Application of lycium ruthenicum murr.polysaccharide in prolonging life and improving exercise capacity of aging individuals

Abstract

The application discloses application of Lycium ruthenicum Murr. polysaccharide in prolonging life span and improving exercise capacity of an aging individual, and belongs to the field of medical biology. The application first finds that the Lycium ruthenicum Murr. polysaccharide can improve the exercise capacity decrease of an aging model organism in the aging process, delay the aging-related behavioral changes such as the decrease of body swing frequency, the decrease of swimming speed and the shortening of sustained exercise capacity, thereby maintaining the body function state and promoting the improvement of healthy life span; compared with the simple prolonging of life span, the application emphasizes more on the intervention of functional decline in the aging process, especially the delay and improvement of exercise capacity decline, and has more definite practical application significance; the Lycium ruthenicum Murr. polysaccharide can be further prepared into food, health products, dietary nutrition supplements or drugs, and is used for improving the aging-related exercise function decrease, maintaining the healthy life span and developing related anti-aging products.

Description

Technical Field

[0001] This invention relates to the application of black goji berry polysaccharides in prolonging lifespan and improving the motor function of aging individuals, and belongs to the field of pharmaceutical biology. Background Technology

[0002] Aging is a complex, gradual, and irreversible physiological degenerative process that occurs in organisms with increasing age. As aging progresses, the body not only experiences systemic changes such as a shortened lifespan, reduced resilience, and imbalanced metabolic homeostasis, but also induces progressive decline in the physiological functions of multiple tissues and organs. Decline in motor function is the earliest, most visually apparent, and most practically significant marker of the aging process. Age-related motor function decline manifests as reduced frequency of voluntary activities, slower movement speed, decreased sustained exercise endurance, and weakened neuromuscular coordination—behavioral changes that are not merely external manifestations of aging but also direct signals of a comprehensive decline in overall physiological function.

[0003] Current anti-aging research has undergone a significant shift, moving beyond simply extending the absolute lifespan of organisms to a greater emphasis on maintaining and improving healthy lifespan. While simply extending lifespan increases survival time, if this increased lifespan is accompanied by problems such as limited mobility, physical weakness, and loss of self-care ability, the practical application of such life-extending interventions is very limited; this is particularly relevant in the case of *Caenorhabditis elegans* (C. elegans). Caenorhabditis elegans In aging biology research, exemplified by [examples of research], there is a significant decoupling between "lifespan extension" and healthy lifespan indicators centered on "motor function" in terms of molecular mechanisms and phenotypic profiles. Past intervention studies tended to equate "improved survival rate" with "successful anti-aging," but increasing evidence suggests that certain longevity interventions may even extend an individual's period of frailty. The literature (BANSALA, ZHU LJ, YEN K, et al. Uncoupling lifespan and healthspan in Caenorhabditis elegans longevity mutants[J / OL]. Proceedings of the National Academy of Sciences, 2015, 112(3). DOI:10.1073 / pnas.1412192112.) used several classic longevity mutants of Caenorhabditis elegans as subjects and proved that total lifespan and health lifespan can be decoupled: although mutations in insulin and mitochondrial pathways can significantly prolong the survival time of nematodes, they cannot effectively delay the decline of movement and physiological functions and the accumulation of aging damage, but only prolong the period of frailty and disability in old age.

[0004] The literature (HAHM JH, KIM S, DILORETO R, et al. C. elegans maximum velocity correlates with healthspan and is maintained in worms with an insulin receptor mutation [J / OL]. Nature Communications, 2015, 6(1). DOI:10.1038 / ncomms9919.) found that altered mitochondrial signaling ( clk-1 ) or inhibit protein translation ( ife-2 Several long-lived mutant nematodes, including [list of mutants], while significantly extending their absolute lifespan, exhibit accelerated decline in motor abilities, such as body bends, in the later stages of aging, leading to a prolonged state of behavioral restriction in their later years. This phenomenon of "longevity without health" suggests that the pathways regulating survival metabolism and maintaining muscle movement integrity may be independent of each other.

[0005] Black goji berries are a unique medicinal and edible plant resource in my country. Unlike regular Ningxia goji berries, they are rich in anthocyanins and other characteristic active substances in addition to polysaccharide components, giving them differentiated advantages in anti-oxidative stress and aging intervention. Black goji berry polysaccharides, as the core active component of black goji berries, are naturally derived, safe, reliable, and biocompatible, with broad prospects for industrial development. Existing research on black goji berry polysaccharides mostly focuses on basic aspects such as extraction and purification process optimization, physicochemical structural characterization, and verification of basic antioxidant activity. While the literature (HSF-1 and SIR-2.1 linked insulin-like signaling is involved in goji berry (Lycium spp.) extracts promoting lifespan extension of Caenorhabditis elegans) discloses that black goji berry extract can extend the lifespan of nematodes, its components are polyphenols and flavonoids, and the final lifespan extension compared to the control group was only 24.28%, failing to effectively extend lifespan and improve age-related decline in motor function.

[0006] Therefore, developing safe, natural, and active substances that improve the decline in motor skills caused by aging and promote healthy lifespan is of great practical significance for healthy aging and improving the quality of life of the elderly population. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides the application of black goji berry polysaccharides in prolonging lifespan and improving the motor function of aging individuals. This invention is the first to discover that black goji berry polysaccharides can improve the decline in motor function observed in aging model organisms during the aging process, delaying age-related behavioral changes such as decreased body swing frequency, slower swimming speed, and shortened sustained exercise capacity, thereby maintaining bodily function and promoting a healthy lifespan. Compared to simply prolonging lifespan, this invention emphasizes intervention in functional decline during the aging process, especially the delay and improvement of motor function decline, which has more explicit practical application significance. Black goji berry polysaccharides can be further prepared into foods, health products, dietary supplements, or pharmaceuticals for improving age-related motor function decline, maintaining a healthy lifespan, and developing related anti-aging products.

