Application of ginseng pectin in prolonging life

Abstract

The invention discloses application of ginseng pectin in prolonging life, and belongs to the technical field of biological medicine. Ginseng pectin and insulin interact with each other, so that the combination process of insulin and a receptor is inhibited, and the activity of an insulin / IGF-1 signal channel is reduced to achieve the effect of prolonging the service life. The low (demethyl) esterified HG structural domain in the ginseng pectin is a key structure for prolonging the life, and is beneficial to the development and application of the pectin in the fields of life-prolonging health care products and functional foods.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of ginseng pectin in prolonging lifespan. Background Art

[0002] Ginseng is a precious traditional Chinese medicinal material, with the effects of prolonging lifespan and strengthening the body, and these health-care effects depend on the bioactive components it contains. Polysaccharides are an important class of bioactive components in ginseng, mainly including amyloid glucan and pectin. Our previous research found that ginseng amyloid glucan has a relatively weak effect on prolonging lifespan, but it is still unclear whether ginseng pectin can prolong lifespan.

[0003] The insulin / IGF-1 signaling pathway is the earliest defined signaling pathway related to lifespan regulation in model organisms and is highly conserved in Caenorhabditis elegans, yeast, Drosophila, and mammals. When the binding of insulin to its transmembrane receptor protein is blocked, the insulin / IGF-1 signaling pathway is inhibited, thereby promoting the expression of target genes of the key transcription factors Forkhead box O (FOXO) / DAF-16 and NF-E2-related factor 2 (Nrf2) / Skinhead-1 (SKN-1) downstream of the pathway, ultimately leading to the prolongation of the lifespan of organisms. However, it is still unknown whether ginseng pectin has the effect of inhibiting the binding of insulin to its transmembrane receptor protein.

[0004] Ginseng pectin is rich in a homogalacturonan (HG) domain with a linear structure, and the methylation substitution of the carboxyl group at the C-6 position of this domain is an important structural feature. Existing research has shown that the methylation modification level of pectin affects the biological activity of pectin to a certain extent. Therefore, clarifying the degree of methylation that determines the lifespan-prolonging function of ginseng pectin provides a new strategy for screening and obtaining ginseng pectin with better lifespan-prolonging effects. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to obtain ginseng pectin with a significantly higher lifespan-prolonging effect than ginseng amyloid glucan. The technical problems to be solved are not limited to the described technical themes, and those skilled in the art can clearly understand other technical themes not mentioned herein through the following description.

[0006] To solve the above technical problems, the present invention provides the application of ginseng pectin in the preparation of drugs for prolonging lifespan. It can interact with insulin, reduce the activity of the insulin / IGF-1 signaling pathway by inhibiting the process of insulin binding to the receptor, and achieve the effect of prolonging lifespan; at the same time, this ginseng pectin exerts its function through its low (de)esterified HG domain.

[0007] The above-mentioned pectin is extracted from the roots of ginseng. The pectin contains an HG domain, and the HG domain is a linear polygalacturonic acid domain.

[0008] The pectin described above is WGPA-2HG or WGPA-2HG-DE. WGPA-2HG-DE is the pectin obtained by demethylating WGPA-2HG. The preparation method of WGPA-2HG includes:

[0009] R1: After extracting the roots of ginseng with water to obtain an extract, adding a 95% ethanol aqueous solution to the extract until the ethanol concentration in the extract is 80%, collecting the precipitate, and obtaining the total polysaccharide of ginseng water extract.

[0010] R2: Performing anion exchange column chromatography on the total polysaccharide of ginseng water extract obtained in R1, and eluting successively with distilled water and a 0.5M sodium chloride solution with a pH of 7.0 to obtain the total pectin of ginseng. The anion exchange group used in the anion exchange column chromatography is DEAE.

[0011] R3: Performing anion exchange column chromatography on the total pectin of ginseng obtained in R2, and eluting successively with distilled water, a 0.1M sodium chloride solution with a pH of 7.0, and a 0.2M sodium chloride solution with a pH of 7.0. Collect the fraction of ginseng pectin eluted with the 0.2M sodium chloride solution with a pH of 7.0. The anion exchange group used in the anion exchange column chromatography is DEAE.

[0012] R4: Purifying the fraction of ginseng pectin obtained in R3 by gel filtration chromatography, eluting with a 0.15M sodium chloride solution with a pH of 7.0, collecting the eluate, and obtaining a pectin fraction with a molecular weight of 32 kDa named WGPA-2HG.

[0013] The specific steps of R1 are as follows: After extracting the roots of ginseng with water to obtain an extract, adding a 95% ethanol aqueous solution to the extract until the ethanol concentration in the extract is 80%, collecting the precipitate, and successively washing and displacing the water in the precipitate with a 95% ethanol aqueous solution and absolute ethanol to obtain the total polysaccharide of ginseng water extract WGP.

[0014] The specific steps of R2 are as follows: Performing anion exchange column chromatography on the total polysaccharide of ginseng water extract WGP obtained in R1, eluting the anion exchange column successively with distilled water and a 0.5M sodium chloride solution with a pH of 7.0, collecting the eluate eluted with the 0.5M sodium chloride solution with a pH of 7.0, concentrating, desalting, and drying to obtain the total pectin of ginseng WGPA. The anion exchange group used in the anion exchange column chromatography is DEAE.

[0015] The specific steps of R3 are as follows: subject the total ginseng pectin WGPA obtained from R2 to anion exchange column chromatography, elute successively with distilled water, 0.1 M sodium chloride solution with pH 7.0, and 0.2 M sodium chloride solution with pH 7.0, collect the eluate obtained by eluting with 0.2 M sodium chloride solution with pH 7.0, concentrate to remove salts and dry to obtain the ginseng pectin fraction WGPA-2. The anion exchange group used in the anion exchange column chromatography is DEAE;

[0016] The specific steps of R4 are as follows: purify the ginseng pectin fraction WGPA-2 obtained from R3 by gel filtration chromatography, elute with 0.15 M sodium chloride solution with pH 7.0, collect the eluate, concentrate to remove salts and dry to obtain a single ginseng pectin fraction named WGPA-2HG with a molecular weight of 32 kDa.

[0017] The operation steps of concentration, desalting and drying in the above steps R2, R3 or R4 are as follows: concentrate under reduced pressure at 60 °C, perform dialysis desalting using a dialysis bag with a cut-off molecular weight of 3500 Da, and then perform freeze-drying (at -40 °C, 0.1 MPa) using a freeze dryer (Christ, Germany) to obtain a freeze-dried powder, and respectively obtain the total pectin or pectin fraction named WGPA, WGPA-2 or WGPA-2HG.

