Polygonatum sibiricum oligosaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of health-care food, and particularly relates to rhizoma polygonati oligosaccharide as well as a preparation method and application thereof. The invention provides the polygonatum kingianum oligosaccharide and proves that the polygonatum kingianum oligosaccharide can prolong the life of nematodes, delay senescence, improve the survival rate and exercise ability of the nematodes, improve the enzyme activity of superoxide dismutase in vivo and the level of reduced glutathione and enhance the defense ability of the nematodes to acute oxidative stress and acute heat stress. The expression level of the genes (daf-2 and daf-16) related to life and the expression level of the genes (gst-4) related to oxidation resistance are influenced. The polygonatum kingianum oligosaccharide has a wide application prospect in preparation of medicines for prolonging life and / or resisting aging or health-care products beneficial to oxidation resistance.

Description

Technical Field

[0001] This invention belongs to the field of health food technology, specifically relating to a Polygonatum oligosaccharide, its preparation method, and its uses. Background Technology

[0002] With changes in dietary habits, environment, and lifestyle in modern society, population aging has become a significant social issue, and age-related diseases are increasingly attracting attention. Aging is a complex process of gradual decline and disease development in various organ systems, often accompanied by multiple diseases such as metabolic diseases, cardiovascular diseases, and nervous system diseases. The search for and development of natural and highly effective compounds to extend lifespan, combat aging, and improve health has become a research hotspot.

[0003] Polygonatum cyrtonema Hua. is one of the original plants of Polygonatum listed in the Chinese Pharmacopoeia. It is considered a plant with both medicinal and edible properties, possessing effects such as nourishing yin and qi, moistening dryness and promoting body fluids, and strengthening the spleen and stomach. The main chemical components of Polygonatum are polysaccharides, steroidal saponins, anthraquinones, and flavonoids. Polysaccharides, as the main active ingredient in Polygonatum, have been reported to have anti-tumor, antioxidant, anti-inflammatory, and life-prolonging effects. Numerous studies have shown that oligosaccharides, compared to polysaccharides, have better water solubility and faster bioavailability, potentially providing higher biological activity and health benefits. Polygonatum oligosaccharides, as natural active substances, are not only highly safe but also rapidly absorbed. However, on the one hand, there are currently no reports of Polygonatum oligosaccharides having life-prolonging effects; on the other hand, the existing methods for obtaining Polygonatum oligosaccharides are costly and unsuitable for industrial production. For example, patent application CN119060209A discloses a method for preparing anti-fatigue oligosaccharides from Polygonatum odoratum. The method involves water extraction and alcohol precipitation of Polygonatum odoratum slices to obtain a small-molecule extract, followed by freeze-drying to obtain a freeze-dried powder. Macroporous resin is then used to enrich the oligosaccharides in the freeze-dried powder to obtain Polygonatum odoratum oligosaccharides. However, the monosaccharides (glucose and fructose) in the Polygonatum odoratum oligosaccharides prepared by this method account for as high as 77.6%, while oligosaccharides (calculated as sucrose and melibiose) account for only about 20%, indicating that this method mainly enriches monosaccharide components rather than oligosaccharides. Furthermore, this method requires first freeze-drying the extract, then using macroporous resin to enrich and purify the Polygonatum odoratum oligosaccharides in the extract, and finally drying again to obtain the target product. This process is costly, cumbersome, and results in a low oligosaccharide content, making it inconvenient for recycling. Summary of the Invention

[0004] In order to solve the above-mentioned problems in the existing technology, the purpose of this invention is to provide a Polygonatum oligosaccharide, its preparation method and uses.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides a Polygonatum oligosaccharide, wherein the relative molecular weight of the Polygonatum oligosaccharide is 1690–1710 Da; the Polygonatum oligosaccharide… 1 In the H-NMR spectrum, resonance peaks were observed at δ5.42, δ5.25, δ4.19, δ3.87, and δ3.67; the oligosaccharide of Polygonatum sibiricum... 13 In the C-NMR spectrum, resonance peak signals are present at δ103.58, δ81.11, δ74.67 and δ69.19;

[0007] The 1 H-NMR signal and 13 The error range of C-NMR signals is ±2%.