[0008] The first objective of this invention is to provide the application of black goji berry polysaccharides in the preparation of products for prolonging lifespan and / or improving the motor function of aging individuals, wherein the preparation method of the black goji berry polysaccharides is as follows: (1) After crushing black goji berries, dissolve them in water, stir, sonicate, centrifuge, and take the supernatant. Extract the precipitate again and combine the two supernatants. (2) The supernatant obtained in step (1) is concentrated to 1 / 2 to 1 / 5 of its original volume to obtain crude extract of black goji berries; (3) Add 10-15 g / 100 mL trichloroacetic acid to the crude black goji berry extract obtained in step (2), let stand for 6-12 h, centrifuge, take the supernatant, repeat the extraction of the precipitate three times in the above method, and combine to obtain the supernatant. (4) Add 4 to 6 times the volume of anhydrous ethanol to the supernatant obtained in step (3) for alcohol precipitation, let stand at 4 to 6°C for 8 to 12 h, centrifuge the solution after standing, discard the supernatant, wash the precipitate, dissolve it, and dialyze it with a dialysis bag with a molecular weight cutoff of 2.8 to 3.3 kDa for 8 to 12 h, and freeze dry to obtain black wolfberry polysaccharide.

[0009] In one embodiment, the method of pulverizing black goji berries in step (1) is to pulverize the dried black goji berries and pass them through a 20-30 mesh sieve to obtain black goji berry powder.

[0010] In one embodiment, the ratio of black goji berry powder to water in step (1) is 1~5 g: 5~20 mL; In one embodiment, the ultrasound conditions in step (1) are 300~400 W ultrasound for 80~100 min.

[0011] In one embodiment, the centrifugation conditions described in steps (1) to (4) are centrifugation at 8000-10000 rpm for 15-20 min.

[0012] In one embodiment, the conditions for the second extraction in step (1) are the same as those for the first extraction, namely, the precipitate is dissolved in water, stirred, sonicated, and centrifuged to obtain the supernatant.

[0013] In one embodiment, the concentration in step (2) is achieved by rotary evaporation concentration at 45-50°C.

[0014] In one embodiment, the monosaccharide composition of black goji berry polysaccharide is fucose (3.63%), rhamnose (2.25%), arabinose (14.99%), galactose (9.26%), glucose (58.42%), xylose (1.91%), mannose (2.61%), and fructose (0.89%).

[0015] In one embodiment, the product includes one or more of the following: pharmaceuticals, food, health products, feed, and feed additives.

[0016] In one embodiment, the dosage form of the drug is a liquid formulation or a solid formulation. Optionally, the dosage form of the drug includes granules, capsules, tablets, pills, or oral liquid; Preferably, the drug further includes pharmaceutically acceptable excipients; Preferably, the pharmaceutical excipients include any one or more of the following: solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, pH adjusters, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release inhibitors.

[0017] In one embodiment, the food includes health food, food for special medical purposes, functional food, or pet food; Preferably, the food products include grain products, starch products, vegetable products, fruit products, meat products, poultry products, egg products, and dairy products.

[0018] In one embodiment, the health product also contains acceptable excipients.

[0019] In one implementation, the extended lifespan and / or improved mobility in aging individuals includes at least one of the following manifestations: (a) Delaying the decline of motor function; (b) Extend lifespan; (c) Increase the level of antioxidant enzyme activity; (d) Reduce the accumulation of lipofuscin.

[0020] In one embodiment, the black goji berry polysaccharide is prepared according to the following method: (1) After crushing black goji berries, dissolve them in water, stir, sonicate, centrifuge, and take the supernatant. Extract the precipitate again and combine the two supernatants. (2) The supernatant obtained in step (1) is concentrated to 1 / 2 to 1 / 5 of its original volume to obtain crude extract of black goji berries; (3) Add 10-15 g / 100 mL trichloroacetic acid to the crude black goji berry extract obtained in step (2), let stand for 6-12 h, centrifuge, take the supernatant, repeat the extraction of the precipitate three times in the above method, and combine to obtain the supernatant. (4) Add 4 to 6 times the volume of anhydrous ethanol to the supernatant obtained in step (3) for alcohol precipitation, let stand at 4 to 6°C for 8 to 12 h, centrifuge the solution after standing, discard the supernatant, wash the precipitate, dissolve it, and dialyze it for 45 to 50 h using a dialysis bag with a molecular weight cutoff of 2.8 to 3.3 kDa. After freeze drying, black wolfberry polysaccharide is obtained.

[0021] A second objective of this invention is to provide a medicament for prolonging lifespan and / or improving the motor function of aging individuals, the medicament comprising black goji berry polysaccharides.

[0022] In one embodiment, the dosage form of the drug is a liquid formulation or a solid formulation. Optionally, the dosage form of the drug includes, but is not limited to, granules, capsules, tablets, pills, or oral liquid; Preferably, the drug further includes pharmaceutically acceptable excipients.

[0023] In one embodiment, the pharmaceutical excipients include any one or more of the following: solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, pH adjusters, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release inhibitors.

[0024] In one implementation, the extended lifespan and / or improved mobility in aging individuals includes at least one of the following manifestations: (a) Delaying the decline of motor function; (b) Extend lifespan; (c) Increase the level of antioxidant enzyme activity; (d) Reduce the accumulation of lipofuscin.

[0025] Beneficial effects This invention utilizes wild-type *Caenorhabditis elegans* in an aging model to explore the applications of black goji berry polysaccharides prepared in this invention in prolonging lifespan, delaying motor function decline, reducing reactive oxygen species (ROS) levels, increasing SOD and CAT activities, and reducing lipofuscin accumulation. Through lifespan experiments, motor evaluation, and measurements of ROS levels, SOD and CAT activities, and lipofuscin levels, it was found that this black goji berry polysaccharide can effectively improve age-related motor function decline, specifically: (1) The black wolfberry polysaccharide prepared by this invention has the effect of effectively prolonging the lifespan of nematodes. Compared with the blank control group, the overall survival curve of nematodes treated with black goji berry polysaccharides showed varying degrees of rightward shift after intervention, with the 100 μg / mL group showing the most significant effect. The median survival time of nematodes in the 100 μg / mL group was extended by 4 days compared with the control group, and the average lifespan was extended by approximately 28%, indicating that black goji berry polysaccharides have a significant life-prolonging effect within an appropriate dosage range.

