Application of lycium ruthenicum murr.polysaccharide in improving fat accumulation, reducing fat, shaping and improving individual exercise capacity products

Abstract

The application discloses application of Lycium ruthenicum Murr. polysaccharide in improving fat accumulation, fat reduction and shaping to improve individual exercise capacity products and belongs to the field of medicine and biology. The application first finds that the Lycium ruthenicum Murr. polysaccharide can optimize in-vivo lipid droplet morphology and fat spatial distribution, improve fat mobilization energy supply efficiency, and delay aging-related behavioral changes such as reduction of body swing frequency, reduction of swimming speed and shortening of sustained exercise capacity. Different from existing simple fat reduction technologies, the application focuses on regulating lipid distribution and utilization efficiency and intervening exercise function 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 medicines and is used for developing related functional products.

Description

Technical Field

[0001] This invention relates to the application of black goji berry polysaccharides in products that improve fat accumulation, reduce fat, and shape the body to improve individual athletic ability, and belongs to the field of pharmaceutical biology. Background Technology

[0002] Recent studies have found that declining athletic performance is not only related to neuromuscular dysfunction but also closely linked to the efficiency of the body's energy storage utilization. Lipids are one of the most important forms of energy storage in organisms, and their ability to be mobilized and converted into energy for exercise directly affects athletic performance and healthy lifespan. Under normal circumstances, lipids are stored in specific tissues in the form of lipid droplets, and fatty acids are released for the body's use through lipolysis during exercise or when energy demands increase. Lipid droplets are not static storage structures but are in a dynamic process of continuous formation, fusion, decomposition, and renewal. The size, morphology, and spatial distribution of lipid droplets in tissues all affect lipid mobilization efficiency.

[0003] With aging or changes in metabolic state, lipid droplets are prone to abnormal enlargement, localized aggregation, and decreased mobility. Although the body still retains a large amount of fat reserves, lipid mobilization efficiency decreases, resulting in insufficient available energy supply and ultimately manifesting as decreased exercise capacity and reduced activity levels. Therefore, improving fat mobilization capacity and maintaining healthy lipid droplet morphology has become an important research direction for improving physical performance. Even if overall lipid levels do not change significantly, abnormal lipid deposition, abnormal lipid droplet enlargement, or ectopic aggregation in non-fat storage tissues can still induce significant lipotoxic reactions, further leading to mitochondrial dysfunction, increased oxidative stress, activated inflammatory responses, and energy metabolism imbalances.

[0004] Under normal physiological conditions, the body's lipid storage and utilization are in a highly dynamic equilibrium. Lipids typically exist in the form of small, uniform droplets, maintaining stable flow and transport between different tissues to meet the body's energy needs. Lipid droplets are not only passive fat storage structures but also important dynamic organelles involved in lipid metabolism regulation. Their size, number, and spatial distribution directly affect lipid mobilization efficiency and metabolic health. When the body is under a high-fat diet, aging, or metabolic stress, lipid droplets gradually transform into "larger particles," "lower mobility," and "abnormal aggregation," resulting in decreased lipid mobilization capacity and ultimately pathological lipid deposition. Especially in non-professional fat storage tissues such as the liver, intestines, and muscles, abnormal lipid aggregation can easily lead to lipotoxic damage, thereby inducing fatty liver, decreased exercise capacity, and various metabolic-related diseases. Therefore, compared to simply reducing total fat volume, improving lipid spatial distribution has gradually become an important direction in the field of lipid metabolism research.

[0005] Black goji berries are a unique medicinal and edible plant native to western my country, rich in polysaccharides, anthocyanins, and various natural active ingredients, possessing high nutritional and development value. Among them, black goji berry polysaccharides (Lycium ruthenicum polysaccharides, LRPs) have attracted widespread attention due to their potential biological activities such as antioxidant, immunomodulatory, and metabolic regulation. Current research on black goji berry polysaccharides mainly focuses on optimizing their extraction process, evaluating their antioxidant capacity, and studying their traditional lipid-lowering effects. Existing studies have shown that they can influence lipid metabolism to some extent. In current technologies, research and applications surrounding lipid metabolism regulation primarily focus on "lipid reduction," that is, improving the body's condition by reducing total lipid levels, decreasing triglyceride content, or mitigating lipid accumulation. However, simply lowering lipid levels is not equivalent to promoting fat mobilization. Lipid-related functional abnormalities are not only related to increased total fat levels but also closely related to the deposition patterns, spatial distribution, local aggregation degree, lipid droplet morphology, and lipid mobilization and utilization efficiency in the body.