[0008] Furthermore, the relative molecular weight of the Polygonatum oligosaccharide is 1702 Da; the Polygonatum oligosaccharide... 1 In the H-NMR spectrum, resonance peaks were observed at δ5.42, δ5.25, δ4.19, δ3.87, and δ3.67; the oligosaccharide of Polygonatum sibiricum... 13 In the C-NMR spectrum, resonance peaks are observed at δ103.58, δ81.11, δ74.67, and δ69.19.

[0009] This invention also provides a method for preparing the above-mentioned Polygonatum oligosaccharides, the method comprising the following steps:

[0010] (1) Prepare Polygonatum powder, add solvent to extract, and obtain Polygonatum crude oligosaccharide extract;

[0011] (2) Mix activated carbon and diatomaceous earth, pour into a chromatography column, and wash to obtain an activated carbon-diatomaceous earth column.

[0012] (3) Take the crude oligosaccharide extract of Polygonatum obtained in step (1), add it to the activated carbon-diatomite column obtained in step (2), elute, collect the eluent, and obtain Polygonatum oligosaccharide.

[0013] Further, in step (1), the ratio of Polygonatum powder to solvent is 1g:(5-50)mL, and the solvent is an alcohol reagent;

[0014] The mass ratio of activated carbon to diatomaceous earth in step (2) is (0.1-10):1, and the activated carbon is activated carbon that has been boiled with an acidic reagent and then washed with water until it is neutral.

[0015] The elution in step (3) is performed by sequentially eluting with water, 5% ethanol, 10% ethanol and high-concentration ethanol, and collecting the eluent obtained by high-concentration ethanol elution; the high-concentration ethanol is 55% to 80% ethanol.

[0016] Further, in step (1), the ratio of Polygonatum powder to solvent is 1g:(10-30)mL, and the solvent is ethanol;

[0017] In step (2), the mass ratio of activated carbon to diatomaceous earth is (0.5-5):1, and the acid reagent is an organic acid.

[0018] Further, the elution in step (3) is performed by gradient elution with 8 column volumes of water, 6 column volumes of 5% ethanol, 6 column volumes of 10% ethanol, and 10 column volumes of high-concentration ethanol.

[0019] Further, in step (1), the ratio of Polygonatum powder to solvent is 1g:20mL, and the ethanol is 50% to 80% ethanol; the concentration of ethanol is volume concentration.

[0020] In step (2), the mass ratio of activated carbon to diatomaceous earth is 2:1, and the organic acid is 10% to 20% acetic acid;

[0021] The high-concentration ethanol mentioned in step (3) is 65% ethanol.

[0022] Further, the organic acid in step (2) is 15% acetic acid.

[0023] Furthermore, the Polygonatum mentioned is Polygonatum multiflorum.

[0024] The present invention also provides the use of the above-mentioned Polygonatum oligosaccharide in the preparation of health products that help with antioxidation.

[0025] The present invention also provides the use of the above-mentioned Polygonatum oligosaccharide in the preparation of drugs for prolonging life and / or anti-aging.

[0026] Furthermore, the drug is prepared using Polygonatum oligosaccharide as the active ingredient, along with pharmaceutically acceptable excipients.

[0027] Furthermore, the drug is a formulation that is either oral or injectable.

[0028] Furthermore, the oral preparation is a tablet, capsule, granule, syrup, or solution; the injectable preparation is an injection solution or sterile powder for injection.

[0029] The present invention has achieved the following beneficial effects:

[0030] This invention provides a Polygonatum oligosaccharide and demonstrates that it can prolong the lifespan of nematodes, delay aging, improve nematode survival rate and motility, increase superoxide dismutase activity and reduced glutathione levels, enhance their defense against acute oxidative stress and acute heat stress, and affect the expression levels of lifespan-related genes (daf-2, daf-16) and antioxidant-related genes (gst-4). This Polygonatum oligosaccharide has broad application prospects in the preparation of drugs for prolonging lifespan and / or anti-aging, or health products that aid in antioxidant activity.