[0018] The preparation method of WGPA-2HG-DE includes:

[0019] Prepare the WGPA-2HG into a liquid with a pectin content of 10 mg / mL with distilled water, then add 0.2 M sodium hydroxide solution with the same volume as the liquid, react at 4 °C for 4 hours to obtain a reaction product, and then add an ice acetic acid solution with a mass percentage content of 10% to the reaction product to obtain a demethylation reaction solution of WGPA-2HG; desalt the demethylation reaction solution, concentrate and freeze-dry to obtain a demethylated pectin fraction named WGPA-2HG-DE.

[0020] In the present invention, there is an interaction between the pectin and insulin. The pectin inhibits the binding of insulin to the receptor by interacting with insulin, reduces the activity of the insulin / IGF-1 signaling pathway to achieve the effect of extending lifespan. Reducing the activity of the insulin / IGF-1 signaling pathway enhances the activity of downstream transcription factors FOXO / DAF-16 and Nrf2 / SKN-1. The enhancement of the activity of the transcription factor FOXO / DAF-16 refers to promoting its nuclear localization and activating the expression of its target genes sod-3 gene and lys-7 gene. The enhancement of the activity of the transcription factor Nrf2 / SKN-1 includes promoting its nuclear localization and activating the expression of its target genes gcs-1 gene, gst-4 gene, gst-7 gene and gst-10 gene.

[0021] Specifically, the ginseng pectin described above exerts its function through the low (de)-esterified HG domain in the ginseng pectin.

[0022] The present invention also provides the application of the above-mentioned ginseng pectin in the preparation of health products and / or foods for prolonging lifespan.

[0023] The present invention also provides the application of the above-mentioned ginseng pectin in the preparation of drugs targeting the insulin / IGF-1 signaling pathway or inhibiting the binding of insulin to its receptor.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The ginseng pectin of the present invention inhibits the insulin / IGF-1 signaling pathway, enhances the activities of transcription factors FOXO / DAF-16 and Nrf2 / SKN-1, and achieves the effect of prolonging lifespan.

[0026] (2) The present invention uses biomembrane interference technology and isothermal titration calorimetry technology to explore that there is an interaction between ginseng pectin and insulin, which blocks the process of insulin binding to its receptor, thereby reducing the activity of the insulin / IGF-1 signaling pathway to achieve the effect of prolonging lifespan.

[0027] (3) The present invention degrades ginseng pectin by endo-polygalacturonase and modifies ginseng pectin by de-methylation / methylation using chemical methods, and explores that the low (de)-esterified HG domain in ginseng pectin is the key structure for prolonging lifespan, providing a new strategy for studying the lifespan-prolonging effect of plant pectin and the structure-activity relationship between methylation modification and pectin activity. And it is beneficial to the development and application of pectin in the fields of lifespan-prolonging health products and functional foods.

[0028] (4) The present invention has confirmed through experiments that compared with ginseng amyloid glucan, the ginseng pectin of the present invention can significantly prolong the lifespan of Caenorhabditis elegans. Description of the Drawings

[0029] Figure 1 It is the molecular weight distribution of the product of ginseng pectin acted on by endo-polygalacturonase.

[0030] Figure 2 It is the infrared spectrogram of the product of ginseng pectin after de-methylation / methylation modification.

[0031] Figure 3 It is the effect of ginseng pectin on the nuclear localization of DAF-16 in Caenorhabditis elegans (A: Nuc means fully nuclear entry, Inter means partial nuclear entry, Cyt means no nuclear entry, and the arrow points to the nucleus expressing DAF-16::GFP; B: The p value is calculated by Chi-test, ***p < 0.001; n represents the number of nematodes counted).

[0032] Figure 4 Effect of ginseng pectin on the nuclear accumulation of SKN-1B / C in the intestine of Caenorhabditis elegans (A: High indicates high nuclear entry, Medium indicates moderate nuclear entry, Low indicates low nuclear entry, the arrow points to the nucleus expressing SKN-1B / C::GFP, B: p value calculated by Chi-test, ***p < 0.001; n represents the number of nematodes counted).

[0033] Figure 5 Effect of ginseng pectin on the mRNA expression levels of DAF-16 and SKN-1 target genes (p value calculated by Student's t-test, *p < 0.05; **p < 0.01; ***p < 0.001, no "*" indicates no significant difference p ≥ 0.05).

[0034] Figure 6 Effect of ginseng pectin on the protein expression levels of SOD-3 and GST-4 in nematodes (A and C are the fluorescence microscopy images of SOD-3::GFP and their fluorescence quantitative analysis results respectively; B and D are the fluorescence microscopy images of GST-4::GFP and their fluorescence quantitative analysis results respectively; p value calculated by Student's t-test, **p < 0.01; ***p < 0.001).

[0035] Figure 7 Effect of ginseng pectin on the binding of insulin to its receptor (binding-dissociation process).

[0036] Figure 8 Interaction between ginseng pectin and insulin. Specific embodiments

[0037] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0038] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0039] In the following embodiments, all quantitative tests are set with three repeated experiments, and the results are averaged.

[0040] Example 1. Preparation of ginseng pectin

[0041] (1) Weigh 1 kg of dry ginseng root raw materials. After washing the surface dust and other impurities with running water, add 16 L of distilled water and soak overnight. The next day, use a multi-functional automatic decocting machine to carry out extraction. The extraction process is as follows: the extraction temperature is 100 °C, the extraction time is 3 h, and the extraction times are 3 times. After each extraction, filter to obtain the extract, and then add 16 L of water to the residue for the next extraction. Combine the extracts of the three times and concentrate to 2 L, centrifuge (4000 rpm, 20 min), take the supernatant, slowly add 95% ethanol while stirring well with a glass rod until the final ethanol concentration reaches 80%, and let it stand overnight. The next day, collect the precipitate by centrifugation (4000 rpm, 20 min), wash and replace the water in the precipitate with 95% ethanol and absolute ethanol in turn, and dry in a 60 °C water bath to obtain total polysaccharides extracted from ginseng with water (hereinafter referred to as WGP).