[0006] In other words, fat reduction does not necessarily mean a more rational lipid distribution, nor does it necessarily lead to more effective improvement in bodily functions. In contrast, if lipid metabolism can be regulated to transform lipids from a state of localized abnormal deposition, coarse aggregation, or low utilization efficiency to a state of more balanced distribution, more stable lipid droplet morphology, more rational mobilization, and more efficient utilization, then it will be more beneficial to promote bodily metabolism and optimize functional status.

[0007] Therefore, improving athletic performance by improving fat spatial distribution differs from simply improving athletic performance by reducing lipid levels in terms of technical issues and mechanisms of action: the former focuses on addressing the problems of irrational lipid deposition patterns and metabolic flow, strongly optimizing lipid distribution; the latter mainly addresses the burden caused by excessive total lipid levels. Based on this, a technical solution is proposed that improves athletic performance by regulating lipid metabolism, improving fat spatial distribution, and lipid droplet state, which has clear practical significance and application value.

[0008] Caenorhabditis elegans, due to its highly conserved lipid metabolism pathways, short lifespan, clear genetic background, and high degree of lipid droplet visualization, is widely used in lipid metabolism and aging-related research. The nematode gut is its main lipid storage site, effectively reflecting lipid droplet size, spatial distribution, and dynamic changes. Through Oil Red O staining, triglyceride quantification, transcriptomics, and metabolomics analysis, the regulatory effects of natural active ingredients on lipid metabolism homeostasis can be systematically evaluated. Therefore, using the Caenorhabditis elegans model to study the effects of black goji berry polysaccharides on lipid spatial distribution and lipid droplet dynamic homeostasis is of great significance for developing novel natural lipid metabolism regulating products.

[0009] Furthermore, a growing body of research indicates that adipose tissue status is closely related to physical performance, healthy lifespan, and metabolic activity. In a healthy state, small, uniform, and highly mobile lipid droplets are more efficient at energy production, while pathologically enlarged lipid droplets can lead to decreased energy utilization efficiency and impaired motor function. Therefore, compared to simply reducing fat storage, maintaining the dynamic turnover capacity of lipids and the healthy morphology of lipid droplets is of greater significance for improving physical performance and maintaining metabolic health.

[0010] Based on this, developing a natural active ingredient that can promote lipid dynamic renewal, optimize lipid droplet morphology, and regulate lipid spatial distribution while maintaining normal energy metabolism in the body has significant application value for preventing and improving abnormal lipid deposition. Currently, there is a lack of relevant technical solutions regarding the role of black goji berry polysaccharides in lipid spatial remodeling and healthy regulation of lipid metabolism. Summary of the Invention

[0011] To address the aforementioned issues, this invention provides the application of black goji berry polysaccharides in products that improve fat accumulation, reduce fat, and shape the body to enhance individual athletic performance. This invention is the first to discover that black goji berry polysaccharides can optimize lipid droplet morphology and fat spatial distribution in the body, improve fat mobilization efficiency, and delay related behavioral changes such as decreased body swing frequency, slower swimming speed, and shortened sustained exercise capacity. Unlike existing simple lipid-lowering technologies, this invention focuses on regulating lipid distribution and utilization efficiency and intervening in the decline of athletic function, thus having more clearly defined practical application significance. Black goji berry polysaccharides can be further prepared into foods, health products, dietary supplements, or pharmaceuticals for the development of related functional products.

[0012] The first objective of this invention is to provide the application of black goji berry polysaccharides in the preparation of products that promote fat mobilization and / or improve individual athletic performance, wherein promoting fat mobilization includes: improving fat accumulation, reducing fat, shaping and slimming the body, tightening the skin, improving cellulite, reducing excess subcutaneous fat, and regulating the body's fat levels; 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.

[0013] 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.

[0014] In one embodiment, the ratio of black goji berry powder to water in step (1) is 1~5 g: 5~20 mL.

[0015] In one embodiment, the ultrasonic conditions in step (1) are 300~400W ultrasound for 80~100 min.

[0016] In one embodiment, the centrifugation conditions described in steps (1) to (4) are centrifugation at 8000-10000 rpm for 15-20 min.