[0031] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0032] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0033] Figure 1 The effects of different conditions on the crude oligosaccharide extract of Polygonatum odoratum. (A) Effect of solid-liquid ratio on the crude oligosaccharide extract of Polygonatum odoratum; (B) Effect of ethanol concentration on the crude oligosaccharide extract of Polygonatum odoratum.

[0034] Figure 2 The effect of ethanol elution concentration on the yield and purity of Polygonatum oligosaccharides. (A) Change in total sugar; (B) Change in reducing sugar.

[0035] Figure 3 This is a chromatogram of the molecular weight of Polygonatum oligosaccharide (PCOP).

[0036] Figure 4 This is a one-dimensional NMR spectrum of Polygonatum oligosaccharide (PCOP). (A) 1 H-NMR spectrum;

[0037] (B) 13 C-NMR spectrum.

[0038] Figure 5 This is a diagram showing the toxicological results of Polygonatum oligosaccharides on Caenorhabditis elegans.

[0039] Figure 6 The figure shows the effect of Polygonatum oligosaccharides on the lifespan of Caenorhabditis elegans.

[0040] Figure 7Figure 1 shows the effect of Polygonatum oligosaccharides on the movement behavior of nematodes. (A) Effect of Polygonatum oligosaccharides on the head-shaking frequency of nematodes; (B) Effect of Polygonatum oligosaccharides on the body bending frequency of nematodes.

[0041] Figure 8 Figure 1 shows the effect of Polygonatum oligosaccharides on the levels of superoxide dismutase (SOD) and glutathione (GSH) in nematodes. (A) Effect of Polygonatum oligosaccharides on the level of superoxide dismutase (SOD) in nematodes;

[0042] (B) Effect of Polygonatum oligosaccharides on the level of glutathione (GSH) in nematodes.

[0043] Figure 9 The effect of Polygonatum oligosaccharides on the acute oxidative stress capacity of nematodes is shown in the figure.

[0044] Figure 10 The effect of Polygonatum oligosaccharides on the acute heat stress resistance of nematodes is shown in the figure.

[0045] Figure 11 The effect of Polygonatum oligosaccharides on the expression of senescence-related genes in nematodes. Detailed Implementation

[0046] The reagents and equipment used in the specific embodiments of this invention are all known products and were obtained by purchasing commercially available products.

[0047] The raw material for Polygonatum multiflorum in this invention was collected from Heping Village, Heping Town, Shaowu City, Fujian Province, China.

[0048] Example 1: Preparation of Polygonatum oligosaccharide extract

[0049] Crude oligosaccharides from Polygonatum sibiricum were extracted using ultrasonic extraction with a certain concentration of ethanol, and the oligosaccharide mixture was separated and purified by activated carbon-diatomaceous earth column chromatography. To improve the total extraction rate of crude oligosaccharides from Polygonatum sibiricum, and to increase the yield and purity of the oligosaccharides, the preparation method of Polygonatum sibiricum oligosaccharides was optimized. The effectiveness of the optimized method was determined by comparing the reducing sugar content in the crude oligosaccharide extract, as well as the elution column volume and oligosaccharide yield under different elution conditions.

[0050] The extraction steps for Polygonatum oligosaccharides are as follows:

[0051] (1) Preparation of crude oligosaccharide extract of Polygonatum sibiricum:

[0052] The Polygonatum odoratum was dried, pulverized, and passed through a 60-mesh sieve to obtain a uniform powder. The powder was then subjected to ultrasonic extraction with ethanol of a specific concentration at a certain material-to-liquid ratio. Each extraction was performed for 30 minutes of sonication followed by centrifugation at 4000 rpm for 10 minutes. This extraction was repeated twice, and the supernatants were combined. The extract was concentrated under reduced pressure at 60°C until no ethanol remained, and then diluted to a final volume of 5 times the sample mass with water to obtain the crude oligosaccharide extract of Polygonatum odoratum.

[0053] The effects of different material-to-liquid ratios (1:10, 1:20, 1:30, g / mL) and different ethanol concentrations (60%, 70%, 80%, v / v) on the extraction rate of crude oligosaccharides from Polygonatum sibiricum were studied. The extraction rate of the crude oligosaccharide extract was determined by measuring the reducing sugar content.