[0026] (2) The black wolfberry polysaccharide prepared by this invention can effectively improve the decline in motor function caused by aging. Taking body swaying frequency as an example, the control group had approximately 90 swaying times / min on day 3, decreasing to approximately 74 swaying times / min on day 6, approximately 52 swaying times / min on day 9, and approximately 40 swaying times / min on day 12; while the black goji berry polysaccharide treatment group had approximately 96, 85, 67, and 62 swaying times / min, respectively. Compared with the control group, the treatment group showed an increase of approximately 15% in body swaying frequency on day 6, approximately 29% on day 9, and approximately 55% on day 12. If the decline in motor ability is calculated with day 3 as the baseline, the control group showed a decrease of approximately 56% by day 12, while the treatment group showed a decrease of approximately 35%, indicating that black goji berry polysaccharide can reduce the decline in body swaying ability by approximately 36%.

[0027] Regarding swimming speed, the control group had approximately 98%, 76%, 65%, and 44% swimming speed on days 6, 9, and 12, respectively, while the treatment group had approximately 114%, 95%, 91%, and 57%. Compared with the control group, the swimming speed of the nematode adults in the treatment group increased by approximately 16%, 25%, 40%, and 30% on days 3, 6, 9, and 12, respectively.

[0028] Regarding swimming duration, the control group had approximately 100%, 70%, 32%, and 12% of the swim duration on days 3, 6, 9, and 12, respectively, while the treatment group had approximately 100%, 79%, 54%, and 20% of the swim duration on days 3, 6, 9, and 12, respectively. Compared with the control group, the treatment group showed an improvement of approximately 13% on day 6, approximately 69% on day 9, and approximately 67% on day 12. Attached Figure Description

[0029] Figure 1The results are the physicochemical characterization of black goji berry polysaccharides; (A) infrared absorption spectrum, (B) ultraviolet absorption spectrum.

[0030] Figure 2 The effect of black goji berry polysaccharide on the lifespan of nematodes; where (A) is the nematode survival curve and (B) is the median survival time.

[0031] Figure 3 The effect of black wolfberry polysaccharide on lipofuscin accumulation in nematodes was shown in Figures A through F. Figures A through F show the fluorescence intensity of lipofuscin in nematodes at black wolfberry polysaccharide concentrations of 0, 50, 100, 200, 400, and 800 μg / mL, respectively. Figure G shows the average quantitative intensity of lipofuscin fluorescence.

[0032] Figure 4 The effect of black goji berry polysaccharide on ROS in nematodes: Figures A to F show the ROS fluorescence intensity in nematodes at black goji berry polysaccharide concentrations of 0, 50, 100, 200, 400, and 800 μg / mL, respectively. Figure G shows the average quantitative intensity of ROS fluorescence.

[0033] Figure 5 The effect of black wolfberry polysaccharide on the SOD and CAT activity levels of nematodes; where (A) represents the SOD activity level and (B) represents the CAT activity level.

[0034] Figure 6 The effect of black goji berry polysaccharide on the motility of aging nematodes; where (A) is the nematode head-shaking frequency, (B) is the nematode swimming speed, and (C) is the nematode swimming duration. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, where specific conditions are not specified, are generally performed under conventional conditions in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0036] Raw materials involved in the examples: The dried black goji berries used in the following examples were produced in Golmud and purchased from Ningxia Qilixiang Goji Berry Co., Ltd.

[0037] Wild-type N2 *C. elegans* and *Escherichia coli* OP50 strain were provided by Professor Zhu Zhen's team from the School of Integrated Circuits, Southeast University; agar powder, sodium chloride, magnesium sulfate, calcium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and trypsin were purchased from Sinopharm Shanghai Laboratory; yeast extract and peptone were purchased from Sigma-Aldrich; cholesterol was purchased from Adamas; ultrapure water was purchased from Wahaha; 5'-FUDR (5' Fluoride-deoxyuridine (FDR) was purchased from Aladdin.

[0038] Culture media involved in the examples: The formulation for LB liquid medium is as follows: per 100 mL system: 1 g tryptone, 1 g NaCl, 0.5 g yeast extract, dissolved in 100 mL ultrapure water by sonication, pH adjusted to 7.0 using 1 M NaOH, and autoclaved at 121℃ for 20 minutes. LB solid medium: Based on the above liquid medium formulation, add 1.5% agar powder.

[0039] Nematode Growth Medium (NGM): 5.1 g agar powder, 0.75 g peptone, 0.9 g NaCl, diluted with distilled water to 300 mL, autoclaved at 121 °C for 30 min, cooled to 60 °C; placed in a clean bench, 1 mol of [unspecified substance] was added to each medium. L -1 MgSO4, 1 mol L -1 CaCl2, 0.01 mol L -1 300 μL each of cholesterol solution and 1 mol L - 1 7.5 mL of KH2PO3 / K2HPO2 buffer solution. The NGM medium used in the experiment needs to have a fertility inhibitor added to suppress nematode oviposition. Therefore, an additional 6 mL of 20 mM 5'-FUDR solution filtered through a 0.22 μm sieve needs to be added to the above system to prepare NGM medium containing 200 μM 5'-FUDR (NGM-FUDR medium).

[0040] Preparation of M9 buffer: For every 100 mL of system, add 0.3 g of KH2PO4, 1.51 g of Na2HPO4·12H2O, and 0.5 g of NaCl to 100 mL of ultrapure water, dissolve by sonication, and autoclave at 121℃ for 20 minutes. Cool to room temperature after sterilization.

[0041] The kits mentioned in the examples: The BCA, SOD, CAT, and DCFH-DA reagent kits were purchased from Beyotime Biotechnology Co., Ltd.

[0042] The measurement methods involved in the examples are as follows: 1. Determination of total sugar content The total sugar content was determined using the phenylsulfuric acid method. Preparation of standard curve: Prepare a 100 μg / mL glucose solution. Take 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of each tube and add water to 2 mL. Add 1 mL of 6% phenol solution to each tube, shake well, and then quickly add 5 mL of concentrated sulfuric acid. Allow the mixture to react for 20 min. Measure the absorbance at 490 nm and plot the standard curve based on the glucose content.