[0017] 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.

[0018] In one embodiment, the concentration in step (2) is achieved by rotary evaporation concentration at 45-50°C.

[0019] 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%).

[0020] In one embodiment, the product includes one or more of the following: pharmaceuticals, food, health products, feed, and feed additives.

[0021] 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.

[0022] In one embodiment, the 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.

[0023] 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.

[0024] In one embodiment, the health product also contains acceptable excipients.

[0025] A second objective of this invention is to provide a drug that promotes fat mobilization and / or improves individual exercise capacity, said drug comprising black goji berry polysaccharide, the preparation method of said black goji berry polysaccharide being 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.

[0026] In one embodiment, the promotion of fat mobilization includes: improving fat accumulation, shaping and slimming the body, tightening the skin, improving cellulite, reducing excess subcutaneous fat, and regulating the body's fat levels.

[0027] 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 liquids.

[0028] In one embodiment, the drug further includes pharmaceutically acceptable excipients.

[0029] In one embodiment, the 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.

[0030] Beneficial effects This invention experimentally investigated the effects of black goji berry polysaccharides on lipid metabolism-related phenotypes and the regulation of fat spatial distribution to improve athletic performance, providing insights for the further application of black goji berry polysaccharides in improving athletic function.

[0031] This invention utilizes wild-type *C. elegans* in a lipid metabolism model to investigate the effects of black goji berry polysaccharide in promoting fat utilization, optimizing fat spatial distribution, and improving motor function. Through Oil Red O staining, exercise evaluation, transcriptomics, and metabolomics analysis, it was found that this black goji berry polysaccharide can effectively regulate fat spatial distribution and efficient fat utilization to promote improved motor performance, specifically: Compared with the control group, the black goji berry polysaccharide treatment group showed higher body wriggling frequencies in adult nematodes on days 4, 5, and 6. The body wriggling frequency in the control group decreased from approximately 87 times / min to 73 times / min, while in the treatment group it decreased from approximately 98 times / min to 88 times / min. Compared with the control group, the treatment group showed an increase of approximately 13% on day 4, approximately 18% on day 5, and approximately 21% on day 6.

[0032] Regarding swimming speed, the percentage of adult nematodes in the control group decreased from approximately 100% on day 4 to approximately 79% on day 6, while the percentage in the black goji berry polysaccharide treatment group decreased from approximately 119% to approximately 96%. Compared with the control group, the treatment group showed an increase of approximately 19% on day 4, approximately 12% on day 5, and approximately 22% on day 6.

[0033] Regarding the duration of movement, the percentage of nematodes in the control group decreased from approximately 100% on day 4 to approximately 76% on day 6, while the percentage in the black goji berry polysaccharide treatment group decreased from approximately 100% to approximately 90%. Compared with the control group, the treatment group showed an increase of approximately 12% on day 5 and approximately 18% on day 6. Attached Figure Description

[0034] Figure 1 The results show the physicochemical characterization of black goji berry polysaccharides; (A) is the infrared absorption spectrum and (B) is the ultraviolet absorption spectrum.

[0035] Figure 2 The effect of black goji berry polysaccharide on the spatial distribution of fat in nematodes.

[0036] Figure 3The effects of black goji berry polysaccharides on the transcriptomics of nematodes are shown in Figure 1. (A) is a PCA analysis diagram of transcriptome samples, and (B) is a statistical diagram of the number of differentially expressed genes upregulated and downregulated.

[0037] Figure 4 The effects of black goji berry polysaccharides on the transcriptomics of nematodes are shown; (A) is a statistical diagram of the KEGG pathway of differentially expressed genes, and (B) is a statistical diagram of the GO pathway of differentially expressed genes.

[0038] Figure 5 The effects of black goji berry polysaccharides on the transcriptomics of nematodes are shown; (A) is a bubble diagram of KEGG enrichment of differentially expressed genes, and (B) is a volcano diagram of differentially expressed genes.

[0039] Figure 6 The effects of black goji berry polysaccharides on the transcriptomics of nematodes are shown in Figure 1. (A) is a heatmap of differentially expressed genes clustering, (B) is a graph of GO enrichment significance analysis of differentially expressed genes, and (C) is a graph of GSEA enrichment.

[0040] Figure 7 The effects of black goji berry polysaccharides on the metabolomics of nematodes are shown in (A), (B), (C), and (D).