[0054] Reducing sugar content determination: The 3,5-dinitrosalicylic acid (DNS) method was used for determination. A standard curve was plotted using glucose standard solution, and the reducing sugar content in the sample was calculated based on the measured absorbance.

[0055] The results are as follows Figure 1 As shown, the extraction rate of the crude oligosaccharide extract from Polygonatum sibiricum was affected by the solid-liquid ratio and the ethanol concentration. The highest reducing sugar content was observed in the crude oligosaccharide extract at a solid-liquid ratio of 1:20 (g / mL). Furthermore, the reducing sugar content in the extract increased with increasing ethanol concentration. Therefore, a solid-liquid ratio of 1:20 (g / mL) and an ethanol extraction concentration of 80% were selected as the preparation conditions for the crude oligosaccharide extract from Polygonatum sibiricum.

[0056] (2) Preparation of activated carbon-diatomaceous earth column: Take activated carbon (200 mesh) and boil it with 15% acetic acid for 30 min, wash it with water until neutral, repeat the above process twice, and filter it to obtain pretreated activated carbon. Mix the pretreated activated carbon and diatomaceous earth in a ratio of 2:1, pour it into the chromatography column, let it settle naturally, rinse it with deionized water until the carbon surface no longer descends, and when the water on the carbon surface is reduced to 2μm~10μm, the activated carbon-diatomaceous earth column is obtained.

[0057] (3) Column chromatography: Take 80 mL of the crude oligosaccharide extract of Polygonatum sibiricum obtained in step (1) and add it to the activated carbon-diatomaceous earth column obtained in step (2). After natural sedimentation, elute sequentially with deionized water, 5% ethanol aqueous solution (v / v), and 10% ethanol aqueous solution (v / v). After no sugar fraction is detected by the phenol-sulfuric acid method, switch to the next gradient. Under the elution conditions of pure water, 5% ethanol aqueous solution, and 10% ethanol aqueous solution, monosaccharides and disaccharides will be preferentially eluted and therefore not collected. The remaining oligosaccharides will be selectively eluted gradually as the ethanol concentration increases. In order to improve the oligosaccharide yield and ensure the oligosaccharide purity, after elution with deionized water, 5% ethanol aqueous solution, and 10% ethanol aqueous solution, the oligosaccharide yield obtained by elution with different ethanol concentrations (55%, 65%, 80%, v / v) was further optimized (elution scheme is shown in Table 1), with reducing sugar content and total sugar content as evaluation indicators. The total polysaccharide content of Polygonatum oligosaccharides was determined by the phenol-sulfuric acid method, with fructose as the standard.

[0058] Table 1. Selection of Elution Protocols

[0059]

[0060] The results are as follows Figure 2As shown, the oligosaccharides eluted with 80% ethanol had the highest total sugar content, but their reducing sugar content was slightly lower than that of the oligosaccharides eluted with 65%. This indicates that the sample eluted with 80% ethanol contained a small amount of non-oligosaccharide polysaccharides. Therefore, to ensure optimal purity of the oligosaccharide mixture, the 65% (v / v) ethanol fraction was ultimately selected for collection (i.e., Scheme 2 in Table 1), yielding Polygonatum oligosaccharides (PCOP) with a yield of 0.61%. Furthermore, if elution with ethanol concentrations lower than 65% (v / v) was used, the oligosaccharide yield decreased significantly, which is not conducive to practical applications. To confirm the molecular characteristics of the collected components, molecular weight determination analysis was further performed in this study.

[0061] (4) Molecular weight analysis of Polygonatum oligosaccharides:

[0062] The molecular weight distribution of PCOP was determined using high-performance liquid chromatography coupled with an electrospray ionization detector. Dextran reference standards with molecular weights of 1000, 5000, 12000, 25000, and 50000 Da were accurately weighed and prepared into standard solutions with a concentration of 2.0 mg / mL. The detection concentration of PCOP was 3.0 mg / mL. Using 0.02 M ammonium acetate as the mobile phase, an Acclaim SEC-300 (5 μm, Analytical, 7.8 × 300 mm) gel chromatography column was used at 40 °C and a flow rate of 0.5 mL / min to separate the standards and samples. Finally, regression analysis was performed on the results, with the logarithm of the relative molecular mass of the reference standards (log Mw) as the ordinate and retention time (Tr) as the abscissa.