[0043] Sample determination: Take 1 mL of a certain concentration of sample solution, add water to 2 mL, and follow the same steps as the standard curve preparation method. Substitute the values ​​into the standard curve to calculate the total sugar content.

[0044] 2. Determination of uronic acid content The content of uronic acid in the sample was determined by the m-hydroxybiphenyl method.

[0045] Preparation of standard curve: Take 6 test tubes and add 0, 0.05, 0.10, 0.15, 0.20, and 0.25 mL of galacturonic acid standard solution (1 mg / mL) respectively. Add distilled water to make up to 0.25 mL, pre-cool in an ice bath, add 1.5 mL of sodium tetraborate-sulfuric acid solution, shake well, boil in a water bath for 5 min, then cool to room temperature in an ice bath, add 25 μL of m-hydroxybiphenyl solution, mix well, and measure the absorbance at 520 nm. Plot the standard curve with galacturonic acid content (μg / mL) on the x-axis and absorbance on the y-axis.

[0046] Sample determination: Take 1 mL of a certain concentration of sample solution, add water to 2 mL, and follow the same steps as the standard curve preparation method. Substitute the values ​​into the standard curve to calculate the total sugar content.

[0047] 3. Determination of protein content The protein content of the sample was determined using the BCA method, following the instructions of the assay kit (Beyotime).

[0048] 4. Determination of the relative molecular mass of polysaccharides: The relative molecular mass of polysaccharides was determined by HPLC. Dextran standards of 5000, 25000, 80000, 150000, 420000, and 670000 Da were accurately weighed and prepared into 2 mg / mL standard solutions, which were then filtered through a 0.22 μm filter. The solutions were injected sequentially according to molecular weight from smallest to largest, and a molecular weight standard curve was plotted with retention time on the x-axis and the logarithm of molecular weight on the y-axis. A 5 mg / mL polysaccharide sample solution was prepared, filtered through a 0.22 μm filter, injected, and the retention time was recorded. The relative molecular mass of the sample was calculated based on the standard curve.

[0049] Chromatographic conditions: LC-20A-HPLC high-performance liquid chromatograph equipped with a differential detector, Ohpak SB-804 HQ column (8.0 mm × 300 mm, Shodex), mobile phase was 0.22 μm permeate ultrapure water, flow rate was 0.6 mL / min, column temperature was 30℃. After elution with ultrapure water for 1 hour to baseline equilibration, polysaccharide samples were analyzed, with an injection volume of 20 μL (polysaccharide concentration 1 mg / mL).

[0050] 5. Monosaccharide composition analysis The monosaccharide composition of the samples was determined by pre-column derivatization HPLC using PMP (Protein-Mechanical Processing). Eight monosaccharide standards (D-Man, L-Rha, D-GlcA, D-GalA, D-Glc, D-Gal, D-Xyl, and L-Ara) and a mixed standard prepared from the monosaccharide standards were subjected to PMP derivatization. Then, the black goji berry polysaccharide sample was analyzed by HPLC under the same conditions, and the monosaccharide composition was determined based on the peak time. Chromatographic conditions: LC20A-HPLC high-performance liquid chromatograph, UV detector, C18 column (250 mm × 4.6 mm, TC-C18, Agilent), mobile phase: 0.1 mol / L pH 6.7 PBS and acetonitrile (83:17 v / v), flow rate: 1.0 mL / min, column temperature: 30℃, detection wavelength: 245 nm, injection volume: 20 μL.

[0051] 6. Ultraviolet spectroscopy analysis Prepare a 1 mg / mL polysaccharide aqueous solution and scan it in the wavelength range of 190~400 nm using a UV-Vis spectrophotometer.

[0052] 7. Infrared spectroscopy analysis Take a small amount of dried sample, mix it with KBr, compress it into tablets, and press it at 4000~400 cm⁻¹. 1 Fourier transform infrared spectroscopy analysis was performed on an infrared spectrometer.

[0053] 8. Detection of nematode motility level Determining the body swaying frequency of nematodes: 15 nematodes were randomly selected and transferred to M9 buffer on a sterile blank plate. The body swaying frequency of the nematodes was observed. One body sway was defined as the head swinging from one side to the other and then swinging backward.

[0054] Swimming speed / movement duration determination: Nematodes were gently picked into M9 buffer on a sterile blank plate, where the M9 buffer immediately triggered swimming. A 3-minute video recording was then used. The swimming speed and movement duration of the nematodes were quantitatively analyzed using a nematode tracker plugin from ImageJ.

[0055] The “median survival time” mentioned in the following examples: also known as the half-survival period, refers to the length of time that 50% of the individuals in the study population survive.

[0056] Example 1: Preparation and Physicochemical Characterization of Black Goji Berry Polysaccharides 1. Preparation of black wolfberry polysaccharides (1) Take 1 kg of dried black goji berries, crush them and pass them through a 20-mesh sieve. Add ultrapure water at a ratio of 1 g: 10 mL. Stir at 95℃ and 30 rpm for 30 min, then extract with ultrasound at 40℃ and 400 W for 60 min. Then centrifuge at 8000 r / min for 15 min and collect the supernatant and precipitate respectively. (2) The precipitate prepared in step (1) was added to ultrapure water at a ratio of 1 g: 10 mL. After stirring at 95℃ and 30 rpm for 30 min, it was extracted by ultrasonication at 40℃ and 400 W for 60 min. Then, it was centrifuged at 8000 r / min for 15 min and the supernatant was collected. (3) Combine the supernatants obtained in steps (1) and (2) and concentrate them to 1 / 3 of the original volume by rotary evaporation at 50°C to obtain crude extract of black goji berries; (4) Add 10 g / 100 mL of trichloroacetic acid (to remove protein) to the crude extract of black goji berries obtained in step (3), let stand for 4 h, and then centrifuge at 4℃ and 8000 r / min for 15 min to remove the precipitate and retain the supernatant; repeat the extraction of the precipitate 3 times in the above manner and combine the supernatant to obtain the supernatant. (5) Add 4 times the volume of anhydrous ethanol to the supernatant obtained in step (4) and precipitate overnight (12 h) at 4℃; then centrifuge at 8000 r / min for 15 min, discard the supernatant, wash the precipitate 3 times with anhydrous ethanol, reconstitute with deionized water, dialyze with a 3 kDa dialysis bag for 48 hours, and then freeze-dry (freeze-dry at -60℃ for 48 h) to obtain black wolfberry polysaccharide.