[0041] Figure 8 The effects of black goji berry polysaccharides on the metabolomics of nematodes are shown; (A) is a heatmap of differential metabolites, and (B) is a bubble diagram of KEGG enrichment of differential metabolites.

[0042] Figure 9 The effects of black goji berry polysaccharides on the metabolomics of nematodes are shown; (A) is a volcano diagram of differential metabolites, and (B) is a network diagram of pathways related to differential metabolites.

[0043] Figure 10 The effects of black goji berry polysaccharide on the motility of nematodes are as follows: (A) the effect of black goji berry polysaccharide on the body swaying ability of nematodes, (B) the effect of black goji berry polysaccharide on the swimming speed of nematodes, and (C) the effect of black goji berry polysaccharide on the swimming duration of nematodes. Detailed Implementation

[0044] 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.

[0045] Raw materials involved in the examples: The dried black goji berries mentioned in the following examples are from Golmud and were purchased from Ningxia Qilixiang Goji Berry Co., Ltd.

[0046] Wild-type N2 Caenorhabditis 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, tryptone, and isopropanol were purchased from Sinopharm Shanghai Laboratory; yeast extract, peptone, 4% paraformaldehyde, and Oil Red O were purchased from Sigma-Aldrich; cholesterol was purchased from Adamas; ultrapure water was purchased from Wahaha; 5-FUDR (5' Fluoride-deoxyuridine and Triton X-100 were purchased from Aladdin.

[0047] The culture medium 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.

[0048] 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 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).

[0049] 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.

[0050] The kits mentioned in the examples: The BCA kit was purchased from Beyotime Biotechnology Co., Ltd.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 2. Determination of uronic acid content The content of uronic acid in the sample was determined by the m-hydroxybiphenyl method.

[0055] 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.

[0056] 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.

[0057] 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).

[0058] 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 membrane. The solutions were injected sequentially according to molecular weight from smallest to largest. A molecular weight standard curve was plotted with retention time on the x-axis and the logarithm of molecular weight on the y-axis.

[0059] Prepare a 5 mg / mL polysaccharide sample solution, filter it through a 0.22 μm filter membrane, inject the sample, record the retention time, and calculate the relative molecular mass of the sample according to the standard curve.

[0060] 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).

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 8. Oil Red Staining ORO working solution preparation: Weigh 0.5 g of powder and add it to 100 mL of isopropanol. Stir magnetically at 300 rpm overnight at 30°C (avoid light). Filter twice through a 0.22 μm sieve to prepare a saturated Oil Red O staining solution. Store in aluminum foil or brown bottles at 4°C. Dilute to 60% with sterile ddH2O before use.

[0065] Collect nematode samples, wash with M9 buffer containing 0.01% Triton X-100, and then fix with 4% paraformaldehyde (PFA) for 30 minutes at room temperature, discarding the supernatant. To enhance cuticle permeability, the fixed nematodes were first treated at -80°C for 3 min, then at room temperature (20-25°C) for 6 min, repeating the freeze-thaw cycle three times. Then, 600 μL of freshly prepared and filtered ORO working solution (Sigma-Aldrich, 60% concentration) was added to each tube, and the tubes were stained with gentle shaking at room temperature in the dark for 2 hours. After staining, excess dye was washed away with M9 buffer, and the nematodes were allowed to settle naturally, discarding as much dye residue as possible. The stained nematodes were placed on a slide and photographed under a stereomicroscope.

[0066] 9. 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.

[0067] 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.

[0068] 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 10 mL / g. 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 trichloroacetic acid (to remove protein) to the crude extract of black goji berries obtained in step (3), let stand for 4 h, 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. (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.

[0069] 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.

[0070] 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 were 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.

[0071] The monosaccharide composition is mainly glucose, and also contains arabinose, galactose, fucose, rhamnose, xylose, mannose and uronic acid components; infrared and ultraviolet spectroscopy show that it has the typical physicochemical characteristics of polysaccharide samples.

[0072] The infrared absorption spectrum shows 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. -1 The peak at 1736 cm⁻¹ indicates the β configuration of these glycosidic bonds. - A distinct peak is visible at ¹, which is the characteristic peak of the C=O stretching vibration of glucuronic acid. 3392 cm⁻¹ -1 The broad absorption band at 818 cm⁻¹ corresponds to the stretching vibration peak of the OH bond, and these characteristic peaks collectively confirm the polysaccharide nature. Furthermore, the 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.

