to break, the dissolution of polysaccharide is facilitated, and the extraction efficiency is improved. Compared with the water extraction and alcohol precipitation method, the method has the advantages that the extraction time is greatly shortened, the structure of the polysaccharide is not changed, and the method has been widely used for extracting the polysaccharide from the traditional Chinese medicine, but the ultrasonic extraction time is not too long, and the polysaccharide structure is possibly changed and sugar chains are broken, so that the polysaccharide yield is affected. The term "microwave-assisted extraction" refers to the extraction of certain components by selective heating, based on differences in the ability to absorb microwaves. The microwave penetrating power is strong, the heating is rapid and uniform, the temperature in the cells can be rapidly increased, the liquid water is rapidly vaporized, the generated strong pressure can break cell membranes and cell walls, and the polysaccharide dissolution is promoted. Compared with other traditional Chinese medicine extraction technologies, the microwave-assisted extraction technology has the characteristics of low required temperature, short working time, low energy consumption, high extraction efficiency, small environmental pollution and the like. Microwave-assisted extraction also has some limitations, such as uneven microwave radiation, which can easily cause local excessive temperature, resulting in denaturation and loss of active ingredients. The term "enzymatic hydrolysis" is a new technique for extracting active ingredients from natural plants in recent years. The principle is that enzyme is utilized to selectively destroy cell walls, so that the intracellular components are easier to dissolve and diffuse, and the method is applied to the extraction of the effective components of the traditional Chinese medicine. Compared with the traditional water extraction method, the technology has the advantages of mild condition, high leaching rate, reduced degradation of thermosensitive components and the like. The term "lye extraction" is to extract the free polysaccharide by dissolving the free polysaccharide further by swelling and breaking the plant cells and cell walls by water absorption under the action of dilute alkali. However, the method has selectivity to materials, is easy to damage polysaccharide structures, and needs to strictly control acid-base concentration. In the specific embodiment of the invention, the extraction method of the penthorum chinense pursh crude polysaccharide is a hot water extraction method.

In the present invention, the term "concentrate" refers to the increase in concentration of a solution by evaporating a solvent, and generally refers to the decrease in unwanted parts and the increase in the relative content of the desired parts. Methods of concentration include, but are not limited to: precipitation, adsorption, ultrafiltration, dialysis, distillation under reduced pressure, and freeze-drying; further, the concentration method is a reduced pressure distillation method.

In the present invention, common methods for removing proteins (deproteinization) from polysaccharides include, but are not limited to: sevag method, trifluorotrichloroethane method, trichloroacetic acid method, enzyme-Sevag method; in a specific embodiment of the invention, the deproteinization process is the Sevag process.

In the invention, the ion exchange chromatographic column is an anion exchange chromatographic column, and the anion exchange chromatographic column comprises a strong anion exchange chromatographic column and a weak anion exchange chromatographic column. The strong anion exchange chromatographic column comprises but is not limited to Q-Sephadex A-25, Q-Sephadex A-50, Q-Sephadex C-25, Q-Sephadex C-50; the weak anion exchange chromatography column includes, but is not limited to, DEAE-Cellulose DE-22, DEAE-Cellulose DE-23, DEAE-Cellulose DE-51, DEAE-Cellulose DE-52, DEAE-Cellulose DE-53, and DEAE-Sepharose Fast Flow. In a specific embodiment of the invention, the ion exchange chromatography column is DEAE-Sepharose Fast Flow.

In the present invention, the term "dialysis" refers to a process of separating molecules of different sizes using a semipermeable membrane. Semipermeable membranes are a class of membranes having a pore size of a certain size. Molecules smaller than their pore size can pass freely. Molecules larger than their pore size cannot pass.

In the present invention, gel chromatography columns include, but are not limited to, sephacrylS-100, sephacrylS-200, sephacrylS-300, sephacrylS-400, superose 12, superose 6 (e.g., sepharose 6Fast Flow), superdex 12, and Superdex 6 from GE company. Gel chromatographic column further comprisesIncluding but not limited to HiLoad Superdex16/600Superdex75pg, superdex Peptide, superdex 200, and Superdex 30 from GE company. In a specific embodiment of the invention, the gel chromatography column is a Sepharose 6Fast Flow. It will be appreciated that any other gel chromatography packing material having a separation range of 500 to 10000 daltons may be used. In general, when using a gel column, ddH may be used first ₂ O balance gel chromatographic column, the flow rate can be determined according to actual conditions. For example, in certain embodiments, the flow rate may be 0.5 to 50ml/min, such as 2ml/min.

In the present invention, the compositions of the present invention comprise a pharmaceutically acceptable carrier, adjuvant or vehicle, as used herein, which includes any and all solvents, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate for the particular dosage form desired. The use of any conventional carrier medium is also considered to be within the scope of the present invention unless it is incompatible with the compounds of the present invention, e.g., due to any undesirable biological effects that may occur or otherwise interact in a deleterious manner with one or more of any of the other components of the pharmaceutically acceptable composition.

The pharmaceutically acceptable carrier may comprise inert ingredients that do not unduly inhibit the biological activity of the compound. The pharmaceutically acceptable carrier should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic, or free of other undesirable reactions or side effects upon administration to a subject. Standard pharmaceutical formulation techniques may be used.

Some examples of substances that may serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as tween 80, phosphate, glycine, sorbic acid or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride or zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, lanolin; sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc powder; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, and colorants, mold release agents, coating agents, sweeteners, flavoring agents, and fragrances, preservatives, and antioxidants can also be present in the composition at the discretion of the formulator.

The composition may be in any orally acceptable dosage form (including, but not limited to, capsules, tablets, aqueous suspensions or solutions) for oral administration. For tablets for oral administration, common carriers include lactose and corn starch. Typically, a lubricant such as magnesium stearate is also added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When an aqueous suspension is required for oral administration, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweeteners, flavoring agents or coloring agents may also be added.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic, c) humectants such as glycerin, d) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glyceryl monostearate, h) absorbents such as kaolin and bentonite, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar, high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also have a composition such that they release the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymers and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar, high molecular weight polyethylene glycols and the like.

Microencapsulated forms with one or more of the above excipients may also be used in the present invention. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings, controlled release coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also contain, as is common practice, additional substances other than inert diluents, such as tabletting lubricants and other tabletting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and may also have a composition such that they release the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymers and waxes.

In the present invention, penthorum chinense pursh polysaccharide or composition can be used for delaying senescence or preventing or treating senescence-associated diseases, and refers to any anti-aging treatment. Anti-aging treatments include, but are not limited to, treatments that result in preventing, ameliorating or reducing the effects of aging, reducing or delaying the increase in biological age, and delaying aging; treating, preventing, ameliorating or reducing the effects of frailty or age-related diseases and conditions or regression, slowing the progression of such regression, prolongation of the healthy phase or life span, rejuvenating, increasing stress or resilience, improving surgery, radiation therapy, disease and/or any other post-stress recovery or other enhancement, prevention and/or treatment of climacteric syndrome, restoring reproductive function, eliminating or reducing the spread of senescent cells, reducing the total cause or causes of all or more of the risk of death or risk of death associated with at least one or at least two age-related diseases or conditions, or delaying such risk increase, reducing the risk of morbidity. Treatment that modulates at least one biomarker of aging to a younger state or slows its transition to an "elderly" state is also considered an anti-aging treatment, including, but not limited to, biomarkers of aging that see signs of aging, such as wrinkles, gray hair, and the like. In some embodiments, the age-related disease or disorder is selected from: atherosclerosis, cardiovascular disease, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, neurodegeneration (including but not limited to Alzheimer's disease, huntington's disease and other dementia with age-related aspects; parkinson's disease; and amyotrophic lateral sclerosis [ ALS ]), stroke, atrophic gastritis, osteoarthritis, NASH, trunk prodromance, chronic obstructive pulmonary disease, coronary artery disease, dopamine imbalance syndrome, metabolic syndrome, fatigue urinary incontinence, hashimoto's thyroiditis, heart failure, senile depression, immune aging (including but not limited to age-related reduced immune response to vaccines, age-related reduced immune response), myocardial infarction, acute coronary syndrome, sarcopenia obesity, senile osteoporosis, urinary incontinence, and the like. Cancer survivors, patients undergoing chemotherapy and radiation therapy, and other comparable stress and HIV patients may accelerate aging, and treatment for such accelerated aging or its consequences is also considered anti-aging treatment and precautions therefor.