[0057] 2. Characterization of black goji berry polysaccharides The black goji berry polysaccharide prepared in step 1 was analyzed for total sugar, uronic acid, protein, infrared spectrum, ultraviolet spectrum, molecular weight, and monosaccharide composition. The results of the infrared and ultraviolet absorption spectra of the black goji berry polysaccharide are as follows: Figure 1 As shown in Table 1, the monosaccharide composition of polysaccharides is as follows.

[0058] The results showed that the total sugar content of the black goji berry polysaccharide sample was 92.92%, the uronic acid content was 44.1%, and the protein content was 2.7%. The molecular weight parameters are Mn 22121, Mw 56187, and Mp 35483; where Mn is the number-average molecular weight, Mw is the weight-average molecular weight, and Mp is the peak molecular weight.

[0059] Infrared and ultraviolet spectroscopy revealed typical physicochemical characteristics of polysaccharide samples. The infrared absorption spectrum showed a value at 1055 cm⁻¹. -1 The absorption peak at 871 cm⁻¹ indicates the presence of pyranose in the form of pyranose, suggesting the existence of pyranoside bonds. - The peak at ¹ indicates the β configuration of these glycosidic bonds. 1736 cm⁻¹ -1 A distinct peak is visible at 3392 cm⁻¹, which is the characteristic peak of the C=O stretching vibration of glucuronic acid. - The broad absorption band at ¹ represents the stretching vibration peak of the OH bond, and these characteristic peaks collectively confirm the polysaccharide nature. Furthermore, at 818 cm⁻¹... -1 The weak peak detected was consistent with the arabinose furan ring vibration, supporting the presence of arabinose in the monosaccharide composition.

[0060] The ultraviolet absorption spectrum shows that the wavelength is between 260 and 280 nm. -1 No characteristic absorption bands of proteins or nucleic acids were observed within the range, confirming the absence of these contaminants in black goji berry polysaccharides.

[0061] Table 1: Composition and distribution of monosaccharide components in black goji berry polysaccharide (LRP)

[0062] As can be seen from Table 1, the monosaccharide composition of the black wolfberry polysaccharide prepared by the present invention is mainly glucose, and also contains arabinose, galactose, fucose, rhamnose, xylose, mannose and uronic acid components. It should be noted that the monosaccharide components and contents in black goji berries from different producing areas are not the same. For example, the literature (Shuang Quan, Zhang Haixia, Lu Yu, et al. Study on chemical components and antioxidant activity of wild black goji berries [J]. Food Industry Technology, 2017, 38(4):94-100.DOI:10.13386 / j.issn1002-0306.2017.04.010.) collected wild black goji berries from different regions and analyzed and compared the nutritional components such as carbohydrates, proteins, fats, ash, amino acids, and fatty acids, as well as the bioactive components such as anthocyanins, polysaccharides, and polyphenols and their antioxidant activities. The study found that the carbohydrate, protein, fat, and ash contents of wild black goji berries from different regions ranged from 69.55% to 77.14%, 10.76% to 14.72%, 3.90% to 6.89%, and 6.63% to 10.99%, respectively, with some differences in the content of each component between different regions. Among them, black goji berries are rich in active ingredients such as polyphenols and polysaccharides, and these contents vary greatly from region to region.

[0063] The literature (Zhang Li, Meng Jing, Gou Chunlin, et al. Research progress on the detection of component characteristics and traceability technology of Lycium barbarum[J]. Journal of Analytical Testing, 2018, 37(7):862-870.) points out that Chen Yanrui determined the monosaccharide composition of Lycium barbarum polysaccharides from Inner Mongolia, Gansu, Qinghai, Hebei, and Xinjiang by gas chromatography-mass spectrometry (GC-MS). Lycium barbarum polysaccharides are mainly composed of monosaccharides such as D-arabinose, L-xylose, L-ribose, D-galactose, D-glucose, and D-mannose. Except for the Lycium barbarum from Hebei, which only detected D-glucose and D-mannose, all six monosaccharides were detected in the other provinces, indicating that the polysaccharide structure of Lycium barbarum from Hebei is different from that of Lycium barbarum from other producing areas and has certain regional characteristics.

[0064] Example 2: Application of black goji berry polysaccharides in extending the lifespan of nematodes 1. Preparation of raw materials Preparation of OP50: *E. coli* OP50 strain was streaked onto LB solid medium and incubated at 37°C for 24 h. Single colonies were then picked and inoculated into LB liquid medium, incubated at 37°C and 180 rpm for 12 h. Subsequently, the culture was centrifuged at 5000 rpm for 2 min, concentrated 10-fold (90% of the supernatant was discarded, and the precipitated OP50 was dissolved in the remaining liquid), and stored for later use, yielding a concentration of 8.8 × 10⁻⁶. 9 OP50 bacterial suspension at CFU / mL.

[0065] Synchronization treatment of wild-type N2 Caenorhabditis elegans: Wild-type N2 Caenorhabditis elegans was inoculated with 0.5 mL of a solution containing 8.8 × 10⁻⁶ cells / mL. 9 CFU / mL OP50 bacterial suspension was cultured in NGM medium at 20°C until the nematodes laid eggs. One hour after the nematodes laid eggs, the adult nematodes were removed, and the eggs were cultured at 20°C for 48 hours to grow into adult nematodes for later use, thus obtaining synchronized nematodes.