[0063] Chromatograms of dextran standards and samples are as follows: Figure 3 As shown: Based on the retention time, the relative molecular weight of the obtained Polygonatum oligosaccharide was calculated to be 1702 Da, indicating that the prepared PCOP sample is indeed an oligosaccharide.

[0064] (5) Nuclear magnetic resonance spectroscopy analysis of Polygonatum oligosaccharides:

[0065] Weigh 30 mg of the Polygonatum oligosaccharide obtained in step (3), dissolve it in 1.0 mL of D2O, let it stand overnight at room temperature, freeze-dry it, then add another 1.0 mL of D2O, repeat 3 times, and finally dissolve it in 1.0 mL of D2O and place it in an NMR tube. Use a Bruker NMR spectrometer to determine the one-dimensional ( 1 H and 13 C) Nuclear magnetic resonance spectrum.

[0066] One-dimensional ( 1 H and 13 C) Nuclear magnetic resonance spectrum, such as Figure 4 As shown. Polygonatum oligosaccharide (PCOP) 1The H-NMR spectrum showed resonance peaks at δ5.42, δ5.25, δ4.19, δ3.87, and δ3.67, indicating the presence of α-D-Glc–(1→, →6)-α-D-Glc-(1→, β-D-Fruf-(2→, →2)-β-D-Fruf-(6→ and →1)-β-D-Fruf-(2→) chemical residues in PCOP. 13 C-NMR spectra showed resonance peaks at δ103.58, δ81.11, δ74.67, and δ69.19, confirming that PCOP is composed of these residues. It is speculated that PCOP is a novel inulin-based fructan.

[0067] The following experimental examples demonstrate the beneficial effects of the present invention.

[0068] Experimental Example 1: Application Research of Polygonatum Oligosaccharides in Prolonging the Lifespan of Caenorhabditis elegans and / or Anti-aging

[0069] Wild-type N2 *C. elegans* was provided by the Caenorha bditis Genetics Center (CGC) at the University of Minnesota. Superoxide dismutase (SOD) and glutathione (GSH) kits were purchased from Solarbio. Culture media reagents were purchased from Maclean's.

[0070] I. Experimental Methods

[0071] Culture and synchronization of *Caenorhabditis elegans*: L4 nematodes were selected and cultured on NGM solid medium containing *E. coli* OP50 at 20°C and 40-60% humidity, with transfer every 3 days. Synchronization was achieved using the sodium hypochlorite lysis method. Nematodes in the oviposition stage were rinsed from the NGM medium with sterile water and transferred to 15 mL centrifuge tubes. Centrifugation was performed at 1200 rpm for 1 min, and this step was repeated twice. The liquid was reduced to 3.5 mL, and then 0.5 mL of 5M NaOH and 1 mL of NaClO were added. The mixture was vortexed for 6 min until all nematodes disappeared. Water was then added to 15 mL to stop the lysis, and centrifugation was performed again at 1200 rpm for 1 min. This step was repeated twice. The supernatant was discarded, and the precipitate (the egg precipitate was white) was collected. The precipitate was washed three times with M9 solution and then cultured in M9 solution at 20°C. Twenty hours later, the L1 nematodes were transferred to NGM plates containing Escherichia coli OP50. After about 48 hours, the nematodes developed to the L4 stage and completed synchronization.

[0072] (1) Evaluation of the toxicity of Polygonatum oligosaccharides to Caenorhabditis elegans

[0073] Synchronized L4 stage wild-type N2 nematodes were transferred to NGM plates containing different mass concentrations (0, 2.5, 5, and 10 mg / mL) of Polygonatum oligosaccharides for culture. Three parallel plates were set up for each concentration, with 50 nematodes per plate. Nematode survival was observed after 24 and 48 hours of culture. Nematode death was defined as no response after 10 seconds of gentle touching with a platinum wire.