In the invention, when the penthorum chinense pursh polysaccharide or the composition is used for preparing a product for delaying aging, the action concentration of the penthorum chinense pursh polysaccharide is selected from 0.5-2.0mg/mL; further, the action concentration of the penthorum chinense pursh polysaccharide is 1mg/mL.

In the present invention, products include, but are not limited to, pharmaceuticals, solid beverages, liquid beverages, dairy products, flavor powders, nutraceuticals. Dosage forms of the medicament include, but are not limited to, solutions, suspensions, emulsions, extracts, elixirs, powders, granules, tablets, capsules.

The route of administration of the drug according to the present invention is not limited as long as it can exert a desired therapeutic effect or prophylactic effect, including (but not limited to): intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intrathecal, intramuscular, intratracheal, subcutaneous, transdermal, pleural, topical, inhalational, transmucosal, dermal, gastrointestinal, intra-articular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic, intratumoral, in some cases systemic, and in some cases topical.

In the present invention, the subject of the drug is not limited and may be any animal including, but not limited to, humans, non-human primates, dogs, cats, rodents, etc., and includes life-span and aging model organism caenorhabditis elegans.

The appropriate dosage of the drug of the present invention can be prescribed in various ways depending on the preparation method, the administration method, the age, weight, sex, pathology, diet, administration time, administration route, excretion rate and response sensitivity of the patient, and the skilled doctor can easily determine the prescription and the dosage of the drug for which the prescription is effective for the desired treatment or prevention.

In the present invention, the terms "comprises" or "comprising" or "having" wherever used herein are not meant to be limited to the elements described below, but rather to include one or more additional elements of no particular mention, with or without functional importance, i.e., the listed steps, elements or options need not be exhaustive. In contrast, it is possible to use "containing", in which the elements are limited to those specified later.

The invention is further illustrated below in conjunction with specific examples, which are intended to illustrate the invention and are not to be construed as limiting the invention. Those of ordinary skill in the art will appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents. The experimental procedure, in which no specific conditions are noted in the examples below, is generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

EXAMPLE 1 extraction and purification of penthorum chinense Pursh polysaccharide

1. Penthorn grass source

The penthorum chinense pursh is purchased from the Chinese medicinal herb market in ancient rush county of Sichuan province in 9 months of 2020.

2. Experimental method

1) Extracting: the Huang Caoquan plants were crushed, sieved through a 100-mesh sieve, and dried to a constant weight in an oven at 60 ℃. 100g of penthorum chinense pursh powder is weighed and placed in a Soxhlet extractor, and petroleum ether (60 ℃ -90 ℃) and acetone and 80% ethanol are sequentially used for repeated extraction to remove substances such as grease, amino acid, monosaccharide, oligosaccharide pigment and the like in the penthorum chinense pursh powder. After drying, the penthorum chinense pursh powder is leached for 3 hours at the temperature of 80 ℃ and the feed-liquid ratio of 1:20. Rotary evaporating supernatant, adding 4 times volume of absolute ethanol to precipitate penthorum chinense pursh crude polysaccharide. After standing overnight at 4 ℃, the precipitate was collected by centrifugation (3500 rpm,10 min), redissolved in water, and deproteinized by the Sevag method (chloroform: n-butanol=4:1). And freeze-drying to obtain the penthorum chinense pursh crude polysaccharide.

2) Separating: preparing a penthorum chinense pursh crude polysaccharide solution with the concentration of 40.0mg/mL, and filtering for later use. Adopting DEAE-agarose gel FF (DEAE-Sepharose Fast Flow) ion exchange chromatographic column, adding herba Penthori chinensis crude polysaccharide solution, gradient eluting with deionized water with flow rate of 2mL/min and 0.1M NaCl solution, and collecting eluate. And combining the polysaccharides eluted by the eluent with the same concentration, dialyzing to remove salt (molecular retention of 3500 Da), and freeze-drying to obtain penthorum chinense Pursh polysaccharide.

3) Purifying: the separated polysaccharide is prepared into a solution with the concentration of 10.0mg/mL and is filtered for standby. The method comprises the steps of adopting an agarose gel 6FF (Sepharose 6Fast Flow) hydrophobic chromatographic column, adding penthorum chinense pursh polysaccharide solution, eluting with deionized water with the Flow rate of 1mL/min, and collecting eluent. And combining the eluates with the same symmetrical peak, filtering, and freeze-drying to obtain uniform penthorum chinense pursh polysaccharide, which is named PCPP.

EXAMPLE 2PCPP chemical ingredient determination

1. Experimental method

1) Neutral sugar content determination: glucose mother liquor with the concentration of 0.20mg/mL is prepared by using glucose, 100 mu L of dilution liquid is respectively transferred into an EP pipe after the glucose mother liquor is respectively diluted into gradient concentrations of 0, 0.02, 0.04, 0.06, 0.08 and 0.10mg/mL by adding water, and 100 mu L of 5% redistilled phenol water solution (w/mL and used for preparation) and 500 mu L of concentrated sulfuric acid are sequentially added and uniformly mixed. After boiling water bath for 30min, absorbance was measured at 490nm to prepare a standard curve. A PCPP solution of 0.1mg/mL was prepared, 100. Mu.L of the PCPP solution was placed in an EP tube, and the absorbance of the reaction was measured at 490nm according to the procedure described above. And calculating the polysaccharide content in the PCPP according to a standard curve.

2) Determination of uronic acid content: the uronic acid mother liquor with the concentration of 0.1mg/mL is prepared by using galacturonic acid, 0, 0.1, 0.2, 0.3, 0.4 and 0.5mL of galacturonic acid solution are respectively absorbed for supplementing water to 0.5mL, 3.0mL of sodium tetraborate-concentrated sulfuric acid solution (0.48 g of sodium tetraborate is dissolved in 100mL of concentrated sulfuric acid) is added for shaking and mixing, the mixture is cooled to room temperature after boiling water bath for 15min, 40 mu L of m-hydroxybiphenyl solution is respectively added for reaction at room temperature for 30min, absorbance is measured at 525nm, and a standard curve is drawn. 1mg/mL of PCPP solution was prepared, 0.5mL of the PCPP solution was aspirated, absorbance of the sample was measured at 525nm according to the above procedure, and uronic acid content in PCPP was calculated from the standard curve.