[0066] NGM containing different concentrations of black goji berry polysaccharides The preparation method of OP50 medium is as follows: The concentration was 8.8 × 10 9 An OP50 bacterial suspension with CFU / mL was mixed with sterile water in an equal proportion to obtain an OP50 bacterial suspension with a final concentration of 0 μg / mL of black goji berry polysaccharide (final concentration of OP50 bacterial suspension: 4.4 × 10⁻⁶). 9 (CFU / mL) The concentration was 8.8 × 10 9 An OP50 bacterial suspension with a concentration of CFU / mL was mixed with a black goji berry polysaccharide solution with a concentration of 100 μg / mL in an equal volume ratio to obtain an OP50 bacterial suspension with a final black goji berry polysaccharide concentration of 50 μg / mL (the final concentration of the OP50 bacterial suspension was 4.4 × 10⁻⁶).9 (CFU / mL) The concentration was 8.8 × 10 9 An OP50 bacterial suspension with a concentration of CFU / mL was mixed with a black goji berry polysaccharide solution with a concentration of 200 μg / mL in an equal volume ratio to obtain an OP50 bacterial suspension with a final black goji berry polysaccharide concentration of 100 μg / mL (the final concentration of the OP50 bacterial suspension was 4.4 × 10⁻⁶). 9 (CFU / mL) The concentration was 8.8 × 10 9 An OP50 bacterial suspension with a concentration of CFU / mL was mixed with a black goji berry polysaccharide solution with a concentration of 400 μg / mL in an equal volume ratio to obtain an OP50 bacterial suspension with a final black goji berry polysaccharide concentration of 200 μg / mL (the final concentration of the OP50 bacterial suspension was 4.4 × 10⁻⁶). 9 (CFU / mL) The concentration was 8.8 × 10 9 An OP50 bacterial suspension with a concentration of CFU / mL was mixed with a black goji berry polysaccharide solution with a concentration of 800 μg / mL in an equal volume ratio to obtain an OP50 bacterial suspension with a final black goji berry polysaccharide concentration of 400 μg / mL (the final concentration of the OP50 bacterial suspension was 4.4 × 10⁻⁶). 9 (CFU / mL) The concentration was 8.8 × 10 9 An OP50 bacterial suspension with a concentration of CFU / mL was mixed with a black goji berry polysaccharide solution with a concentration of 1600 μg / mL in an equal volume ratio to obtain an OP50 bacterial suspension with a final black goji berry polysaccharide concentration of 800 μg / mL (the final concentration of the OP50 bacterial suspension was 4.4 × 10⁻⁶). 9 (CFU / mL) 0.5 mL of each of the above-mentioned OP50 bacterial suspensions containing different concentrations of black goji berry polysaccharides was added dropwise to NGM medium containing 200 mM 5'-FUDR. After drying in a clean bench, the suspensions were stored at 4°C for a short period of time to obtain NGM medium containing different concentrations of black goji berry polysaccharides. OP50 medium.

[0067] 2. Effects of black wolfberry polysaccharides on the lifespan of wild-type N2 Caenorhabditis elegans The synchronized nematodes obtained in step 1 were randomly divided into six groups, and 200 nematodes / 10 cm petri dish were randomly transferred to NGM containing different concentrations of black goji berry polysaccharides prepared in step 1. In OP50 medium, three replicates were prepared for each group and cultured at 20°C until the organisms died.

[0068] Starting from the day of transfer (Day 0), the number of surviving nematodes was counted daily, excluding those that escaped, died accidentally, or had eggs hatching inside the nematodes, until all nematodes died. Survival curves were then plotted, and the lifespan experimental data were analyzed using Kaplan-Meier survival analysis. The Log-rank test was used to compare the processed Kaplan-Meier survival curves, and the median survival time was determined.

[0069] Simultaneously, the average lifespan of the population is measured, and the mathematical expression for the average lifespan of the population is:

[0070] Among them, t i d: The time of the i-th record i : in t i The number of deaths at any given time. The calculation method is: the number of survivors at the current time point minus the number of survivors at the next time point (d...). i = N i -N i+1 N: Initial total number of individuals (i.e., the number of survivors on day 1);

[0071] The results of median survival time, population mean lifespan, and nematode survival time measurements are as follows: Figure 2 And as shown in Table 2: Table 2 Median survival time of nematodes

[0072] The results show: Depend on Figure 2 (A) It can be seen that, compared with the blank control group, the overall survival curve of nematodes after intervention with black goji berry polysaccharide showed a rightward shift to varying degrees, with the 100 μg / mL group showing the most significant effect.

[0073] Depend on Figure 2 (B) It can be seen that the median survival time of nematodes in the 100 μg / mL group was extended by 4 days compared with the control group, and the average lifespan was extended by about 28%, indicating that black goji berry polysaccharide has a significant life-prolonging effect within the appropriate dosage range.

[0074] Meanwhile, it was observed that the 50 μg / mL and 200 μg / mL groups also showed a certain life-extending trend, but the effect was weaker than that of the 100 μg / mL group; the life-extending effect of the 400 μg / mL group was weakened; and the 800 μg / mL group showed a life-shortening trend, but the difference was not statistically significant compared with the control group.

[0075] The above results indicate that the anti-aging effect of black goji berry polysaccharides is dose-dependent and has an optimal concentration range. Moderate doses, especially 100 μg / mL, are more conducive to delaying aging, while excessively high doses may weaken its beneficial effects.

[0076] 3. Effects of black wolfberry polysaccharides on lipofuscin levels in nematodes The synchronized nematodes obtained in step 1 were randomly divided into six groups, with three replicates in each group, and transferred to the NGM containing different concentrations of black goji berry polysaccharides prepared in step 1. After 7 days of culture at 20°C in OP50 medium, the lipofuscin content of the 7th cultured anthracid was measured.

[0077] The specific measurement method is as follows: Nematodes cultured for 7 days were anesthetized with 10 mM levamisole hydrochloride, placed on 2% agar pads, the pads were cut off and placed on glass slides, and the autofluorescence of lipofuscin in the nematode intestine was excited by purple excitation light under a fluorescence microscope and recorded. The fluorescence intensity was analyzed by ImageJ.

[0078] The measurement results are as follows Figure 3 And as shown in Table 3: Table 3 Quantitative analysis results of lipofuscin fluorescence intensity

[0079] The results show: Depend on Figure 3 (A) to Figure 3 (F) It can be seen that with the intervention of black wolfberry polysaccharide, the overall fluorescence of lipofuscin in nematodes was weakened, with the decrease being more obvious in the 100 μg / mL group and the 200 μg / mL group.