[0074] (2) Life test

[0075] L4-stage N2 nematodes were transferred to bacterial culture dishes containing the Polygonatum oligosaccharide group (10 mg / mL) and the control group, and incubated at 20°C. This was recorded as day 0 of the nematode lifespan. Eight plates were prepared for each of the Polygonatum oligosaccharide and control groups, with 50 nematodes per plate. To eliminate contamination between the tested nematodes and their newly produced larvae, ensure sufficient food for the nematodes, and prevent sample degradation throughout the experiment, live nematodes were transferred to new culture plates containing the same sample concentration every 24 hours for continued incubation. Each day, the nematodes were transferred to new culture dishes and gently stimulated with platinum wire, and the number of surviving nematodes was recorded, continuing until the last nematode died.

[0076] (3) Measurement of Nematode Movement Behavior

[0077] Head oscillation frequency measurement: During the determination of the lifespan of *C. elegans*, on days 5, 10, and 15, five nematodes of good growth and similar condition were selected from each group to observe and record the head oscillation. 10 μL of M9 solution was added to a glass slide, and one nematode was randomly selected each time and placed in the M9 solution. After acclimatization for about 1 minute, the number of head oscillations within 30 seconds was recorded (one head oscillation is defined as the nematode's head swinging from one direction to another and then back).

[0078] Measurement of nematode body bending frequency: The measurement method is the same as in 4.1. Record the number of times the body bends within 30 seconds (the movement of the nematode relative to one wavelength in the direction of the body's long axis is considered as one body bend). The number of nematodes measured in each group of experiments shall not be less than 5.

[0079] (4) Levels of superoxide dismutase (SOD) and glutathione (GSH) in vivo

[0080] On day 5 of nematode culture, nematodes from different treatment groups were collected, washed three times with M9, and then thoroughly ground and broken up on ice. The supernatant was collected by centrifugation at 12,000 rpm / min for 5 min at 4°C. The SOD activity, GSH level, and protein content of the supernatant were determined according to the kit instructions.

[0081] (5) Effects on lifespan of *C. elegans* under oxidative stress

[0082] An oxidative damage model was established using H2O2. Synchronized L4-stage nematodes were placed in NGM plates (3 plates per group, 30 nematodes per plate). 100 μL of Polygonatum oligosaccharide (10 mg / mL) or NGM liquid (blank control) was added. After 3 days of treatment, the nematodes in each group were transferred to NGM containing 0.05% H2O2. The survival status of the nematodes was recorded every 10 hours until all nematodes died.

[0083] (6) Effects of Polygonatum oligosaccharides on acute heat stress in nematodes

[0084] Larvae synchronized to the L4 stage were placed in NGM plates (6 plates in total, 30 larvae per plate). They were divided into two groups: each group was treated with either 100 μL of Polygonatum oligosaccharide (10 mg / mL) or NGM liquid (blank control). After treatment at 20℃ for 2 days, the nematodes in each group were transferred to a 35℃ incubator for acute heat stress. Survival rates were observed and recorded every 1 hour until all nematodes died.

[0085] (7) Effects of Polygonatum oligosaccharides on aging-related genes

[0086] Wild-type N2 nematodes synchronized to the L4 stage were inoculated into treatment culture medium containing 10 mg / mL Polygonatum oligosaccharide and a blank control group (NGM plate), and cultured at 20℃ for 5 days. After collecting the nematodes from both groups, total RNA was extracted, cDNA was synthesized, and qPCR was performed. The expression differences of daf-2, daf-16, and gst-4 genes between the two groups were determined using actin as an internal control.

[0087] II. Experimental Results

[0088] (1) Toxicity evaluation of Polygonatum oligosaccharides

[0089] Toxicological experiments of Polygonatum oligosaccharides on Caenorhabditis elegans, such as... Figure 5 As shown, concentrations of Polygonatum oligosaccharides below 10 mg / mL have no toxic effect on Caenorhabditis elegans, therefore, Polygonatum oligosaccharides at a concentration of 10 mg / mL were selected for subsequent experiments.