[0042] (2) Load WGP onto a DEAE-cellulose column (12×43 cm, the column volume of this chromatography column is 4 L, and the elution flow rate is 25 mL / min) for ion exchange chromatography. First, elute with distilled water for 4 column volumes to separate amyloid glucan in WGP. Concentrate under reduced pressure at 60 °C using a rotary evaporator (Shanghai Sunsir Technology Co., Ltd.), and then carry out freeze-drying under reduced pressure (at -40 °C, 0.1 MPa) using a freeze dryer (Christ, Germany) to obtain a freeze-dried powder, named WGPN (ginseng amyloid glucan). Then elute with 0.5 M sodium chloride solution with a pH of 7.0 (the solute is sodium chloride and the solvent is distilled water), collect the eluate with an elution volume of the 5th column volume - the 8th column volume, concentrate under reduced pressure at 60 °C using a rotary evaporator, dialyze to remove salt using a dialysis bag with a cut-off molecular weight of 3500 Da, and then carry out freeze-drying under reduced pressure (at -40 °C, 0.1 MPa) using a freeze dryer to obtain total ginseng pectin (hereinafter referred to as WGPA).

[0043] (3) Load WGPA onto a DEAE-cellulose column (8×20 cm, the column volume of this chromatography column is 1 L, and the elution flow rate is 12.5 mL / min) for ion exchange chromatography. First, elute with distilled water for 2 column volumes, and then carry out gradient elution with 0.1 M sodium chloride solution with a pH of 7.0 and 0.2 M sodium chloride solution with a pH of 7.0 (the solute is sodium chloride and the solvent is distilled water) in turn. Collect the eluate with an elution volume of the 5th column volume - the 8th column volume eluted with 0.2 M sodium chloride solution with a pH of 7.0, concentrate under reduced pressure at 60 °C using a rotary evaporator, dialyze to remove salt using a dialysis bag with a cut-off molecular weight of 3500 Da, and then carry out freeze-drying under reduced pressure (at -40 °C, 0.1 MPa) to obtain a ginseng pectin fraction with a uniform charge distribution (hereinafter referred to as WGPA-2).

[0044] (4) Using a Sepharose CL-6B gel column (GE-healthcare, USA), with a column volume of 500 mL (2.6×100 cm), gel filtration chromatography was performed to purify WGPA-2. Elution was carried out with a 0.15 M sodium chloride solution (solute: sodium chloride, solvent: distilled water) at pH 7.0, and the flow rate was 0.4 mL / min. The elution peak (eluate with an elution volume of 0.8 to 1 column volume) was collected, concentrated under reduced pressure at 60 °C using a rotary evaporator, desalted by dialysis using a dialysis bag with a cut-off molecular weight of 3500 Da, and then freeze-dried under reduced pressure (at -40 °C, 0.1 MPa) to obtain a HG-type pectin fraction with a uniform molecular weight distribution, named WGPA-2HG.

[0045] Example 2: Structural modification of ginseng pectin and preparation of corresponding fractions

[0046] (1) Removal of the HG domain of ginseng pectin

[0047] A: Take 5 mg of WGPA-2HG prepared in Example 1 and dissolve it in 1 mL of acetic acid-sodium acetate buffer (100 mM, pH 4.2). To remove the HG domain as completely as possible, add endo-polygalacturonase (Endo-polygalacturonase M2 (Endo-PG), E.C. 3.2.1.15, Megazyme, Ireland) to the sample (acetic acid-sodium acetate buffer dissolved with WGPA-2HG) at an enzyme addition amount of 2 U / mL. The sample after adding the enzyme was reacted at 40 °C for 12 hours, heated at 100 °C for 10 minutes to inactivate the enzyme, centrifuged to obtain the supernatant, and the enzyme addition reaction was repeated once according to the above steps to obtain the Endo-PG hydrolysis product of WGPA-2HG. The change in the molecular weight distribution of the Endo-PG hydrolysis product was detected using a Shimadzu high-performance gel liquid chromatography system (HPGPC, infusion pump: LC-20Ai; differential refractive index detector: RID-20A; chromatographic column: TSK-gel G3000PW XL 、7.8×300 mm, Shimadzu, Japan).

[0048] B: Add 3 volumes of absolute ethanol (75% alcohol precipitation) to the Endo-PG hydrolysis product of WGPA-2HG in step A, shake repeatedly several times and then centrifuge to obtain the precipitate, and use this method to remove oligosaccharides and monosaccharides in the sample. Add distilled water to redissolve the precipitate, desalt and freeze-dry to obtain the remaining polysaccharide part in the enzymatically hydrolyzed sample: Endo-PG hydrolysis fraction WGPA-2HG-EndoPG. By pre-column derivatization with PMP and a Shimadzu high-performance liquid chromatography system (HPLC, infusion pump: LC-20AT; ultraviolet detector: SPD-20A; reversed-phase C18 chromatographic column: COSMOSIL 5C18 Analyze the monosaccharide composition of the above-mentioned Endo-PG hydrolysis fraction using a high-performance liquid chromatograph (HPLC, Shimadzu LC-20A, Japan) with a PAQ column (4.6×250 mm).

[0049] The change in the molecular weight distribution of the Endo-PG hydrolysis product is as Figure 1 shown. As Figure 1 can be seen, after the hydrolysis of WGPA-2HG by Endo-PG, the main structure of the pectin sample (i.e., the main peak at a retention time of 9 - 13 minutes) was almost completely degraded into low-molecular-weight oligosaccharide fragments with different degrees of polymerization, and these oligomers were eluted at a retention time of 14 - 16 minutes. In addition, lower elution peaks were observed at retention times of 10 - 11 minutes and 12 - 14 minutes in the HPGPC-RI elution profile, indicating that a small amount of high-molecular-weight pectin structures were eluted, presumably the RG-I and RG-II domains released from the pectin sample with the degradation of the HG domain.

[0050] Table 1 Monosaccharide composition of the Endo-PG hydrolysis fraction of ginseng pectin

[0051]

[0052] The analysis results of the monosaccharide composition of the Endo-PG hydrolysis fraction are shown in Table 1. As can be seen from Table 1, the monosaccharide composition of WGPA-2HG-EndoPG is mainly galacturonic acid, indicating that the sample contains an RG-II domain with a linear polygalacturonic acid backbone. In addition, the sample also contains a small amount of rhamnose, arabinose, and galactose representing the RG-I domain. From the analysis results of the monosaccharide composition, it can be known that after hydrolysis by Endo-PG, the remaining polysaccharide structures in the ginseng pectin sample WGPA-2HG are mainly the RG-I and RG-II domains.