3) And (3) measuring the flavone content: weighing 21mg of rutin reference substance, and fixing the volume in a 100mL volumetric flask with 50% ethanol to obtain rutin mother solution. Respectively sucking standard 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6mL rutin mother liquor into a 10.0mL centrifuge tube, supplementing 50% ethanol to 4.0mL, respectively adding 5% sodium nitrite to 0.3mL, shaking uniformly, standing at room temperature for 6min, adding 10% aluminum nitrate solution to 0.3mL, shaking uniformly, standing for 6min, adding 1M NaOH solution to 4mL, shaking uniformly, standing for 6min, diluting with 50% ethanol to a 10mL scale mark, standing at room temperature for 15min, measuring absorbance at a wavelength of 510nm by an ultraviolet spectrophotometer, and making a standard curve. 10mg of PCPP was weighed out and the volume was determined by a 50mL volumetric flask. 4.0mL of the polysaccharide solution was placed in an EP tube, absorbance was measured at a wavelength of 510nm according to the procedure described above, and the PCPP flavone content was calculated from the standard curve.

4) Protein content determination: preparing bovine serum albumin mother liquor with the concentration of 0.1mg/mL, respectively sucking 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL of bovine serum albumin mother liquor, supplementing water to 1.0mL, adding 5mL of Coomassie brilliant blue G-250 (0.05% dissolved by 95% ethanol), uniformly mixing, reacting for 20min at room temperature, measuring absorbance at 595nm, and drawing a standard curve. A polysaccharide solution of 0.1mg/mL was prepared, 1.0mL of the polysaccharide solution was placed in an EP tube, absorbance was measured at 595nm according to the procedure described above, and the protein content in PCPP was calculated from the standard curve.

5) The calculation formula of the chemical component content in PCPP is as follows:

PCPP chemical content (%) = [ (chemical mass x dilution ratio)/sample mass ] ×100%.

6) Ultraviolet spectrum scanning and measuring: preparing 1.0mg/mL PCPP solution, and carrying out ultraviolet spectrum analysis on the PCPP by using an ultraviolet spectrophotometer at a wave band of 200-400 nm to detect the contents of nucleic acid and protein.

2. Experimental results

The experimental result of chemical component measurement shows that the crude polysaccharide yield of penthorum chinense pursh is 2.84+/-0.20%, the protein in the purified PCPP is not detected, and the neutral sugar content and uronic acid content of the PCPP are 86.97 +/-1.72% and 13.02+/-0.23% in sequence.

The results of the ultraviolet spectrum scanning are shown in FIG. 1, and the results show that no strong absorption peaks at 260nm and 280nm indicate that the content of nucleic acid and protein in PCPP is extremely low.

EXAMPLE 3PCPP molecular weight determination

1. Experimental method

5mg of dextran standard substances and PCPP with different molecular weights (1-700 kDa) are respectively weighed, 0.05mol/L NaCl solution is added to obtain a sample solution with the final concentration of 5.0mg/mL, and the sample solution are mixed and filtered by a 0.22 mu m water system microporous filter membrane for standby. Molecular weight was determined using gel permeation chromatography and a linear regression curve was drawn to give a standard curve of weight average molecular weight (Mw) (y= -0.1889x+12.007, R) ² = 0.9943) and a standard curve of number average molecular weight (Mn) (y= -0.1752x+11.304, r ² = 0.9931). The width of the molecular weight distribution of the polysaccharide is measured using the ratio of Mw and Mn, i.e., the polydispersity index (PDI). Chromatographic conditions: the chromatographic column is BRT105-104-102 series gel column (8×300 mm); column temperature is 40 ℃; the sample injection amount is 20 mu L; the mobile phase is 0.05mol/L NaCl solution; the flow rate was 0.6mL/min.

2. Experimental results

The results are shown in FIG. 2, which shows that PCPP peaks at 41.46min, and that PCPP has a Mw of 14.96kDa and a Mn of 10.97kDa according to the standard curve. The PDI of PCPP is 1.36, the molecular weight distribution of PCPP is narrow, and the PCPP has good uniformity, which shows that the agarose gel 6FF has good purification effect.

EXAMPLE 4PCPP monosaccharide composition determination

1. Experimental method

10mg of PCPP was weighed and dissolved in 2mL of deionized water, sealed with 2mL of 2mol/L trifluoroacetic acid (TFA), and hydrolyzed at 120℃for 4h. After hydrolysis, the water was evaporated to dryness at 50 ℃ using a rotary evaporator and the residual TFA was removed by repeated washing with methanol. The PCPP hydrolysate and the monosaccharide standard were dissolved with 2mL of deionized water, respectively, and then filtered with a 0.22 μm aqueous microporous membrane for use. Monosaccharide composition was determined using High Performance Liquid Chromatography (HPLC), HPLC conditions: the column was Dionex CarboPac PA (150X 3.0mm,6 μm); the sample injection amount is 5 mu L; the mobile phases were 0.1M NaOH and 0.2M NaAC (95:5, v/v); the flow rate was 0.5mL/min.

2. Experimental results

The results are shown in FIG. 3, which shows that PCPP consists of 7 monosaccharides, galactose (Gal) (37.12%), arabinose (Ara) (35.21%), glucose (Glc) (9.14%), mannose (Man) (9.98%), xylose (Xyl) (3.75%), galacturonic acid (GalA) (2.62%), and glucuronic acid (Glc-UA) (2.18%), with the highest galactose and arabinose monosaccharide content.

EXAMPLE 5PCPP methylation analysis

1. Experimental method

Methylation reaction: 10mg of PCPP was weighed into a test tube with a stopper, and 2mL of anhydrous dimethyl sulfoxide and 20mg of NaOH were added thereto, followed by sealing reaction for 12 hours. 1.5mL of methyl iodide (CH) ₃ I) After 3h of reaction under nitrogen, 1mL of deionized water was added to terminate the reaction. 2mL of chloroform (CHCl) was added ₃ ) Recovering the methylation product. Dialyzing with distilled water for 24h, and freeze-drying the dialysate to obtain a first methylated sample. Repeated methylation was carried out until the sample was detected by infrared spectroscopy at 3700cm ^-1 ～3100cm ^-1 no-OH characteristic absorption peak appears nearby, indicating complete methylation.

Acetylation reaction: the methylated PCPP was hydrolyzed with 1mL of TFA at 120℃for 2h, dried by spin-drying under reduced pressure with the addition of 3mL of methanol, and repeated several times until the residual TFA was completely removed to give a hydrolysate. The hydrolysate was treated with sodium borodeuterium (NaBD) ₄ ) After 30min of reduction the reaction was quenched with acetic acid. Adding methanol, repeatedly washing, and removing borate ion. After emulsification at 85℃for 4h, 2mL of pyridine was addedAnd 1mL of n-propylamine, derivatization for 30min at 55 ℃, 1mL of each of pyridine and acetic anhydride was added, and the temperature was turned to 95 ℃ for continuous derivatization for 2h. Evaporating to dryness, adding CHCl into the residue ₃ Dissolving. GC-MS analysis was performed after passing through an organic series 0.22 μm filter.

Methylation analysis was performed using gas chromatography-mass spectrometry tandem, GC-MS conditions: the chromatographic column is DB-5MS (30 m×0.25mm×0.25 μm, carrier gas is He, flow rate is 1mL/min, sample injection amount is 1 μL, split ratio is 10:1, sample inlet temperature is 250 ℃, ion source temperature is 230 ℃, temperature of four-stage rod is 50 ℃, initial temperature of a column incubator is 140 ℃, the temperature is kept for 2.0min, temperature is raised to 230 ℃ by a program of 3 ℃/min, and the scanning range of m/z is kept between 30 and 600.