[0080] Figure 3 (G) Quantitative analysis of the fluorescence intensity of lipofuscin showed that the accumulation of lipofuscin in the low-to-medium dose treatment group was lower than that in the control group, while the 100 μg / mL group showed the best reduction effect. Lipofuscin is considered a typical marker of the gradual accumulation of recalcitrant metabolic waste during aging, and its elevated content usually indicates accelerated aging.

[0081] Black goji berry polysaccharides can reduce lipofuscin levels, indicating that they can not only improve short-term stress damage, but also reduce the deposition of age-related metabolic waste, thereby delaying the formation of aging phenotypes.

[0082] 4. Effects of black goji berry polysaccharides on ROS levels in nematodes The synchronized nematodes obtained in step 1 were randomly divided into six groups, with three replicates in each group, and transferred to the NGM containing different concentrations of black goji berry polysaccharides prepared in step 1. After culturing in OP50 medium at 20°C for 5 days, the ROS levels of the fifth anthelmintic were measured using the Beyotime reactive oxygen species detection kit.

[0083] The specific measurement method is as follows: Nematodes were collected and washed with M9 buffer, centrifuged at 3000 rpm for 2 min, and the supernatant was discarded. 990 μL of M9 buffer was mixed with 10 μL of DCFH-DA to obtain a mixed solution. 1 mL of the mixed solution was added to each tube of nematodes and stored at room temperature in the dark for 2 hours. After 2 hours, the nematode suspension was washed five times with M9 buffer. The suspension was then transferred to an agar plate, and the fluorescence intensity was measured at 488 nm. The fluorescence intensity was analyzed using ImageJ.

[0084] The measurement results are as follows Figure 4 And as shown in Table 4: Table 4 Results of quantitative analysis of ROS fluorescence intensity

[0085] The results show: Compared with the control group, the green fluorescence signal in nematodes was weakened overall after treatment with black goji berry polysaccharide, especially in the 50 μg / mL, 100 μg / mL and 200 μg / mL groups, suggesting that black goji berry polysaccharide can reduce ROS accumulation in nematodes.

[0086] Figure 4 (G) Quantitative analysis of fluorescence intensity showed that the 100 μg / mL group had the lowest ROS level, which showed a significant decreasing trend compared with the control group. Excessive accumulation of ROS is an important factor leading to oxidative damage and accelerated aging in the body.

[0087] Black goji berry polysaccharides significantly reduced ROS levels in nematodes, indicating that black goji berry polysaccharides can maintain redox homeostasis in the body by reducing oxidative stress load, thereby playing a role in maintaining motility.

[0088] 5. Effects of black wolfberry polysaccharides on the activity levels of SOD and CAT enzymes in nematodes The synchronized nematodes obtained in step 1 were randomly divided into six groups, with three replicates in each group, and transferred to the NGM containing different concentrations of black goji berry polysaccharides prepared in step 1. After culturing in OP50 medium at 20°C for 5 days, nematodes were collected using M9 buffer and the supernatant was discarded. The nematodes were then washed 3-5 times with pre-cooled M9 buffer to remove residual OP50 and drugs attached to the nematodes and intestinal wall. After discarding the supernatant, Western blotting and IP cell lysis buffer (Beyotime) were added to lyse the nematodes. The SOD and CAT contents were then measured according to the assay kit (Beyotime) instructions.

[0089] The measurement results are as follows Figure 5 And as shown in Table 5: Table 5. Determination of SOD and CAT enzyme activities

[0090] The results show: Compared with the control group, the SOD activity in nematodes was increased overall after treatment with black goji berry polysaccharides, with the 100 μg / mL group showing the most significant increase and a statistically significant difference; the 50 μg / mL and 200 μg / mL groups also showed varying degrees of increase, while the 400 μg / mL and 800 μg / mL groups did not show a significant advantage.

[0091] The trend of CAT activity was basically consistent with that of SOD. The CAT activity was highest in the 100 μg / mL group, which was significantly higher than that in the control group, suggesting that at this concentration, black goji berry polysaccharide is more effective in enhancing the ability of nematodes to remove peroxides.

[0092] Superoxide dismutase (SOD) and catalase (CAT) are important antioxidant enzymes in the body. SOD can disproportionate superoxide anions into hydrogen peroxide, while CAT further decomposes hydrogen peroxide, thereby reducing oxidative damage. Black goji berry polysaccharides significantly enhance the activity of SOD and CAT, indicating that their anti-aging effect is at least partially related to enhancing the function of the endogenous antioxidant enzyme system.

[0093] Example 3: Application of black goji berry polysaccharides in improving the motility of aging nematodes The results from Example 2 show that the nematodes treated with 100 μg / mL black goji berry polysaccharide showed the best average lifespan extension and other indicators. Therefore, a black goji berry polysaccharide concentration of 100 μg / mL was selected for the motility test.

[0094] The synchronized nematodes obtained in step 1 of Example 2 were randomly divided into two groups, with three replicates in each group, and were transferred to the NGM containing 0 and 100 μg / mL black goji berry polysaccharides prepared in step 1 of Example 2, respectively. In OP50 medium (wherein, 0 μg / mL of black goji berry polysaccharide NGM) OP50 medium was used as a control. The nematodes were cultured at 20°C, and changes in their motility, including body swaying frequency, swimming speed, and swimming duration, were detected on days 3, 6, 9, and 12.

[0095] The measurement results are as follows Figure 6 And as shown in Table 6: Table 6. Determination of nematode locomotion ability

[0096] Note: Swimming speed and swimming duration are based on the Control group on day 3 as 100% baseline.

[0097] The results show: As nematodes in the control group aged, all their motor behavior indicators gradually declined, exhibiting typical age-related motor capacity decline; while the black goji berry polysaccharide treatment group maintained a high level of motor activity throughout the senescence process. According to Figure 6 The results showed that black goji berry polysaccharide (100 μg / mL) significantly delayed the decline in the motility of aging nematodes.