[0073] The ultraviolet absorption spectrum shows that the wavelength is between 260 and 280 nm. - ¹No characteristic absorption bands of proteins or nucleic acids were observed within the range, confirming the absence of these contaminants in black goji berry polysaccharides.

[0074] Table 1: Composition and distribution of monosaccharide components in black goji berry polysaccharide (LRP)

[0075] 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.

[0076] 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.

[0077] Example 2: Application of black goji berry polysaccharides in improving athletic performance by regulating lipid metabolism 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.

[0078] 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.

[0079] 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) 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 μM 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 with black goji berry polysaccharide concentrations of 0 and 100 μg / mL. OP50 medium, containing NGM at a concentration of 0 μg / mL for black goji berry polysaccharide. OP50 medium was used as a blank control group, with NGM containing black goji berry polysaccharide at a concentration of 100 μg / mL. OP50 medium was used as the experimental group.

[0080] 2. Effects of black wolfberry polysaccharides on the spatial distribution of lipids in wild-type N2 Caenorhabditis elegans The synchronized nematodes obtained in step 1 were randomly divided into two groups, and 200 nematodes / 10 cm petri dish were randomly transferred to NGM prepared in step 1 with black goji berry polysaccharide concentrations of 0 and 100 μg / mL, respectively. In OP50 medium, NGM was inoculated with black goji berry polysaccharide at a concentration of 0. OP50 medium was used as a blank control group, and NGM was inoculated with black goji berry polysaccharide at a concentration of 100 μg / mL. OP50 medium was used as the experimental group. Each group had three replicates. After culturing at 20°C for 5 days, the body fat level of the nematodes on the 5th day of culture was measured. The oil red stained nematodes were placed on a glass slide and photographed under a stereomicroscope.

[0081] The measurement results are as follows Figure 2 As shown, the results indicate that the Oil Red O staining signal was more concentrated in the nematodes of the blank control group, and obvious dark red lipid deposition was visible in the intestinal region. The local staining distribution was uneven, suggesting that lipids tend to accumulate in a relatively concentrated manner in the body.

[0082] In contrast, the oil red O staining distribution in nematodes treated with black goji berry polysaccharides was more uniform overall, and the lipid staining in the intestines showed a relatively continuous and gradual distribution, with fewer localized high-intensity aggregation areas. This suggests that the lipid deposition pattern in nematodes was adjusted after black goji berry polysaccharide intervention. These results indicate that the effect of black goji berry polysaccharides is not only reflected in changes in total lipid content, but may also be manifested in optimizing the spatial distribution of lipids, i.e., reducing localized abnormal aggregation and resulting in a more balanced lipid distribution in the body.

[0083] Meanwhile, morphologically, the stained areas in the control group nematodes showed a relatively dense and concentrated lipid particle-like appearance, while the stained morphology in the treatment group was relatively finer and more uniform. This suggests that black goji berry polysaccharides may have a regulatory effect on lipid droplet morphology homeostasis, transforming lipid droplets from a relatively concentrated, coarse, or aggregated state to a more dispersed and uniform state. This improvement in lipid droplet morphology is beneficial to the dynamic mobilization and utilization of lipids, indicating that black goji berry polysaccharides may promote the rational distribution of lipids by regulating lipid deposition patterns, improving lipid droplet morphology, and promoting lipid metabolism.

[0084] 3. Regulatory effects of black wolfberry polysaccharides on lipid metabolism-related transcriptomics in nematodes The synchronized nematodes obtained in step 1 were randomly divided into two groups, and 200 nematodes / 10 cm petri dish were randomly transferred to NGM prepared in step 1 with black goji berry polysaccharide concentrations of 0 and 100 μg / mL, respectively. In OP50 medium, NGM was inoculated with black goji berry polysaccharide at a concentration of 0. OP50 medium was used as a blank control group, and NGM was inoculated with black goji berry polysaccharide at a concentration of 100 μg / mL. OP50 medium was used as the experimental group, with four biological replicates per group. The blank control group was numbered A1, A2, A3, and A4, and the experimental groups were numbered B1, B2, B3, and B4. After culturing at 20°C for 5 days, the nematodes were collected using M9 buffer, the supernatant was discarded, and the nematodes were washed 3-5 times with pre-cooled M9 buffer to remove residual OP50 and the drug attached to the nematode body and intestinal wall. Total RNA was then extracted from the nematodes. Transcriptome sequencing was performed after total RNA extraction, and differentially expressed genes were analyzed using GO, KEGG, and GSEA.