[0053] (2) Demethylation and methylation modification of ginseng pectin

[0054] A: To remove the methylation modification of ginseng pectin, weigh 10 mg of ginseng pectin sample WGPA-2HG and place it in a dry acid hydrolysis vial. Add 1 mL of distilled water, and stir magnetically until the pectin is fully dissolved, then cool it at 4 °C. Then add 1 mL of 0.2 M sodium hydroxide solution (solute: sodium hydroxide, solvent: distilled water) pre-cooled at 4 °C, and keep stirring during the addition. Place the mixed solution at 4 °C and react for 4 hours. After the reaction, add 10% glacial acetic acid solution dropwise in an ice bath to neutralize, and keep stirring until the neutralization is complete to obtain the demethylation reaction solution of WGPA-2HG. Use a Sephadex G-10 column (GE-healthcare, USA), with a column volume of 20 mL, to desalt the aforementioned demethylation reaction solution. Load 1 mL of the demethylation reaction solution of WGPA-2HG onto the equilibrated Sephadex G-10 desalting column. After the sample completely enters the gel, add 700 μL of distilled water for elution to remove the acetate in the sample. Then add 1.2 mL of distilled water for elution, collect the eluate, and freeze-dry it under reduced pressure (at -40 °C, 0.1 MPa) to obtain the demethylated fraction WGPA-2HG-DE.

[0055] B: To methylate ginseng pectin, weigh 10 mg of ginseng pectin sample WGPA-2HG and place it in a dry acid hydrolysis vial, and cool it at 4 °C. Then add 1 mL of pre-prepared and cooled 2 M sulfuric acid-methanol salt solution (solute: concentrated sulfuric acid, solvent: methanol), and react the mixture at 4 °C for 4 days, with continuous stirring during the reaction. After the reaction, centrifuge the mixture at 12000 rpm for 10 minutes, discard the supernatant, and collect the precipitate. Wash the precipitate repeatedly with methanol aqueous solution (methanol:water = 3:1) to remove the excess sulfuric acid. Redissolve the washed precipitate in distilled water, and add 0.1 M sodium hydroxide (solute: sodium hydroxide, solvent: distilled water) dropwise to adjust the pH to about 6 - 7 to obtain the methylation reaction solution of WGPA-2HG. Use a Sephadex G-10 column to desalt the aforementioned methylation reaction solution. Load 1 mL of the methylation reaction solution of WGPA-2HG onto the equilibrated Sephadex G-10 desalting column. After the sample completely enters the gel, add 700 μL of distilled water for elution to remove the sodium salt in the sample. Then add 1.2 mL of distilled water for elution, collect the eluate, and freeze-dry it under reduced pressure (at -40 °C, 0.1 MPa) to obtain the methylated fraction WGPA-2HG-E.

[0056] C: Detect the change in the degree of methylation of ginseng pectin after demethylation and methylation modification. The instrument used is: PerkinElmer Spectrum Two infrared spectrometer (FT-IR, wavenumber 4000 - 400 cm -1, (from the United States, PE), and using OMNIC infrared spectroscopy analysis software, calculate the degree of esterification of the pectin sample according to the ratio of the vibration absorption peak areas of free carboxyl groups and methyl-esterified carboxyl groups.

[0057] The results are shown in Figure 2 , the degree of methylation (DM) of the untreated original sample WGPA-2HG is 15.8%, which is a low-esterified pectin. In the demethylated fraction WGPA-2HG-DE, the stretching vibration characteristic peak of the methyl-esterified carboxyl group of galacturonic acid near 1740 cm -1 disappears, while the peak area of the stretching vibration characteristic peak of the free carboxyl group of galacturonic acid near 1630 cm -1 becomes significantly larger compared with the untreated original sample, indicating that the methyl groups in the pectin sample have been completely removed and the degree of methylation is 0%. In the infrared spectrum of the methyl-esterified fraction WGPA-2HG-E, the peak area of the stretching vibration characteristic peak of the methyl-esterified carboxyl group of galacturonic acid increases significantly, and the peak area of the stretching vibration characteristic peak of the free carboxyl group is relatively small, indicating that the degree of methylation of the sample has increased significantly compared with the untreated original sample. After calculation, the degree of methylation of WGPA-2HG-E is 72.6%.

[0058] Example 3. Effect of ginseng pectin fraction on the lifespan of Caenorhabditis elegans

[0059] 1. Effect of ginseng pectin on the lifespan of Caenorhabditis elegans

[0060] (1) Preparation of culture medium

[0061] Nematode growth medium (NGM): Weigh 1.2 g of sodium chloride, 1.0 g of peptone, and 8.0 g of agar. First, add 300 mL of distilled water to dissolve, then add 400 μL of 1 M magnesium sulfate (solute: magnesium sulfate, solvent: distilled water) and 400 μL of 1 M calcium chloride solution (solute: calcium chloride, solvent: distilled water) that have been prepared, and make up the volume to 390 mL with distilled water. Sterilize at 121 °C under high-pressure steam for 30 minutes. After sterilization, add 400 μL of 5 mg / mL cholesterol solution (solute: cholesterol, solvent: 95% ethanol) and 10 mL of sterile 1 M potassium hydrogen phosphate solution (pH 6.0, solute: potassium dihydrogen phosphate, dipotassium hydrogen phosphate, solvent: distilled water) in a laminar flow hood, mix well and pour into petri dishes, let it solidify and dry in the laminar flow hood (2 - 3 days), and store at 4 °C.

[0062] Experimental group NGM medium: Prepare solutions of WGPN and WGPA-2HG at 1 mg / mL with distilled water, filter them through a 0.22 μm filter in a laminar flow hood, and then separately mix them with Escherichia coli OP50 bacterial solution (OD 600nm= 0.7 - 0.9, including Escherichia coli OP50 and LB liquid medium) were mixed at a volume ratio of 1:9 to make the final concentration of WGPN or WGPA-2HG 0.1 mg / mL, obtaining a bacterial solution mixed with WGPN or WGPA-2HG. The bacterial solution mixed with WGPN or WGPA-2HG was evenly spread on nematode growth medium (NGM), and air-dried overnight in a laminar flow hood, thus obtaining the experimental group NGM medium containing WGPN or WGPA-2HG.

[0063] Control group NGM medium: Distilled water with the same volume as the WGPN or WGPA-2HG solution and Escherichia coli OP50 bacterial solution (OD 600nm = 0.7 - 0.9, including Escherichia coli OP50 and LB liquid medium) were mixed at a volume ratio of 1:9 to obtain a diluted bacterial solution. The diluted bacterial solution was evenly spread on nematode growth medium (NGM), and air-dried overnight in a laminar flow hood, thus obtaining the control group NGM medium.

[0064] (2) Synchronized wild-type nematodes at the Day1 stage (adult worm age of 1 day) (N2 Bristol, sourced from Caenorhabditis Genetics Center (CGC)) were cultured on the experimental group NGM medium and the control group NGM medium at a temperature of 20 °C, and the experiment was repeated three times. Record the number of nematodes that died daily, and transfer the remaining surviving nematodes to fresh NGM medium daily until all nematodes died.