2. Experimental results

The results are shown in FIG. 4 and Table 1, and show that t- α -Araf- (1→5) - α -Araf- (1→4) - α -Galp- (1→composed AG region, →3, 6) - β -Galp- (1→3, 6) - α -Galp- (1→composed HG region, which is 67% in proportion to the skeleton of PCPP, →2) - β -Manp- (1→3, 4) - β -Galap- (1→3, 6) - β -Galp- (1→t- α -Glcp- (1→4) - α -Glcp- (1→2) - α -GlcAp- (1→4) - α -Galp- (1→3) - α -Galp- (1→4) - α -Galp- (1→3, 6) - β -Galp- (1→1→4) - α -Galp- (1→4) - β -Xylep- (1→1→located in the branched chain of PCPP.

TABLE 1 methylation analysis of PCPP

EXAMPLE 6PCPP Nuclear magnetic resonance Spectrometry

1. Experimental method

30mg of PCPP was reacted with 20mM NaOD at D ₂ O was subjected to three deuterium exchanges and then dissolved in 0.5mL D ₂ O, and is filled into a nuclear magnetic tube. 1 DNMR% ¹ H-NMR ¹³ C-NMR) and 2DNMR ¹ H- ¹ H COSY、 ¹ H- ¹ H TOCSY、 ¹ H- ¹ HNOESY、 ¹ H- ¹³ CHSQC ¹ H- ¹³ C HMBC) was tested and recorded on a 600MHz nuclear magnetic resonance spectrometer (Bruker AV II-600MHz, bruker company, switzerland). Based on the glycosidic bond information, P is establishedSchematic structure of CPP to demonstrate putative linker sequences.

2. Experimental results

The test results are shown in FIG. 5 and Table 2, and the results are shown in ¹ In the H NMR spectrum, the anomeric proton signal was distributed between 5.39 and 4.42 ppm. The other signal of protons in the sugar ring (H2-H6) is 4.15 to 3.15ppm. In the 13C NMR spectrum, the anomeric carbon signal range was 107.14 to 96.84ppm. The C2-C6 signal is 4.14 to 1.18ppm. A signal around 172ppm indicates the presence of uronic acid. The C-1 signal is correlated with the H-1 signal by the cross-signal on the HSQC spectrum and the H-2 signal is distributed by the cross-peak of H-1/H-2 on the COSY spectrum. The H-3 to H-6 signals can be deduced from COSY, TOCOSY. Corresponding C-2 to C-6 signals were assigned according to the HSQC spectrum.

The anomeric signals of the a-Araf residues A and E were 5.39/105.86 and 5.12/104.72ppm, respectively. A2 The chemical shifts of H-2/C-2 for (4.14/79.22 ppm) and E2 (4.06/72.44 ppm) can be assigned based on the correlation of the cross-peaks of H-1/H-2 in the COSY spectrum and H-2/C-22 in the HSQC. In combination with COSY, TOCOSY and NOESY, residues A and E, H-3 to H-5, were assigned. The pattern of linkage to adjacent glycosyl residues was deduced by HMBC spectroscopy. The cross peaks at 5.39/74.23, 5.12/74.23 and 5.12/77.97ppm were assigned to the α -Araf- (1→5) - α -Araf- (1→and→5) - α -Araf- (1→4) - α -Galp- (1→indicating the presence of arabinose segments and arabinogalactan regions in PCPP.

The anomeric signals of α -Galp residues D, G, H and K were 5.09/104.50, 5.02/105.17, 5.17/107.14 and 4.98/105.68ppm, respectively. Based on the analogy to the alpha-Araf residues, H2-H6 and C2-C6 were assigned and listed in table 2. The downward field shifts of the glycosidic sites at G-3, H-4, K-4 and K-6 compared to other alpha-Galp residues are shown. Linkage patterns between adjacent glycosyl residues were deduced from the cross-peak signals at 5.17/77.97, 5.17/74.23, 5.17/66.84, 5.09/81.44, 5.02/74.23, 5.02/81.44, 5.02/74.19, 4.98/81.44 and 4.98/74.30ppm in the HMBC spectra. These 1H and 13C correlations are attributed to alpha-Galp- (1.fwdarw.3) -alpha-Galp- (1.fwdarw.3) 3) -alpha-Galp- (1- > 3, 6) -alpha-Galp- (1- > 4) -alpha-Galp- (1- > 5) -alpha-Araf- (1- > 4) -alpha-Galp- (1- > 4, 6) -alpha-Galp- (1- > 3) -alpha-Galp- (1- > and 4, 6) -alpha-Galp- (1- > 3, 4) -beta-Galp- (1-, in addition, arabinogalactan (AG) regions were identified by cross-linking between Araf and α -Galp.

HG fragments consisting of beta-Galp residues can infer the way in which the branches are linked by residual cross-signals of 4.45/67.61, 5.17/74.19, 5.17/67.51 and 4.45/67.51. In the branches, various glycosidic linkages with 2-. Beta. -Manp, β -Xylp, α -Gal, β -GalA, α -GlcA and α -GlcP residues were identified at the O-3, O-4 and O-6 sites of the different Galp residues.

The ligation sequence is shown in FIG. 6, and the results show that the α -Araf residues and α -Galp residues mainly constitute a "smooth" neutral AG region, which is linked to the HG region consisting of the β -Galp residues on the other side, and that the HG region is linked to the 2- β -Manp, β -Xylp, α -Gal, β -GalA, α -GlcA and α -GlcP residues to form a "dense" branch.

TABLE 2 glycosidic bond and NMR chemical shifts of PCPP

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EXAMPLE 7PCPP Fourier IR Spectroscopy

1. Experimental method

PCPP and KBr were baked to constant weight in an oven at 60 ℃. A small amount of PCPP sample and KBr powder were placed in an agate mortar, thoroughly mixed and ground into superfine powder. After the die iS pressed into tablets, the spectrum of PCPP iS scanned by a Fourier transform infrared spectrometer (Nicolet iS10, thermofisher science and technology Co., USA), and the signal acquisition range iS 400-4000 cm ^-1 。

2. Experimental results

The results are shown in FIG. 7, which shows that PCPP is at 3394.79cm ^-1 And 1618.24cm ^-1 The spectral band at is due to the O-H stretching and bending vibrations of PCPP; 2937.46cm ^-1 And 1401.30cm ^-1 Is C-H stretching vibration and C-O symmetrical stretching vibration of PCPP; 1234.20cm ^-1 And 1055.37cm ^-1 The peak at which corresponds to the C-O-C symmetric tensile vibration of PCPP; indicating that PCPP has an absorption peak typical of polysaccharides.

Example 8PCPP three-dimensional helix Structure determination

1. Experimental method

The three-dimensional helix structure of PCPP was determined by congo red test. 1mL of 2.0mg/mL PCPP and 1.5mL of 0.2mM Congo red reagent are mixed, and 1mol/L NaOH solution is added to change the final concentration gradient of NaOH in the EP tube from 0 to 0.5 mol/L. The change in the maximum absorption wavelength of the sample in the range of 400 to 700nm was recorded by an ultraviolet spectrophotometer (UV-1800, shimadzu corporation, japan).