[0090] (2) Evaluation of the effect of Polygonatum oligosaccharide on prolonging the lifespan of nematodes

[0091] The effect of Polygonatum oligosaccharides on the lifespan of Caenorhabditis elegans, such as Figure 6 As shown in the figure, compared with the control group, Polygonatum oligosaccharides significantly shifted the survival curve of nematodes to the right. All nematodes in the control group died by day 24, while those treated with Polygonatum oligosaccharides had their lifespan extended to 27 days.

[0092] (3) Effects of Polygonatum oligosaccharides on nematode locomotion behavior

[0093] Head twitching frequency and body flexion frequency can serve as indicators reflecting basic nervous system functions. The movement behavior of nematodes in the treated group and the control group was observed on days 5, 10, and 15 after drug administration. The experimental results are as follows: Figure 7 As shown. During the three time periods of the test, the head-swinging frequency and body-bending frequency of nematodes in the Polygonatum oligosaccharide group were both higher than those in the control group. Specifically, on day 10, the head-swinging frequency of nematodes in the PCOP group was 21 times per 30 seconds (i.e., 21 head swings during a 30-second test), and the body-bending frequency was 13.8 times per 30 seconds; both significantly higher than those in the control group (17.6 times per 30 seconds and 9.4 times per 30 seconds). On day 5, there was no significant difference in head-swinging frequency and body-bending frequency between the two groups.

[0094] (4) Effects of Polygonatum oligosaccharides on the levels of superoxide dismutase (SOD) and glutathione (GSH) in nematodes

[0095] Experimental results are as follows Figure 8 As shown, compared with the control group, Polygonatum oligosaccharides significantly increased SOD enzyme activity and GSH levels in nematodes. This indicates that Polygonatum oligosaccharides can effectively enhance the antioxidant activity of enzymes and non-enzymes in nematodes, thereby mitigating oxidative damage caused by peroxidation during aging, improving their survival ability, and prolonging their lifespan.

[0096] (5) Effects of Polygonatum oligosaccharides on acute oxidative stress capacity of nematodes

[0097] Acute stress responses (such as heat stress and oxidative stress) are closely related to lifespan. Moderate stress can activate cellular defense mechanisms; however, aging individuals typically experience a decline in stress tolerance. Therefore, assessing an organism's tolerance to acute stress can serve as an important indicator of its aging status and lifespan. Figure 9 As shown, under 0.05% H2O2 stimulation, the longest survival time of nematodes in the control group was 40 hours. After treatment with Polygonatum oligosaccharides, their survival time was extended to 50 hours. Therefore, the experimental results indicate that Polygonatum oligosaccharides can alleviate the damage caused by H2O2-induced oxidative stress to nematodes and prolong their survival rate.

[0098] (6) Effects of Polygonatum oligosaccharides on acute heat stress in nematodes

[0099] Studies have shown that certain genes related to nematode lifespan not only control the average lifespan of nematodes but also affect their acute heat stress tolerance. The effects of Polygonatum oligosaccharides on the acute heat stress tolerance of nematodes are as follows: Figure 10 As shown, the survival rate of nematodes treated with Polygonatum oligosaccharides was significantly improved under acute heat stress (35℃). In the acute heat stress experiment, the longest survival time of nematodes in the control group was 10 hours. After treatment with Polygonatum oligosaccharides, their survival time was extended to 12 hours.

[0100] (7) Effects of Polygonatum oligosaccharides on aging-related genes

[0101] The aforementioned experiments have demonstrated that Polygonatum oligosaccharides can slow down the aging process of nematodes and prolong their lifespan. This experiment further explores the molecular mechanism of its action. DAF-2, DAF-16, and GST-4 are genes that play a major regulatory role in the insulin / insulin-like growth factor (IIS) signaling pathway. The effects of Polygonatum oligosaccharides on aging-related genes are as follows: Figure 11 As shown, Polygonatum oligosaccharides can affect the expression levels of genes related to lifespan (daf-2, daf-16) and genes related to antioxidant activity (gst-4), specifically decreasing the expression level of daf-2 and increasing the expression levels of daf-16 and gst-4. It is speculated that Polygonatum oligosaccharides exert their lifespan-extending effect by regulating aging-related genes in the IIS pathway.