[0065] (3) Identification criteria for nematode death: Gently poke the nematodes with a platinum wire. If the nematodes show no response, they are considered dead. Nematodes burrowing into the bottom of the medium, crawling to the edge of the medium, and the appearance of the Wormbag phenomenon (i.e., the phenomenon of larvae hatching from eggs inside the mother) are all regarded as lost and not included in the data statistics. The results are expressed as mean lifespan ± SEM, and the significant difference is calculated by the Log-rank test. An average lifespan data of nematodes can be obtained from each experiment. The average lifespan in the table is the average of the average lifespans obtained from three experiments, and the error is SEM. The total number is the total number of nematodes used in the three experiments. The number of deaths is the number of nematodes that meet the data statistics in the three experiments (since there are generally cases of nematode loss in the experiment, the number of deaths will be less than the total number).

[0066] Table 2 Effects of ginseng pectin on the lifespan of Caenorhabditis elegans

[0067] Group Average lifespan ± SEM (days) p-value Number of deaths / Total number (animals) Control group 16.521±0.149 - 247 / 259 WGPN 17.668±0.113 <![CDATA[0.0442 a > 248 / 258 WGPA-2HG 19.094±0.122 <![CDATA[<0.0001 a > 260 / 262 <![CDATA[0.0076 b >

[0068] Note: The p-value represented by a is the value obtained by comparing with the control group. The p-value represented by b is the value obtained by comparing WGPA-2HG with WGPN.

[0069] As can be seen from Table 2, compared with the control group of wild-type Caenorhabditis elegans fed with distilled water (16.521 days), although feeding with WGPN can slightly extend the average lifespan of C. elegans (17.668 days, p < 0.05), feeding with WGPA-2HG more significantly extends the lifespan of C. elegans (19.094 days, p < 0.0001), increasing the average lifespan of C. elegans by 15.57%. The lifespan-extending effect of WGPA-2HG is significantly better than that of WGPN (p < 0.01).

[0070] 2. Effects of enzymolysis and modified fractions of ginseng pectin on the lifespan of Caenorhabditis elegans

[0071] The difference from "1. Effects of ginseng pectin on the lifespan of Caenorhabditis elegans" in this example lies in the different types of pectin fractions (the types of pectin fractions are shown in Tables 3.1 - 3.2, prepared from Example 1 and Example 2), and the rest of the steps are the same.

[0072] Table 3.1 Effects of ginseng pectin and its enzymolysis fractions on the lifespan of Caenorhabditis elegans

[0073] Group Average lifespan ± SEM (days) p-value Number of deaths / Total number (animals) Control group 16.215±0.086 - 246 / 250 WGPA-2HG 19.126±0.044 <![CDATA[<0.0001 a > 251 / 255 WGPA-2HG-EndoPG 17.913±0.140 <![CDATA[0.0230 a > 253 / 256 <![CDATA[0.0093 b >

[0074] Note: The p-value represented by a is the value obtained by comparison with the control group, and the p-value represented by b is the value obtained by comparison between WGPA-2HG-EndoPG and WGPA-2HG.

[0075] Table 3.2 Effects of ginseng pectin and its modified fractions on the lifespan of Caenorhabditis elegans

[0076] Group Average lifespan ± SEM (days) p-value Number of deaths / Total number (animals) Control group 16.334±0.134 - 255 / 260 WGPA-2HG 19.216±0.108 <![CDATA[<0.0001 a > 254 / 260 WGPA-2HG-DE 19.117±0.085 <![CDATA[<0.0001 a > 250 / 268 <![CDATA[0.7818 b > WGPA-2HG-E 16.668±0.116 <![CDATA[0.9405 a > 255 / 264 <![CDATA[<0.0001 b >

[0077] Note: The p-value represented by a is the value obtained by comparison with the control group, and the p-value represented by b is the value obtained by comparison between WGPA-2HG-DE or WGPA-2HG-E and WGPA-2HG.

[0078] As can be seen from Tables 3.1 - 3.2, the fraction WGPA-2HG-EndoPG obtained by removing the HG domain with endo-polygalacturonase (Endo-PG) has a significantly lower effect on extending the lifespan of nematodes than the untreated original sample (i.e., WGPA-2HG), indicating that the HG domain mainly determines the lifespan-extending effect of ginseng pectin.

[0079] WGPA-2HG-DE is the product obtained by treating WGPA-2HG with sodium hydroxide to remove the methyl esterification modification of the sample. WGPA-2HG-E is the product obtained by treating WGPA-2HG with sulfuric acid-methanol to increase the methyl esterification modification of the sample. WGPA-2HG-DE can significantly extend the lifespan of nematodes, and there is no significant difference in the effect compared with the untreated original sample (the untreated original sample is low-esterified pectin). However, WGPA-2HG-E cannot extend the lifespan of nematodes. It can be seen that high methyl esterification leads to a significant decrease in the lifespan-extending activity of ginseng pectin, while de-methyl esterification modification does not change the original lifespan-extending activity of ginseng pectin, indicating that the low (de-)esterified HG domain is the key structure determining the lifespan-extending effect of ginseng pectin.

[0080] 3. The lifespan extension of ginseng pectin depends on the insulin / IGF-1 signaling pathway

[0081] Prepare the control group and experimental group NGM media according to "1. Effect of ginseng pectin on the lifespan of Caenorhabditis elegans" in this example. Mutant strains of Caenorhabditis elegans with loss-of-function of the insulin receptor DAF-2 gene in the insulin / IGF-1 signaling pathway at the Day1 stage (adult worm age of 1 day) after synchronization, namely daf-2(e1370) (CB1370, from Caenorhabditis Genetics Center (CGC)), daf-2(e1368) (DR1572, from Caenorhabditis Genetics Center (CGC)), mutant strains of Caenorhabditis elegans with loss-of-function of the ribosomal protein S6 kinase RSKS-1 gene in the TOR signaling pathway, rsks-1(ok1255) (RB1206, from Caenorhabditis Genetics Center (CGC)), mutant worms of daf-16 gene with loss-of-function, daf-16(mu86) (CF1038, from Caenorhabditis Genetics Center (CGC)), and mutant worms of skn-1 gene with loss-of-function, skn-1(zu135) (LG340, from Caenorhabditis Genetics Center (CGC)) were cultured on the nematode growth medium (NGM) of the control group and experimental group respectively (the preparation method refers to "1. Effect of ginseng pectin on the lifespan of Caenorhabditis elegans" in this example. The difference between the experimental group and the control group of the same nematode is that the experimental groups contain the pectin components shown in Table 4, and the control group contains the same volume of distilled water. The rest of the operations are the same as "1. Effect of ginseng pectin on the lifespan of Caenorhabditis elegans" in this example), at a temperature of 20 °C, and the experiment was repeated three times. Record the number of dead nematodes every day, and transfer the remaining surviving nematodes to fresh NGM medium until all nematodes died. Statistically analyze the death situation of nematodes in different media according to the nematode death identification criteria shown in "1. Effect of ginseng pectin on the lifespan of Caenorhabditis elegans" in this example.