2. Experimental results

The results are shown in fig. 8, showing that in dilute alkali solution, λmax of the PCPP and congo red mixture shows a significant red shift from 488 to 496nm, indicating the presence of a helical conformation of the PCPP-congo complex. With increasing NaOH concentration, the alkaline environment influences the binding of the PCPP-Congo complex, and λmax remains around 495 nm.

EXAMPLE 9PCPP scanning electron microscope analysis

1. Experimental method

PCPP is fixed on a sample stage by conductive adhesive and then placed in a sample chamber of a multifunctional scanning electron microscope (Quanta 450, FEI Co., USA). The PCPP surface morphology was observed using a high vacuum mode with an acceleration voltage of 20kV and magnifications of 50X, 300X, 500X and 1000X, respectively.

2. Experimental results

The results are shown in fig. 9, which shows that the PCPP surface has a loose cellular structure similar to honeycomb.

EXAMPLE 10PCPP thermodynamic Property analysis

1. Experimental method

The thermodynamic properties of PCPP were measured by differential scanning calorimetry. After 2mg of PCPP was sealed in an aluminum pan, it was placed on a sample stage of a differential scanning calorimeter (TA-Q20, TA instruments Co., USA). Under nitrogen atmosphere, the temperature was raised from 50℃to 400℃at 10℃per minute, and the empty plate was used as a control, and the metal indium was calibrated.

2. Experimental results

The results are shown in FIG. 10, which shows that the endothermic peak at around 100deg.C is caused by evaporation of free water and bound water in PCPP, while the exothermic transition peak at around 320 deg.C is caused by degradation of PCPP; the PCPP showed good thermal stability.

Example 11PCPP in vitro antioxidant assay

1. Experimental method

1) DPPH clearance measurement: DPPH-ethanol solution (0.4 mmol/L) and PCPP and Trolox solutions with different concentration gradients (0.25, 0.5, 1.0, 2.0 and 5.0 mg/mL) were prepared. 100. Mu.L of DPPH solution and 100. Mu.L of sample solution are removed and placed in a 96-well plate, and after shaking, the reaction is carried out in a dark place for 30min, and the absorbance is measured at 517 nm. DPPH clearance was calculated.

2) Iron ion reducing force measurement: different concentrations (0.25, 0.5, 1.0, 2.0, 5.0 mg/mL) of PCPP solution and Trolox solution were formulated, each 1.0mL. 1.0mL of 0.2mol/L phosphate buffer and 1mL of 1% potassium ferricyanide solution are respectively added, after reaction is carried out for 20min at 50 ℃, 1mL of 10% trichloroacetic acid solution is added, centrifugation is carried out at 1000rpm/min for 5min, 2.5mL of supernatant is sucked and transferred to a new EP tube, 0.5mL of 0.1% ferric trichloride solution and 2.5mL of deionized water are respectively added, and the mixture is uniformly mixed. The absorbance was measured at 700 nm.

3) Total antioxidant power measurement: a series of concentrations (0.25, 0.5, 1.0, 2.0, 5.0 mg/mL) of PCPP solution and Trolox solution were formulated. Preparing 0.3mol/L acetic acid buffer solution, 10 mmol/L2, 4, 6-tripyridyl-S-triazine (TPTZ) solution and 20mmol/L FeCl ₃ The solution was mixed at a ratio of 10:1:1 to form the FRAP working solution. PCPP and Trolox solutions with different concentrations of 0.1mL are sucked, 2.9mL of FRAP working solution is added respectively, the mixture is subjected to light-shielding reaction at room temperature for 10min, and the absorbance at 593nm is measured.

2. Experimental results

As a result, as shown in FIG. 11, the DPPH/scavenging ability measurement experiment showed that, when the concentration was in the range of 0.25 to 2.0mg/mL,the clearance rate of PCPP to DPPH is obviously increased from 66.52 +/-3.30% to 84.74+/-0.67%, and the IC thereof is rapidly increased ₅₀ At 0.19mg/mL, this indicates that PCPP has good DPPH scavenging ability and has the potential to be an antioxidant. The experimental result of iron ion reducing force measurement shows that when the concentration is 0.25-2.0 mg/mL, the absorbance of PCPP is rapidly increased from 0.13 to 1.10, and the EC is improved ₅₀ 1.43mg/mL. However, the iron ion reducing power of PCPP was relatively weak compared to the Trolox positive control group. The test result of the total antioxidant capacity test shows that when the concentration is 0.25-1.0 mg/mL, the EC50 of the total antioxidant of PCPP is 0.97mg/mL, and the total antioxidant has good antioxidant activity similar to the total antioxidant trend of the positive control Trolox.

Example 12 protection of PCPP against cellular oxidative stress

1. Experimental method

1) Effect of PCPP on HHL-5 cell viability: HIL-5 cell density was adjusted to 10 ⁵ After each mL, 90. Mu.L of the cell suspension was removed and cultured in a 96-well cell culture plate for 12 hours by adherence, and 10. Mu.L of PCPP at different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0 mg/mL) was added to each well and cultured for 24 hours. After 30min of staining with CCK-8, absorbance was measured at 450 nm. Cell viability (%) was calculated.

2) Effect of PCPP on HHL-5 cell oxidative damage protection: HIL-5 cell density was adjusted to 10 ⁵ After each mL, 90. Mu.L of the cell suspension was removed and cultured in a 96-well cell culture plate for 12 hours by adherence, and 10. Mu.L of PCPP at different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0 mg/mL) was added to each well and cultured for 24 hours. The medium was removed and 90. Mu.L of whole medium and 10. Mu.L of paraquat oxidative stress at a concentration of 1mM were added for 3h. After 30min of staining with a solution containing 10% CCK-8, the absorbance was measured at 450 nm. Cell viability (%) was calculated.

3) Effect of PCPP on HHL-5 cell Reactive Oxygen Species (ROS): HIL-5 cell density was adjusted to 10 ⁵ 1.8mL of the cell suspension was removed and cultured in a 6-well cell culture plate (with a cell slide placed thereon) for 12 hours, and 0.2mL of PCPP solution was added for 24 hours. The medium was removed and oxidative stress was performed by adding 1.8mL of whole medium and 0.2mL of paraquat at a concentration of 1mM for 3 hours. Adding H2DCF-DA fluorescent dye, reacting for 30min in dark place, and performing fluorescence microscopy ROS levels of HHL-5 cells were visualized with a mirror.

4) Effect of PCPP on HHL-5 cell mitochondrial membrane potential change: HIL-5 cell density was adjusted to 10 ⁵ 1.8mL of the cell suspension was removed and cultured in a 6-well plate (with a cell slide placed thereon) for 12 hours, and 0.2mL of PCPP solution was added for 24 hours. The medium was removed and oxidative stress was performed by adding 1.8mL of whole medium and 0.2mL of paraquat at a concentration of 1mM for 3 hours. After the JC-1 fluorescent dye is added and the cells are stained for 25min under dark condition, the mitochondrial membrane potential of the cells is observed by a fluorescence microscope.

5) Effect of PCPP on HHL-5 apoptosis: modulation of HIL-5 cells to 10 ⁵ 1.8mL of the cell suspension was removed and cultured in a 6-well cell culture plate for 12 hours at a volume of one mL/mL, and 0.2mL of PCPP solution was added for 24 hours. The medium was removed and oxidative stress was performed by adding 1.8mL of whole medium and 0.2mL of paraquat at a concentration of 1mM for 3 hours. After 25min of reaction by Annexin V-FITC/PI apoptosis kit reagent, apoptosis was measured by flow cytometry (Accuri C6, BD Co., USA).