[0102] In summary, this invention provides a Polygonatum oligosaccharide and demonstrates that it can prolong the lifespan of nematodes, delay aging, improve nematode survival rate and motility, increase superoxide dismutase activity and reduced glutathione levels, enhance their defense against acute oxidative stress and acute heat stress, and affect the expression levels of lifespan-related genes (daf-2, daf-16) and antioxidant-related genes (gst-4). This Polygonatum oligosaccharide has broad application prospects in the preparation of drugs for prolonging lifespan and / or anti-aging, or health products that aid in antioxidant activity.

Claims

1. A Polygonatum oligosaccharide, characterized in that, The relative molecular weight of the Polygonatum oligosaccharide is 1690–1710 Da; the relative molecular weight of the Polygonatum oligosaccharide is... 1 In the H-NMR spectrum, resonance peaks were observed at δ5.42, δ5.25, δ4.19, δ3.87, and δ3.67; the oligosaccharide of Polygonatum sibiricum... 13 In the C-NMR spectrum, Resonance peak signals are present at δ103.58, δ81.11, δ74.67 and δ69.19; The 1 H-NMR signal and 13 The error range of C-NMR signals is ±2%.

2. The Polygonatum oligosaccharide according to claim 1, characterized in that, The relative molecular weight of the Polygonatum oligosaccharide is 1702 Da; the molecular weight of the Polygonatum oligosaccharide is... 1 In the H-NMR spectrum, resonance peaks were observed at δ5.42, δ5.25, δ4.19, δ3.87, and δ3.67; the oligosaccharide of Polygonatum sibiricum... 13 In the C-NMR spectrum, resonance peaks are observed at δ103.58, δ81.11, δ74.67, and δ69.

19.

3. The method for preparing Polygonatum oligosaccharides according to claim 1 or 2, characterized in that, The preparation method includes the following steps: (1) Prepare Polygonatum powder, add solvent to extract, and obtain Polygonatum crude oligosaccharide extract; (2) Mix activated carbon and diatomaceous earth, pour into a chromatography column, and wash to obtain an activated carbon-diatomaceous earth column. (3) Take the crude oligosaccharide extract of Polygonatum obtained in step (1), add it to the activated carbon-diatomite column obtained in step (2), elute, collect the eluent, and obtain Polygonatum oligosaccharide.

4. The preparation method according to claim 3, characterized in that, In step (1), the ratio of Polygonatum powder to solvent is 1g:(5-50)mL, and the solvent is an alcohol reagent. The mass ratio of activated carbon to diatomaceous earth in step (2) is (0.1-10):1, and the activated carbon is activated carbon that has been boiled with an acidic reagent and then washed with water until it is neutral. The elution in step (3) is performed by sequentially eluting with water, 5% ethanol, 10% ethanol and high-concentration ethanol, and collecting the eluent obtained by high-concentration ethanol elution; the high-concentration ethanol is 55% to 80% ethanol.

5. The preparation method according to claim 4, characterized in that, In step (1), the ratio of Polygonatum powder to solvent is 1g:(10-30)mL, and the solvent is ethanol; In step (2), the mass ratio of activated carbon to diatomaceous earth is (0.5-5):1, and the acid reagent is an organic acid.

6. The preparation method according to claim 5, characterized in that, In step (1), the ratio of Polygonatum powder to solvent is 1g:20mL, and the ethanol is 50% to 80% ethanol; In step (2), the mass ratio of activated carbon to diatomaceous earth is 2:1, and the organic acid is 10% to 20% acetic acid, preferably 15%. The high-concentration ethanol mentioned in step (3) is 65% ethanol.

7. Use of the Polygonatum oligosaccharide of claim 1 in the preparation of health products that help with antioxidation.

8. Use of the Polygonatum oligosaccharide of claim 1 in the preparation of a medicament for prolonging life and / or anti-aging.

9. The use according to claim 8, characterized in that, The drug is prepared using Polygonatum oligosaccharide as the active ingredient, along with pharmaceutically acceptable excipients.

10. The use according to claim 9, characterized in that, The drug is a formulation that is either an oral or injectable preparation; preferably, the oral preparation is a tablet, capsule, granule, syrup, or solution; and the injectable preparation is an injection solution or sterile powder for injection.

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