[0082] Table 4 Effect of ginseng pectin on the lifespan of mutant Caenorhabditis elegans

[0083]

[0084] Note: The p values represented by a, b, c, d, and e are the values obtained by comparing the feeding of WGPA-2HG with the control group in the mutant nematodes of daf-2(e1370), daf-2(e1368), rsks-1(ok1255), daf-16(mu86), and skn-1(zu135).

[0085] As can be seen from Table 4, WGPA-2HG cannot extend the lifespan of the nematode daf-2(e1370) and daf-2(e1368) with loss-of-function mutations in the insulin receptor DAF-2 gene in the insulin / IGF-1 signaling pathway, but can significantly extend the lifespan of the nematode rsks-1(ok1255) with loss-of-function mutations in the ribosomal protein S6 kinase RSKS-1 gene in the TOR signaling pathway. Moreover, WGPA-2HG can hardly extend the lifespan of nematodes with DAF-16 gene mutations daf-16(mu86) (loss of DAF-16 gene function) and nematodes with SKN-1 gene mutations skn-1(zu135) (loss of SKN-1 gene function), indicating that the lifespan-extending effect of ginseng pectin depends on the transcription factors DAF-16 and SKN-1 (key transcription factors downstream of the insulin / IGF-1 signaling pathway). These results suggest that the lifespan-extending effect of ginseng pectin depends on the insulin / IGF-1 signaling pathway and does not depend on the TOR signaling pathway.

[0086] Example 4. Nuclear localization analysis of transcription factors and mechanism of ginseng pectin in extending lifespan

[0087] Synchronized transgenic nematodes DAF-16::GFP (TJ356, zls356, sourced from Caenorhabditis Genetics Center (CGC), which expresses the transcription factor DAF-16 tagged with green fluorescent protein GFP) and SKN-1B / C::GFP (LD1, ldls7, sourced from Caenorhabditis Genetics Center (CGC), which expresses the transcription factor SKN-1 tagged with green fluorescent protein GFP) were cultured on nematode growth medium (NGM) in the control group and the experimental group respectively (for the preparation method, refer to "1. Effect of ginseng pectin on the lifespan of Caenorhabditis elegans" in Example 3. The difference between the experimental group and the control group of the same nematode is that the experimental groups contain the pectin component WGPA-2HG, and the control group contains the same volume of distilled water. The remaining operations are the same as the preparation method of the medium in "1. Effect of ginseng pectin on the lifespan of Caenorhabditis elegans" in Example 3) until the L4 (fourth instar larvae) or Day0 (young adult) stage at a temperature of 20 °C. Nematodes of the DAF-16::GFP (TJ356) strain were fixed with 1% formaldehyde, and nematodes of the SKN-1B / C::GFP (LD1) strain were fixed with 2 mM levamisole. Then, the GFP expression was observed under a laser confocal microscope (Carl Zeiss, Germany), and photographs were taken under the same exposure conditions. The experiment was repeated three times. The localization patterns of DAF-16::GFP and SKN-1B / C::GFP in the nucleus of the intestine are defined as follows: "Nuc" or "High" means that strong GFP signals were observed in the nuclei of the head, body, and tail of the nematode. "Inter" or "Medium" means that weak GFP signals were observed in the nuclei of the head, body, and tail of the nematode, or strong GFP signals were only observed in the head or tail of the nematode. "Cyt" or "Low" indicates that almost no GFP signals were detected in the nuclei of the head, body, and tail of the nematode.

[0088] It can be seen from Figure 3 and Figure 4 that compared with the control group, after feeding with WGPA-2HG, the proportions of the "Nuc" pattern of DAF-16::GFP and the "High" pattern of SKN-1B / C::GFP both increased significantly. Thus, it can be seen that ginseng pectin significantly promoted the nuclear localization of the transcription factor DAF-16 and the transcription factor SKN-1.

[0089] Example 5. Reverse transcription reaction and real-time fluorescence quantitative PCR (RT-qPCR) analysis of the mechanism of ginseng pectin in extending lifespan

[0090] Synchronized wild-type N2 nematodes were cultured from eggs to Day 1 (adult age of 1 day) at 20 °C on nematode growth medium (NGM) in the control group and the experimental group (for the preparation method, refer to "1. Effect of ginseng pectin on the lifespan of Caenorhabditis elegans" in Example 3. The difference between the experimental group and the control group is that the experimental groups contain the pectin component WGPA-2HG, and the control group contains the same volume of distilled water. The remaining operations are the same as the preparation method of the medium in "1. Effect of ginseng pectin on the lifespan of Caenorhabditis elegans" in Example 3). Total RNA of the above nematodes was extracted using a Trizol kit (TaKaRa, Japan), and cDNA was obtained using reverse transcriptase (Promega, USA). The RT-qPCR reaction was carried out on a QuantStudio 3 real-time fluorescence quantitative PCR instrument (Thermo, USA) using the premixed TB Green Premix ExTaq enzyme mixture (TaKaRa, Japan) with the cDNA obtained by reverse transcription as the template, to detect the mRNA expression levels of the DAF-16 target genes (including: superoxide dismutase sod-3 gene, metallothionein mtl-1 gene, lysozyme lys-7 gene, heat shock protein hsp-16.2 gene), and the SKN-1 target genes (including: glutamylcysteine synthetase gcs-1 gene, glutathione S-transferase gst-4, gst-7, and gst-10 genes), with act-1 as the internal reference gene. The experiment was repeated three times.

[0091] The results are as Figure 5 shown. Compared with the control group, after feeding with WGPA-2HG, the mRNA expression levels of the DAF-16 target genes (including: superoxide dismutase sod-3 gene, lysozyme lys-7 gene), and the SKN-1 target genes (including: glutamylcysteine synthetase gcs-1 gene, glutathione S-transferase gst-4, gst-7, and gst-10 genes) in wild-type N2 nematodes were significantly increased. This indicates that ginseng pectin activates the transcriptional activities of the transcription factors DAF-16 and SKN-1 through the insulin / IGF-1 signaling pathway.