2. Experimental results

The cell viability detection experimental result is shown in figure 12, and shows that when the concentration range of the penthorum chinense pursh polysaccharide is 0.1-0.4 mg/mL, compared with a control group, the PCPP has no obvious influence on HHT-5 cell viability; when the concentration of PCPP is 0.4-1.0 mg/mL, the activity of HIL-5 cells is reduced, indicating that a concentration of PCPP greater than 0.4mg/mL is toxic to cells. The cell oxidative damage protection experimental result shows that compared with a damaged group, PCPP of 0.1-0.5 mg/mL can obviously improve the activity of HIL-5 cells; among them, 0.2mg/mL PCPP has the best effect on resistance to oxidative stress, and this concentration is not toxic to cells.

The results of the cellular ROS level assay are shown in FIG. 13, which shows that paraquat treatment induced ROS levels in HIL-5 cells (injured group) 3.3 times greater than the blank (p < 0.001); compared with the injured cell group, the ROS of the PCPP treated cell of 0.20mg/mL is obviously reduced by 1.7 times (p < 0.01), which shows that the PCPP can effectively inhibit the excessive generation of ROS in cells and restore the redox steady state.

The experimental results of the change of the mitochondrial membrane potential of the cells are shown in fig. 14, and the results show that compared with a blank control, JC-1 of HHT-5 cells after paraquat stress changes from a polymerization state to JC-1 monomer dispersed on the inner membrane of the cells in mitochondria, so that the modeling effect is good, and compared with a damaged group, the MMP of a PCPP (prestressed high-molecular-weight polyethylene) experimental group added in a culture medium is obviously increased by 13.31+/-5.976% (p < 0.01), so that the PCPP can effectively maintain the mitochondrial membrane potential of the cells, and the mitochondrial membrane damage of the paraquat-induced cells is reduced.

The results of the apoptosis experiments are shown in fig. 15, and the results show that the percentage of early and late apoptosis of PCPP treatment is significantly reduced by 9.62+/-0.31% (p < 0.01) under oxidative stress, which indicates that PCPP can effectively improve paraquat-induced apoptosis.

EXAMPLE 13 influence of PCPP on the stress resistance of C.elegans

1. Experimental method

1) Heat stress experiment: c.elegans N2 at the egg stage was transferred to NGM medium containing varying concentrations of PCPP (0.25, 0.5, 1.0, 2.0, 5.0 mg/mL) and incubated at 20℃for 48h, and then transferred to 35℃for heat stress. The head or tail of the nematodes were touched with platinum wires and if no response was noted as dead, the number of nematodes surviving was recorded every 2h until all nematodes died. The test group without PCPP was a blank control group, 40 nematodes were picked up in each group, and 3 replicates were set.

2) Oxidative stress experiment: the test concentration of the heat stress test group (1.0 mg/mL) with the best test result was selected as the subsequent test concentration. The C.elegans N2 in the egg stage were transferred to NGM medium with or without PCPP and incubated for 48h at 20 ℃. Nematodes were transferred to acute oxidative stress in NGM medium supplemented with paraquat (150 mM). The number of nematodes surviving was recorded every 1h until all nematodes died. The test group without PCPP was a blank control group, 40 nematodes were picked up in each group, and 3 replicates were set.

2. Experimental results

The results of the heat stress experiments are shown in FIG. 16, and the results show that the average life span of the C.elegans is prolonged by 14.53+ -2.14% (p < 0.01), 38.25+ -3.73% (p < 0.0001) and 14.45+ -1.50% (p < 0.01) respectively by 0.5, 1.0 and 2.0mg/mL of PCPP compared with the blank group, which indicates that the PCPP significantly improves the N2 heat stress resistance of the wild type nematodes.

The results of the oxidative stress experiments are shown in FIG. 17, and the results show that the average life span of the caenorhabditis elegans under oxidative stress can be prolonged by 19.82+/-1.10% (p < 0.001) by 1.0mg/mL of PCPP compared with the control group, so that the PCPP has stronger in-vivo antioxidant activity.

Example 14 Effect of PCPP on caenorhabditis elegans ROS levels

1. Experimental method

After transferring the C.elegans N2 in the egg stage to the NGM medium with or without PCPP for 48h at 20 ℃, heat stress is carried out in an incubator at 35 ℃ for 4h. The nematodes were stained with H2DCF-DA fluorochrome for 30min and assayed for ROS levels in vivo using a fluorescence microscope. Fluorescence intensity was quantified using Image J software. The test group without PCPP was a blank control group, and 3 independent tests were set up to determine 25 nematodes per group.

2. Experimental results

The results are shown in fig. 18, which shows that the intracellular ROS content of caenorhabditis elegans is significantly reduced by 50.96 ±6.95% (p < 0.0001) compared to the control group, indicating that PCPP may either directly scavenge ROS or maintain ROS radical balance by increasing antioxidant enzyme activity in vivo.

Example 15 Effect of PCPP on DAF-16 Nuclear localization

1. Experimental method

After the TJ356 nematode mutant carrying daf-16:GFP had been contemporaneous, it was heat-stressed for 4h in an incubator at 35℃after incubation for 24h at 20℃in NGM medium with or without PCPP. The distribution of GFP in the cells was observed by fluorescence microscopy. The test group without PCPP was a blank control group, and 3 independent tests were set up, each group was photographed for 25 nematodes.

2. Experimental results

The results are shown in FIG. 19, which shows that 1.0mg/mL of PCPP treated group showed significant change in DAF-16 distribution, and the distribution of DAF-16 in the nucleus was increased to 38.88.+ -. 6.04% (p < 0.001), indicating that PCPP might activate DAF-16 directly or gene located upstream thereof, thereby enhancing the oxidative stress resistance of C.elegans.

Example 16 effect of PCPP on SOD-3:: GFP and HSP-16.2:: GFP expression

1. Experimental method

1) After synchronizing the CF1553 nematode mutant carrying SOD-3:GFP, it was incubated for 48h at 20℃in NGM medium with or without PCPP and then heat-stressed for 4h in an incubator at 35 ℃. Fluorescent photographs were taken by a fluorescent microscope, and SOD-3 was analyzed by Image J software for fluorescence intensity of GFP, and the expression level of SOD-3 was evaluated. The test group without PCPP was a blank control group, and 3 replicates were set, each group was photographed for at least 25 nematodes.

2) After the TJ375 nematode mutant carrying HSP-16.2:GFP has been contemporaneous, it is incubated for 48h at 20℃in NGM medium with or without PCPP. And (3) thermally stressing the mixture for 4 hours in an incubator at 35 ℃. Fluorescence photographs were taken with a fluorescence microscope, and the expression level of HSP-16.2:: GFP was evaluated by analyzing the fluorescence intensity of HSP-16.2:: GFP by Image J software. The test group without PCPP was a blank control group, and 3 replicates were set, each group was photographed for at least 25 nematodes.

2. Experimental results

The results are shown in figure 20, and the results show that PCPP significantly increases the expression of the caenorhabditis elegans SOD-3 by 26.32+/-3.07 percent (p < 0.001), which indicates that PCPP can possibly promote DAF-16 to enter the nucleus, further up-regulate the expression of the SOD-3, enhance the activity of SODs, eliminate ROS in the body and improve the oxidative stress survival rate of caenorhabditis elegans; compared with the control group, 1.0mg/mL PCPP significantly increases the expression of the caenorhabditis elegans HSP-16.2 by 53.38+/-4.60 percent (p < 0.0001), which shows that the HSP-16.2 is taken as a DAF-16 downstream regulatory gene, and the PCPP can promote DAF-16 to enter the nucleus and also can regulate the expression of the HSP-16.2.