[0085] The specific measurement method is as follows: Total RNA was extracted from each sample, and its quality was assessed. Quality checks included RNA integrity, purity, and concentration, using methods such as agarose gel electrophoresis, Nanodrop, Qubit, and capillary electrophoresis. After passing quality checks, library construction was performed. An mRNA enrichment strategy was employed, using Oligo(dT) magnetic beads to enrich mRNA with polyA tails. This was followed by reverse transcription to synthesize cDNA, end repair, A-tail addition, adapter ligation, PCR amplification, and magnetic bead purification to obtain the sequencing library. After quantification and quality control, the libraries were sequenced using the Illumina high-throughput sequencing platform in PE150 mode.

[0086] Raw sequencing data were identified using Bcl2fastq software to obtain FASTQ format raw data. FastQC was then used for quality assessment, and Fastp was used to remove adapter sequences, low-quality bases, and excessively short reads to obtain clean data. The filtered clean data was aligned to the *C. elegans* reference genome using HISAT2 software to obtain alignment results. Further gene expression quantification was performed using HTSeq, calculating the read count and FPKM value of each gene in different samples. Based on the read count, differential expression analysis was performed using DESeq2 software to screen for differentially expressed genes that showed changes in expression after black goji berry polysaccharide treatment. Subsequently, GO enrichment analysis, KEGG enrichment analysis, and GSEA analysis were performed on the differentially expressed genes to clarify the functional pathways and molecular regulatory networks affected by black goji berry polysaccharide intervention.

[0087] The measurement results are as follows Figures 3-6 As shown in the results, compared with the control group, a large number of differentially expressed genes were screened from nematodes treated with black goji berry polysaccharide. Among them, the number of upregulated genes was significantly greater than that of downregulated genes, suggesting that black goji berry polysaccharide can significantly reshape the transcriptional state of nematodes. According to the default threshold, the treatment group had a total of 640 upregulated genes and 224 downregulated genes compared with the control group.

[0088] GSEA analysis showed that, compared with the control group, black goji berry polysaccharide treatment significantly enriched (upregulated) several pathways and their precursor genes closely related to antioxidation, detoxification, and metabolic homeostasis maintenance. These mainly included: the glycine, serine, and threonine metabolic pathway (GLYCINE, SERINE, AND THREONINE METABOLISM), with precursor genes including cth-2, cth-1, cbs-2, gldc-1, gcsh-1, gcsh-2, agxt-1, and alh-9; the sulfur metabolic pathway (SULFUR METABOLISM), with precursor genes including cysl-3, cysl-4, cysl-2, cysl-1, mpst-3, mpst-4, ethe-1, and sqrd-1; and the cysteine ​​and methionine metabolic pathway (CYSTEINE, AND METHIONINE). METABOLISM, whose leading genes include cysl-3, cysl-4, cth-2, cth-1, cbs-2, cdo-1, mpst-3, mpst-4, etc.; and the drug metabolism-other enzyme pathway (DRUGMETABOLISM-OTHER ENZYMES), whose leading genes include gst-10, gst-5, gst-2, gst-4, gst-1, gst-3, gst-6, gst-7, gst-36, gst-38, ugt-23, ugt-46, ugt-48, ugt-58, ugt-62, gsto-1, etc.

[0089] Further analysis based on the original differentially expressed gene results revealed that black goji berry polysaccharides have a clear regulatory trend on lipid metabolism, mainly manifested in the following aspects: (1) Promotes the mobilization of stored lipids Compared with the control group, the lipase genes lipl-1 and lipl-3, which are related to lipid droplet degradation and lipid release, were significantly upregulated after treatment with black goji berry polysaccharide. The enhanced expression of lipl-1 and lipl-3 suggests that black goji berry polysaccharide can promote the transformation of stored lipids from a static, accumulated state to a mobilized state, thereby facilitating lipid redistribution and utilization.

[0090] (2) Enhances fatty acid breakdown and β-oxidation Compared with the control group, fatty acid degradation-related genes acox-1.5, ech-9, and acdh-8 were significantly upregulated after treatment with black goji berry polysaccharides.