[0092] Example 6. Detection and analysis of the protein expression levels of SOD-3 and GST-4 to explore the mechanism of ginseng pectin in extending lifespan

[0093] Synchronized transgenic nematodes SOD-3::GFP (CF1553, muIs84, sourced from Caenorhabditis Genetics Center (CGC), which expresses superoxide dismutase SOD-3 with a green fluorescent protein GFP tag in the nematodes), and GST-4::GFP (CL2166, dvIs19, sourced from Caenorhabditis Genetics Center (CGC), which expresses glutathione S-transferase GST-4 with a green fluorescent protein GFP tag in the nematodes) were cultured from eggs to the Day0 stage (Young-adult stage) at 20 °C on nematode growth medium (NGM) in the control group and the experimental group (for the preparation method, refer to "1. Effect of ginseng pectin on the lifespan of Caenorhabditis elegans" in Example 3. The difference between the experimental group and the control group of the same nematode is that the experimental groups contain the pectin component WGPA-2HG, and the control group contains an equal volume of distilled water. The remaining operations are the same as the preparation method of the medium in "1. Effect of ginseng pectin on the lifespan of Caenorhabditis elegans" in Example 3). The intensity of the fluorescent protein was observed and detected. A part of the nematodes was taken from the control groups of the above two transgenic nematode strains and cultured in NGM medium containing tert-butyl hydroperoxide (tBHP, purchased from Sigma) (tBHP-NGM medium, preparation method: add 9.125 mM tert-butyl hydroperoxide to the nematode growth medium (NGM)) for 2 hours as the positive control group (tBHP). The remaining transgenic nematodes SOD-3::GFP (CF1553) and GST-4::GFP (CL2166) that were not treated with tBHP (only cultured in the control group NGM medium) were used as the negative control group (control). The nematodes in the negative control group (control), positive control group (tBHP), and experimental group were fixed with 1% formaldehyde. Then, the GFP expression was observed under a laser confocal microscope (Carl Zeiss, Germany), and photos were taken under the same exposure conditions. A multifunctional microplate reader (Tecan, Switzerland) was used to quantitatively analyze the fluorescence expression of SOD-3::GFP and GST-4::GFP in the nematodes. The excitation wavelength of the light was 488 nm, and the emission wavelength of the light was 535 nm. The experiment was repeated three times.

[0094] As Figure 6 shown, the results of microscopic observation of the fluorescent protein and fluorescence quantitative analysis by the multifunctional microplate reader showed that compared with the control group without feeding pectin, after feeding WGPA-2HG, the protein expression levels of SOD-3 and GST-4 in the nematodes were significantly increased. It was further demonstrated that ginseng pectin activated the transcriptional activities of the transcription factors DAF-16 and SKN-1, resulting in a significant increase in the expression levels of their downstream target genes superoxide dismutase SOD-3 and glutathione S-transferase GST-4.

[0095] Example 7. Analysis of the mechanism of ginseng pectin in prolonging lifespan by biolayer interferometry

[0096] (1) The insulin receptor protein with His-tag (Bio-Techne, USA) was prepared into a 100 μg / mL solution with PBST (PBS solution at pH 7.4 added with 0.05% Tween-20) as the protein immobilized on the biosensor (Ni-NTA, ForteBio, USA). The WGPA-2HG was prepared into a 10 mg / mL WGPA-2HG solution with PBST, and then the WGPA-2HG solution was gradient-diluted to 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, and 0.625 mg / mL respectively. Insulin (Sigma-Aldrich, USA) was prepared into a 2000 nM solution with PBST and then gradient-diluted to 1000 nM, 500 nM, 250 nM, 125 nM, and 62.5 nM. All samples were filtered through a 0.22 μm filter membrane.

[0097] When detecting the interaction between insulin and insulin receptor, the concentrations of insulin used were 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, and 0 nM (i.e., PBST without insulin).

[0098] When detecting the effect of pectin on the affinity between insulin and insulin receptor, ginseng pectin and insulin were mixed to prepare a "pectin + insulin" mixed sample. The "pectin + insulin" mixed sample was used as the solution interacting with the insulin receptor protein immobilized on the biosensor. The specific preparation method is as follows:

[0099] The ginseng pectin WGPA-2HG solutions with concentrations of 10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, and 0 mg / mL (i.e., PBST without pectin) (solvent: PBST) were respectively mixed with an equal volume of the insulin solution with a concentration of 2000 nM (solvent: PBST) to obtain the "pectin + insulin" mixed samples. The concentrations of pectin in the mixed samples were 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.3125 mg / mL, and 0 mg / mL respectively, and the concentration of insulin was 1000 nM.

[0100] (2) Add PBS, PBST, protein sample, "pectin + insulin" mixed sample (mixed with ginseng pectin WGPA-2HG and insulin), and regeneration reagent into a black 96-well plate in a specific order, 200 μL per well. The first column: PBST; the second column: 100 μg / mL insulin receptor protein solution; the third column: PBST; the fourth column: 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 0 nM insulin solutions; the fifth column: "pectin + insulin" mixed sample; the ninth column: glycine-hydrochloric acid (pH 2.0); the tenth column: PBS; the eleventh column: 10 mM nickel chloride solution (pH 4.0); the twelfth column: PBS. Place the black 96-well plate and the biosensor into a Biofilm Interferometer Fortebio Octet Red 96 (ForteBio, USA).

[0101] (2) Set the program through the instrument kinetic online detection software: Baseline process for 60 seconds, the biosensor runs to the first column to stabilize the biosensor in the solvent PBST; Loading protein process for 240 seconds, the biosensor runs to the second column to bind the insulin receptor protein to the biosensor; Baseline process for 60 seconds, the biosensor runs to the third column to wash away the non-specifically bound or overly bound insulin receptor protein during the loading process; Association process for 120 seconds, the biosensor runs to the fourth / fifth column to allow the insulin receptor protein to interact with insulin in the "pectin + insulin" mixed sample (mixed with ginseng pectin WGPA-2HG and insulin) or insulin without pectin; Disassociation process for 120 seconds, the biosensor runs back to the third column to dissociate the insulin in the above "pectin + insulin" mixed sample or insulin without pectin in PBST; Regeneration process for 30 seconds, the biosensor runs to the ninth column, in glycine-hydrochloric acid at pH 2.0, the nickel ions and the proteins and pectin samples bound to it are washed away, then it runs to the tenth column of PBS at pH 7.4, repeated 3 times to thoroughly wash the biosensor; Quenching for 120 seconds, the biosensor runs to the eleventh column, in 10 mM nickel chloride solution, the nickel ions bind to the biosensor again to enable it to have the ability to bind proteins again; Baseline process for 60 seconds, the biosensor runs to the twelfth column to wash away the excess nickel chloride. Run the program according to the above steps. After running, use the instrument data processing software ForteBioDataAnalysis Software 8.0 for data analysis.