EXAMPLE 17 effect of PCPP on caenorhabditis elegans antioxidant enzyme Activity and malondialdehyde accumulation

1. Experimental method

The activity of the caenorhabditis elegans SODs, CATs and MDA contents was determined according to the kit instructions. After culturing C.elegans N2 in the egg phase in NGM medium with or without PCPP at 20℃for 48h, heat stress was applied to the culture in an incubator at 35℃for 4h. The nematodes were broken up, centrifuged, and the supernatant was collected. The specific SODs, CATs and MDA content determination methods were operated according to the kit instructions.

2. Experimental results

The results are shown in FIG. 21, which shows that the SODs and CATs activities of the nematodes treated by PCPP are significantly improved by 14.34% + -1.60% (p < 0.01) and 98.03+ -6.93% (p < 0.001), respectively, indicating that the ability of the caenorhabditis elegans to resist oxidative stress is related to the expression levels of SODs and CATs in vivo, and that the improvement of the activity of the antioxidant enzyme can enhance the oxidative stress tolerance of the caenorhabditis elegans and further prolong the life thereof; PCPP reduces MDA in caenorhabditis elegans by 31.44+ -3.64% (p < 0.001), which indicates that penthorum chinense pursh polysaccharide can relieve lipid peroxidation in caenorhabditis elegans under oxidative stress conditions, and is related to PCPP enhancing activities of different enzymes in caenorhabditis elegans and inhibiting generation of free radicals.

The result shows that the penthorum chinense pursh polysaccharide can regulate the transcription factor DAF-16 in the caenorhabditis elegans to enter the cell nucleus from cytoplasm, further up-regulate the expression of resistance genes, promote the activity of antioxidant enzyme and induce the production of small heat shock protein, balance the in vivo ROS level and further improve the environmental hypochondriac survival rate of the caenorhabditis elegans.

EXAMPLE 18 influence of PCPP on the healthy longevity of caenorhabditis elegans

1. Experimental method

1) Life test: the C.elegans N2 at the egg stage were transferred to NGM medium with or without PCPP and incubated at 20℃in an incubator, and the nematodes were transferred daily to freshly prepared medium to ensure that the concentration of poly PCPP was unchanged throughout the experiment. The number of caenorhabditis elegans survivors was recorded daily for the same period of time until all deaths. 40 nematodes were selected and 3 replicates were set.

2) Body length and body width test: the C.elegans N2 at the egg stage were cultured in an NGM medium with or without 1.0mg/mL PCPP at 20℃in an incubator, replaced daily with fresh medium, and counted for length and width at 2d and 5 d. Each group was randomly selected from 20 nematodes and 3 replicates were set.

3) Exercise ability and pharyngeal pumping experiments: the C.elegans N2 at the egg stage were cultured in an NGM medium with or without 1.0mg/mL PCPP at 20℃in an incubator. At 2, 5 and 10d, after transferring the nematodes to fresh S-bascal buffer to adapt to the new environment for 2min, the number of body curves per nematode per 30S was counted with an electron microscope. After transferring nematodes to fresh NGM medium to adapt to the new environment for 2min, the number of pharyngeal pumping per 30s of caenorhabditis elegans was counted by electron microscopy. Each group was randomly selected from 20 nematodes and 3 replicates were set.

4) Lipofuscin accumulation assay: caenorhabditis elegans N2 in stage L1 is cultivated in an NGM medium with or without PCPP at 20 ℃. The lipofuscin content in nematodes was determined by fluorescence microscopy at excitation wavelength 380nm and emission wavelength 485nm at 5 and 10 d. Each group was randomly selected from 20 nematodes and 3 replicates were set.

2. Experimental results

The life test results are shown in FIG. 22, and the results show that 1.0mg/mL PCPP can prolong the average life of the caenorhabditis elegans by 16.29 +/-4.21% (p < 0.001) compared with the control group, thereby showing that the PCPP has the potential of prolonging the biological life.

The experimental results of body length and body width are shown in fig. 23, and the results show that compared with the control group, the PCPP can reduce the body length and body width of the caenorhabditis elegans in the larval stage (corresponding to Day 2) by 12.15% +/-2.71% (p < 0.001) and 16.81+/-5.39% (p < 0.001), respectively, and the body length and body width of the caenorhabditis elegans in the adult stage (corresponding to Day 5) experimental group and the blank control group have no significant difference, so that the PCPP can reduce the growth rate of the caenorhabditis elegans in the L1-L4 stages, regulate the growth period and further influence the service life of the caenorhabditis elegans.

The exercise capacity and pharyngeal pumping experiment results are shown in fig. 24, and the results show that in the larval stage and the early stage of the adult (respectively corresponding to Day2 and Day 5) of the caenorhabditis elegans, the influence of PCPP on the exercise capacity and pharyngeal pumping frequency of the caenorhabditis elegans is not obvious, so that the muscle function and the integrity of the caenorhabditis elegans at the stage are good, and the good state of the caenorhabditis can be maintained without adding an antioxidant; the PCPP can obviously improve the body bending frequency and pharyngeal pumping frequency of the caenorhabditis elegans in the old age (corresponding to Day 10), and 29.18 +/-7.18 percent (p < 0.01) and 22.25+/-3.76 percent (p < 0.01) respectively, which shows that the PCPP can maintain the muscle function of the caenorhabditis elegans and prolong the healthy life of the caenorhabditis elegans.

The results of lipofuscin accumulation experiments are shown in fig. 25, and the results show that PCPP significantly inhibits the accumulation of lipofuscin of the 5d nematode, which is consistent with the inhibition effect of PCPP on MDA (lipid peroxidation product), and indicate that PCPP can reduce the accumulation of lipofuscin, improve the exercise capacity, enhance the pharyngeal pumping frequency, prolong the healthy life and exhibit good biological activity by moderately delaying the growth and development of larvae.

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims

1. The penthorum chinense pursh polysaccharide PCPP is characterized by mainly comprising 7 monosaccharides of galactose, arabinose, glucose, mannose, xylose, galacturonic acid and glucuronic acid;

preferably, the galactose content is 37.12%;

preferably, the arabinose content is 35.21%;

preferably, the glucose content is 9.14%;

preferably, the mannose content is 9.98%;

Preferably, the xylose content is 3.75%;

preferably, the galacturonic acid content is 2.62%;

preferably, the glucuronic acid content is 2.18%.