[0098] With the frequency of body swaying ( Figure 6 Taking A) as an example, the control group had approximately 90 vibrations / min on day 3, decreasing to approximately 74 vibrations / min on day 6, approximately 52 vibrations / min on day 9, and approximately 40 vibrations / min on day 12; while the black goji berry polysaccharide treatment group had approximately 96, 85, 67, and 62 vibrations / min, respectively. Compared with the control group, the treatment group showed an increase of approximately 15% in body swaying frequency on day 6, approximately 29% on day 9, and approximately 55% on day 12. If the decline in motor function is calculated with day 3 as the baseline, the control group showed a decrease of approximately 56% by day 12, while the treatment group showed a decrease of approximately 35%, indicating that black goji berry polysaccharide can reduce the decline in body swaying ability by approximately 36%.

[0099] In swimming speed ( Figure 6 In aspect B), the control group had approximately 98%, 76%, 65%, and 44% necrosis rates on days 6, 9, and 12, respectively, while the treatment group had approximately 114%, 95%, 91%, and 57%. Compared with the control group, the swimming speed of adult nematodes in the treatment group increased by approximately 16%, 25%, 40%, and 30% on days 3, 6, 9, and 12, respectively.

[0100] During swimming duration ( Figure 6 In aspect C), the control group had approximately 100%, 70%, 32%, and 12% on days 3, 6, 9, and 12, respectively, while the treatment group had approximately 100%, 79%, 54%, and 20% on days 3, 6, 9, and 12. Compared with the control group, the treatment group showed an improvement of approximately 13% on day 6, approximately 69% on day 9, and approximately 67% on day 12.

[0101] The above results indicate that black goji berry polysaccharides can significantly slow down the decline in nematode motility during aging, especially in the middle and late stages of aging, where their effect on improving body swinging ability, swimming speed, and sustained exercise ability is more significant, thus helping to maintain the healthy lifespan-related phenotypes of aging individuals.

[0102] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of black goji berry polysaccharides in the preparation of products for prolonging life and / or improving the motor function of aging individuals, characterized in that, The preparation method of the black goji berry polysaccharide is as follows: (1) After crushing black goji berries, dissolve them in water, stir, sonicate, centrifuge, and take the supernatant. Extract the precipitate again and combine the two supernatants. (2) The supernatant obtained in step (1) is concentrated to 1 / 2 to 1 / 5 of its original volume to obtain crude extract of black goji berries; (3) Add 10-15 g / 100 mL trichloroacetic acid to the crude black goji berry extract obtained in step (2), let stand for 6-12 h, centrifuge, take the supernatant, repeat the extraction of the precipitate three times in the above method, and combine to obtain the supernatant. (4) Add 4 to 6 times the volume of anhydrous ethanol to the supernatant obtained in step (3) for alcohol precipitation, let stand at 4 to 6°C for 8 to 12 h, centrifuge the solution after standing, discard the supernatant, wash the precipitate, dissolve it, and dialyze it with a dialysis bag with a molecular weight cutoff of 2.8 to 3.3 kDa for 8 to 12 h, and freeze dry to obtain black wolfberry polysaccharide.

2. The application according to claim 1, characterized in that, The products include one or more of the following: pharmaceuticals, food, health products, feed, and feed additives.

3. The application according to claim 2, characterized in that, The drug is in the form of a liquid or solid formulation. Optionally, the dosage form of the drug includes granules, capsules, tablets, pills, or oral liquid; Preferably, the drug further includes pharmaceutically acceptable excipients; Preferably, the pharmaceutical excipients include any one or more of the following: solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, pH adjusters, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release inhibitors.

4. The application according to claim 2, characterized in that, The food products mentioned include health foods, foods for special medical purposes, functional foods, or pet foods. Preferably, the food products include grain products, starch products, vegetable products, fruit products, meat products, poultry products, egg products, and dairy products.

5. The application according to claim 2, characterized in that, The health products also contain acceptable excipients.

6. The application according to any one of claims 1 to 5, characterized in that, The extended lifespan and / or improved mobility in aging individuals include at least one of the following: (a) Delaying the decline of motor function; (b) Extend lifespan; (c) Increase the level of antioxidant enzyme activity; (d) Reduce the accumulation of lipofuscin.

7. A drug for prolonging lifespan and / or improving the motor function of aging individuals, characterized in that, The medicine contains black goji berry polysaccharide, and the preparation method of the black goji berry polysaccharide is as follows: (1) After crushing black goji berries, dissolve them in water, stir, sonicate, centrifuge, and take the supernatant. Extract the precipitate again and combine the two supernatants. (2) The supernatant obtained in step (1) is concentrated to 1 / 2 to 1 / 5 of its original volume to obtain crude extract of black goji berries; (3) Add 10-15 g / 100 mL trichloroacetic acid to the crude black goji berry extract obtained in step (2), let stand for 6-12 h, centrifuge, take the supernatant, repeat the extraction of the precipitate three times in the above method, and combine to obtain the supernatant. (4) Add 4 to 6 times the volume of anhydrous ethanol to the supernatant obtained in step (3) for alcohol precipitation, let stand at 4 to 6°C for 8 to 12 h, centrifuge the solution after standing, discard the supernatant, wash the precipitate, dissolve it, and dialyze it for 45 to 50 h using a dialysis bag with a molecular weight cutoff of 2.8 to 3.3 kDa. After freeze drying, black wolfberry polysaccharide is obtained.

8. The medicament according to claim 7, characterized in that, The drug is in the form of a liquid or solid formulation. Optionally, the dosage form of the drug includes, but is not limited to, granules, capsules, tablets, pills, or oral liquid; Preferably, the drug further includes pharmaceutically acceptable excipients.

9. The drug according to claim 7, characterized in that, The pharmaceutical excipients include any one or more of the following: solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, pH adjusters, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release inhibitors.

10. The drug according to any one of claims 7 to 9, characterized in that, The extended lifespan and / or improved mobility in aging individuals include at least one of the following: (a) Delaying the decline of motor function; (b) Extend lifespan; (c) Increase the level of antioxidant enzyme activity; (d) Reduce the accumulation of lipofuscin.

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