[0102] As Figure 7As shown, compared with the insulin solution in combination, the degree of change in the membrane thickness of the biosensor when the insulin receptor binds to the insulin solution mixed with ginseng pectin WGPA-2HG is significantly reduced. The equilibrium dissociation constant K calculated by the data processing software D can further analyze the difference in affinity between the sample and the insulin receptor. The data are shown in Table 5. The results show that the K D value of the insulin solution mixed with ginseng pectin interacting with the insulin receptor is about two orders of magnitude higher than that of the insulin solution without pectin. From these results, it can be seen that WGPA-2HG reduces the affinity between insulin and the insulin receptor, indicating that ginseng pectin inhibits the binding of insulin to the receptor.

[0103] Table 5 K value of the interaction between insulin mixed with ginseng pectin and the insulin receptor detected by the biomembrane interference technique D value

[0104] Sample <![CDATA[K D (μM)]]> Insulin 0.7 WGPA-2HG + Insulin 19.7

[0105] Example 8. Analysis of the mechanism of ginseng pectin in extending lifespan by isothermal titration calorimetry

[0106] (1) Preparation of samples: Prepare a 0.05 mM insulin solution with PBS (pH 7.4), and prepare a 2.0 mM WGPA-2HG solution with PBS (pH 7.4). Degas the insulin solution and the WGPA-2HG solution by ultrasonic treatment for 10 minutes.

[0107] (2) Perform experiments using an isothermal titration microcalorimeter Nano ITC (Waters, USA). Titrate the aforementioned WGPA-2HG solution into the sample cell (previously filled with 0.05 mM insulin solution) at 30 °C, with each titration volume of 2 μL, a titration time interval of 300 seconds, and a total of 25 titrations. During the entire experiment, the contents of the sample cell were stirred at a speed of 300 rpm to ensure sufficient mixing of pectin and protein. Titrate ginseng pectin into PBS solution under the same conditions as a blank control. After the titration reaction is completed, use the instrument data processing software NanoAnalyzeTM Software for data analysis.

[0108] As Figure 8 shown, titrating insulin with WGPA-2HG is an endothermic reaction. Use the instrument software to convert the original data peaks in the figure, and then subtract the blank control to obtain a graph of enthalpy change versus molar ratio, and calculate the equilibrium binding constant K D of the sample according to the fitted curve, as shown in Table 6. The results show that the K D value of the interaction between WGPA-2HG and insulin is within the range of K for detecting the interaction between samples by the isothermal titration microcalorimeter Nano ITCD Within the range of values (0.001 - 1000 μM), it indicates that there is a certain affinity between WGPA - 2HG and insulin. This result shows that ginseng pectin inhibits the binding of insulin to the receptor by interacting with insulin.

[0109] Table 6 K for the interaction between ginseng pectin and insulin detected by isothermal titration calorimetry D value

[0110] Sample <![CDATA[K D (μM)]]> WGPA-2HG 10.6

[0111] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any variations, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made by conventional techniques known in the art. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. Use of ginseng pectin in the preparation of drugs for prolonging lifespan; The ginseng pectin is WGPA-2HG or WGPA-2HG-DE, and the WGPA-2HG-DE is the pectin after the demethylation of WGPA-2HG. The preparation method of the WGPA-2HG includes: R1. Extract the roots of ginseng with water to obtain an extract, add 95% ethanol aqueous solution to the extract until the ethanol concentration in the extract is 80%, collect the precipitate, and obtain the total polysaccharide extracted from ginseng with water; R2. Perform anion exchange column chromatography on the total polysaccharide extracted from ginseng obtained in R1, and elute successively with distilled water and 0.5M sodium chloride solution with a pH of 7.0 to obtain total ginseng pectin. The anion exchange group used in the anion exchange column chromatography is DEAE; R3. Perform anion exchange column chromatography on the total ginseng pectin obtained in R2, and elute successively with distilled water, 0.1M sodium chloride solution with a pH of 7.0, and 0.2M sodium chloride solution with a pH of 7.

0. Collect the ginseng pectin fraction eluted with 0.2M sodium chloride solution with a pH of 7.

0. The anion exchange group used in the anion exchange column chromatography is DEAE; R4. Purify the ginseng pectin fraction obtained in R3 by gel filtration chromatography, elute with 0.15M sodium chloride solution with a pH of 7.0, collect the eluate, and obtain a pectin fraction with a molecular weight of 32 kDa named WGPA-2HG.

2. The application according to claim 1, characterized in that The preparation method of the WGPA-2HG-DE includes: Prepare the WGPA-2HG into a liquid with a pectin content of 10 mg / mL with distilled water, then add 0.2M sodium hydroxide solution with the same volume as the liquid, place it at 4°C and react for 4 hours to obtain a reaction product, and then add a 10% glacial acetic acid solution by mass percentage to the reaction product to obtain a demethylation reaction solution of WGPA-2HG; Desalt the demethylation reaction solution, concentrate and freeze-dry it to obtain a demethylated pectin fraction named WGPA-2HG-DE.

3. The application according to any one of claims 1 or 2, characterized in that There is an interaction between the pectin and insulin. By inhibiting the binding of insulin to the receptor, the activity of the insulin / IGF-1 signaling pathway is reduced to prolong lifespan.

4. The application according to claim 3, characterized in that, The reduction of the insulin / IGF-1 signaling pathway activity includes enhancing the activities of transcription factors FOXO / DAF-16 and Nrf2 / SKN-1.

5. The application according to claim 4, characterized in that, The enhancement of the activity of transcription factor FOXO / DAF-16 includes promoting the nuclear localization of transcription factor FOXO / DAF-16 and activating the expression of the target genes of transcription factor FOXO / DAF-16. The target genes of FOXO / DAF-16 include the sod-3 gene and the lys-7 gene. The enhancement of the activity of transcription factor Nrf2 / SKN-1 includes promoting the nuclear localization of transcription factor Nrf2 / SKN-1 and activating the expression of the target genes of transcription factor Nrf2 / SKN-1. The target genes of activating transcription factor Nrf2 / SKN-1 include the gcs-1 gene, the gst-4 gene, the gst-7 gene, and the gst-10 gene.

6. Use of the pectin according to any one of claims 1 to 5 in the preparation of health products and / or foods for extending lifespan.

7. Use of the pectin according to any one of claims 1 to 5 in the preparation of a drug for targeting the insulin / IGF-1 signaling pathway or inhibiting the binding of insulin to its receptor.

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