[0091] Among them: acox-1.5 participates in the initial step of fatty acid oxidation; ech-9 participates in the intermediate process of fatty acid β oxidation; and acdh-8 participates in the dehydrogenation process of acyl-CoA.

[0092] The synergistic upregulation of the above genes indicates that black goji berry polysaccharides not only promote the release of lipids from lipid droplets, but also promote the further entry of fatty acids into the oxidative decomposition and energy utilization pathways.

[0093] (3) Improve the environment related to lipid homeostasis oxidation and detoxification Compared with the control group, after treatment with black goji berry polysaccharides, glutathione transferase-related genes such as GST-2, GST-10, GST-5, and GST-1 were significantly upregulated, and sulfur-containing metabolism and transsulfur metabolism-related genes such as CYSL-3, CYSL-4, CTH-2, CTH-1, and CBS-2 were significantly enriched. This suggests that black goji berry polysaccharides can enhance the detoxification and antioxidant capacity of nematodes, improve the lipid peroxidation and membrane damage environment, and thus provide a more suitable intracellular environment for the normal mobilization, distribution, and utilization of lipids.

[0094] (4) Autophagy / nutrient sensing pathways are remodeled GSEA analysis showed that, compared with the control group, the nematodes treated with black goji berry polysaccharides exhibited changes in other autophagy pathways (AUTOPHAGY-OTHER), with significant upregulation of genes such as atg-2, atg-7, atg-9, atg-18, vps-34, vps-15, daf-15, and let-363. This suggests that black goji berry polysaccharides may also participate in the regulation of related lipid metabolism by modulating cell maintenance and nutrient sensing processes.

[0095] In summary, transcriptomics results indicate that black goji berry polysaccharides can promote the transformation of lipids in nematodes from "abnormal accumulation" to "orderly mobilization and efficient utilization" by upregulating lipid mobilization genes, fatty acid oxidation and decomposition genes, and antioxidant / detoxification-related genes, thereby providing a molecular basis for improving motility.

[0096] 4. Regulatory effects of black wolfberry polysaccharides on lipid metabolism-related metabolomics in aging nematodes The synchronized nematodes obtained in step 1 were randomly divided into two groups, and 200 nematodes / 10 cm petri dishes were randomly inoculated into NGM prepared in step 1 with black goji berry polysaccharide concentrations of 0 and 100 μg / mL, respectively. In OP50 medium, NGM was inoculated with black goji berry polysaccharide at a concentration of 0. OP50 medium was used as a blank control group, and NGM was inoculated with black goji berry polysaccharide at a concentration of 100 μg / mL. OP50 medium was used as the experimental group, with six biological replicates in each group. The blank control group was numbered A1, A2, A3, A4, A5, and A6, and the experimental group was numbered B1, B2, B3, B4, B5, and B6. After culturing at 20°C for 5 days, the cultured nematodes were collected using M9 buffer, the supernatant was discarded, and the nematodes were washed 3-5 times with pre-cooled M9 buffer to remove residual OP50 and the drug attached to the nematode body and intestinal wall, thus obtaining different test samples.

[0097] Metabolites were extracted from nematodes and subjected to metabolomics analysis. The analysis method is as follows: Approximately 25 mg of each sample was weighed and added to 0.5 mL of pre-cooled extraction buffer, a mixture of methanol, acetonitrile, and water in a volume ratio of 2:2:1, along with a mixed isotope-labeled metabolite internal standard. After adding steel beads, the sample was homogenized using a tissue homogenizer, followed by ultrasonic extraction (200 W for 5 min) under ice-water bath conditions to fully release metabolites from the nematodes. After extraction, the sample was allowed to stand at low temperature to precipitate proteins, centrifuged (10000 rpm for 3 min), and the supernatant was collected and filtered through a membrane to obtain the test sample. Simultaneously, equal volumes of supernatant from each sample were mixed as a QC control sample to monitor instrument stability and data quality. Metabolomics analysis was performed using an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-MS / MS) platform. The liquid chromatography section employed a Vanquish UHPLC system with a Waters ACQUITY UPLC BEH Amide column (2.1 mm × 50 mm, 1.7 μm). Mobile phase A consisted of an aqueous phase containing ammonium acetate and ammonia, while mobile phase B was acetonitrile. The sample pan temperature was 4 °C, and the injection volume was 2 μL. Mass spectrometry was performed using an Orbitrap Exploris 120 mass spectrometer for primary and secondary mass spectrometry data acquisition. Raw mass spectrometry data were converted to mzXML format using ProteoWizard software. Metabolite identification was performed using a self-built database, and peak extraction, alignment, normalization, and data preprocessing were conducted using R software. The treatment group and control group were compared to screen for differentially expressed metabolites. Further KEGG enrichment analysis, metabolite network analysis, and GSEA analysis were performed to clarify the affected metabolic pathways and key metabolite changes in nematodes after treatment with black goji berry polysaccharides.