2. The penthorum chinense pursh polysaccharide PCPP of claim 1, characterized in that the penthorum chinense pursh polysaccharide PCPP comprises an arabinogalactan region, a galactohomoregion, a mannose segment side chain, a glucose segment side chain, a galactohomosegment side chain, and a xylose segment side chain;

preferably, the skeleton of the arabinogalactan region is formed by sequentially cross-linking 12 galactose residues and 12 arabinose residues;

preferably, the skeleton of the homogalactose region is formed by sequentially connecting 12 galactose residues;

preferably, the mannose segment branches consist of 1 mannose residue, 1 galactose residue and 1 galacturonic acid residue;

preferably, the glucose segment branches consist of 2 glucose residues, 1 glucuronic acid residue and 1 galactose residue;

preferably, the galactosylated branches consist of 4 galactose residues;

preferably, the xylose segment branches consist of 1 xylose residue and 1 galactose residue;

preferably, the penthorum chinense pursh polysaccharide PCPP is formed by sequentially connecting an arabinogalactan region and a homogalactosyl region;

Preferably, the skeleton of the homogalactose region is sequentially connected with a mannose segment branched chain, a glucose segment branched chain, a homogalactose segment branched chain and a xylose segment branched chain respectively;

preferably, the penthorum chinense pursh polysaccharide PCPP comprises the following sugar residues: α -Araf- (1→β -Xylp- (1→α -Glcp- (1→α -Galp- (1→α -5) - α -Araf- (1→2) - β -Manp- (1→3, 4) - α -Galp- (1→4) - α -Glcp- (1→2) - α -glc ap- (1→4, 6) - α -Galp- (1→4, 6) - β -Galp- (1→3, 4) - β -GalpA- (1→4);

preferably, the Mw molecular weight of the penthorum chinense pursh polysaccharide PCPP is 14.96kDa;

preferably, the Mn molecular weight of the penthorum chinense pursh polysaccharide PCPP is 10.97kDa.

3. The penthorum chinense pursh polysaccharide PCPP according to claim 1, wherein the extraction method of the penthorum chinense pursh polysaccharide PCPP is carried out according to the following steps:

(1) Extracting penthorum chinense pursh crude polysaccharide;

4. The penthorum chinense pursh polysaccharide PCPP of claim 3, wherein the specific step of step (1) comprises: extracting penthorum chinense pursh powder, concentrating, precipitating with ethanol, deproteinizing, and drying to obtain penthorum chinense pursh crude polysaccharide;

Preferably, the method for obtaining the penthorum chinense pursh powder comprises the steps of crushing penthorum chinense pursh, degreasing and drying;

preferably, the degreasing comprises petroleum ether, acetone and/or ethanol degreasing;

preferably, the extraction method comprises a hot water extraction method, an ultrasonic assisted extraction method, a microwave assisted extraction method, an enzymolysis method or an alkali liquor extraction method;

preferably, the extraction method is a hot water extraction method;

preferably, the extraction temperature of the hot water extraction method is 80 ℃;

preferably, the extraction time of the hot water extraction method is 3 hours;

preferably, the ratio of the feed liquid of the hot water leaching method is 1:20;

preferably, the concentration method comprises precipitation method, adsorption method, ultrafiltration method, dialysis method, reduced pressure distillation method, and freeze drying method;

preferably, the concentration method is a reduced pressure distillation method;

preferably, the deproteinization method comprises a Sevag method, a trifluorotrichloroethane method, a trichloroacetic acid method, an enzyme-Sevag method;

preferably, the deproteinization method is a Sevag method;

preferably, the method of drying comprises freeze drying, vacuum drying, atmospheric drying or spray drying;

preferably, the method of drying is freeze drying.

5. The penthorum chinense pursh polysaccharide PCPP of claim 3, wherein the specific step of step (2) comprises: separating the penthorum chinense pursh crude polysaccharide in the step (1) by adopting an ion exchange chromatographic column, performing gradient elution, collecting eluent, dialyzing and drying to obtain penthorum chinense pursh polysaccharide;

preferably, the ion exchange chromatography column comprises an anion exchange chromatography column and a cation exchange chromatography column; the anion exchange chromatographic column comprises a strong anion exchange chromatographic column and a weak anion exchange chromatographic column; the strong anion exchange chromatographic column comprises Q-Sephadex A-25, Q-Sephadex A-50, Q-Sephadex C-25 and Q-Sephadex C-50; the weak anion exchange chromatographic column comprises DEAE-Cellulose DE-22, DEAE-Cellulose DE-23, DEAE-Cellulose DE-51, DEAE-Cellulose DE-52, DEAE-Cellulose DE-53 and DEAE-Sepharose Fast Flow;

preferably, the ion exchange chromatography column is DEAE-Sepharose Fast Flow;

preferably, the gradient elution method comprises the steps of sequentially adopting deionized water and/or salt solution for gradient elution;

preferably, the salt solution comprises a sodium chloride solution, a sodium sulfate solution, a magnesium sulfate solution, or an ammonium sulfate solution;

Preferably, the flow rate of the deionized water is 2mL/min;

preferably, the concentration of the sodium chloride solution is 0.1M;

preferably, the method of drying is freeze drying.

6. The penthorum chinense pursh polysaccharide PCPP of claim 3, wherein the specific step of step (3) comprises: purifying the penthorum chinense pursh polysaccharide in the step (2) by adopting a hydrophobic chromatography column, performing gradient elution by adopting deionized water, collecting eluent, filtering and freeze-drying to obtain penthorum chinense pursh polysaccharide PCPP;

preferably, the hydrophobic chromatography column comprises SephacrylS-100, sephacrylS-200, sephacrylS-300, sephacrylS-400, superose 12, superose 6, superdex 12 or Superdex6; the Superose 6 comprises Sepharose 6Fast Flow;

preferably, the hydrophobic chromatography column is a Sepharose 6Fast Flow;

preferably, the flow rate of the deionized water is 1mL/min.

7. A composition, characterized in that the composition comprises penthorum chinense pursh polysaccharide PCPP according to any one of claims 1-6;

preferably, the composition further comprises a pharmaceutically acceptable carrier, adjuvant or excipient.

8. Any of the following applications:

(1) Use of the penthorum chinense pursh polysaccharide PCPP of any one of claims 1-6 or the composition of claim 7 in the preparation of an antioxidant product;

(2) Use of the penthorum chinense pursh polysaccharide PCPP of any one of claims 1-6 or the composition of claim 7 in the manufacture of a product for use in delaying aging, preventing or treating aging-related diseases;

(3) Use of the penthorum chinense pursh polysaccharide PCPP of any one of claims 1-6 or the composition of claim 7 in the preparation of a product for scavenging free radicals;

(4) Use of penthorum chinense pursh polysaccharide PCPP of any one of claims 1-6 or the composition of claim 7 for the preparation of a product for reducing ROS levels;

(5) Use of the penthorum chinense pursh polysaccharide PCPP of any one of claims 1-6 or the composition of claim 7 in the preparation of products for enhancing SODs or CATs activity;

(6) Use of penthorum chinense pursh polysaccharide PCPP of any one of claims 1-6 or the composition of claim 7 for the preparation of a product for reducing MDA levels;

(7) Use of the penthorum chinense pursh polysaccharide PCPP of any one of claims 1-6 or the composition of claim 7 in the preparation of a product that promotes expression of DAF-16 or HSP-16.2.

9. The use according to claim 8, wherein the free radicals comprise superoxide anion radicals, H ₂ O ₂ Singlet oxygen, HO. Alkyl peroxide radical, lipid peroxide radical, ABTS+, DPPH;

preferably, the free radical is DPPH.

10. The use according to claim 8, wherein the aging-related disorders include atherosclerosis, cardiovascular diseases, arthritis, cataracts, osteoporosis, type 2 diabetes mellitus, hypertension, neurodegeneration, stroke, atrophic gastritis, NASH, trunk prodromal disease, chronic obstructive pulmonary disease, coronary artery disease, dopamine imbalance syndrome, metabolic syndrome, fatigue urinary incontinence, hashimoto thyroiditis, heart failure, senile depression, immune aging, myocardial infarction, acute coronary syndrome, sarcopenia, obesity, senile osteoporosis, urinary incontinence.