[0098] The measurement results are as follows Figures 7-9As shown, the results indicated that treatment with black goji berry polysaccharides significantly altered the overall metabolic profile of nematodes, demonstrating its ability to remodel age-related metabolic states. GSEA analysis of metabolites revealed changes in multiple pathways closely related to lipid homeostasis after black goji berry polysaccharide treatment, with glycerophospholipid metabolism being the most important, showing positive enrichment. This suggests that membrane lipid and structural lipid metabolism was significantly reprogrammed in the treated group.

[0099] Further analysis of the differential metabolite classification results revealed that after treatment with black goji berry polysaccharides, lipid differential metabolites were significantly enriched (upregulated), mainly including: (1) Glycerophospholipids Compared with the control group, the black goji berry polysaccharide treatment group showed significant enrichment (upregulation) of various representative metabolites, including PC, PE, PG, LPC, and LPE, indicating an adjustment in the lipid environment related to cell membrane lipids and lipid droplets. This result suggests that black goji berry polysaccharides not only affect total lipid content but may also alter lipid packaging, distribution, and membrane stability.

[0100] (2) Carnitine and acylcarnitines Compared with the control group, the black goji berry polysaccharide treatment group showed a significant enrichment of representative differential metabolites, including Acetylcarnitine, Propionylcarnitine, Butyrylcarnitine, Hexanoylcarnitine, Octanoylcarnitine, Heptanoylcarnitine, Nonanoylcarnitine, Palmitoleylcarnitine, and Tetradecadienoylcarnitine.

[0101] Acylcarnitine is a key intermediate in the process of fatty acid entering the mitochondria for β-oxidation. Its significant changes indicate that after treatment with black goji berry polysaccharides, fatty acids are transformed from a stored state to an oxidative utilization state, which further supports the enhancement of fatty acid decomposition and energy utilization.

[0102] (3) Fatty acids and conjugates Compared with the control group, the black goji berry polysaccharide treatment group showed enrichment of various fatty acids and their derivatives, suggesting that the balance of fatty acid synthesis, transport and oxidation was readjusted.

[0103] (4) Glycerides and other lipids module (Glycerolipids, sphingolipids, etc.) Compared to the control group, changes were observed in glycerol and other lipid modules in the black goji berry polysaccharide treatment group, suggesting that black goji berry polysaccharide affects the dynamic balance of energy storage lipids, membrane lipids, and signaling lipids. In addition to lipid pathways, GSEA metabolomics also showed that, compared to the control group, the black goji berry polysaccharide treatment group exhibited negative enrichment (downregulation) of the pyrimidine metabolism pathway and negative enrichment (downregulation) of the nucleotide metabolism pathway. These results suggest that black goji berry polysaccharide can alleviate the high-load anabolic state of aging nematodes, shifting metabolic allocation from "synthesis and accumulation" to "maintenance and utilization," thereby creating a metabolic background more conducive to fat mobilization and maintenance of motor function.

[0104] Comprehensive metabolomics network analysis revealed that the differential metabolites induced by black goji berry polysaccharides were not isolated changes, but rather formed an overall regulatory relationship within the corresponding lipid metabolism network, indicating that they can systematically intervene in lipid metabolism at the pathway level.

[0105] In summary, metabolomics results indicate that black goji berry polysaccharides can improve the fat utilization level of nematodes by remodeling metabolic networks such as glycerophospholipids, acylcarnitines, and fatty acids, thereby promoting the transformation of lipids from an abnormal accumulation state to an orderly mobilization, distribution, and oxidative utilization state, and making fat distribution more uniform, thus achieving the dual purpose of fat reduction and body shaping.

[0106] 5. Effects of black goji berry polysaccharides on the motility of nematodes The synchronized nematodes obtained in step 1 were randomly divided into two groups, and 200 nematodes / 10 cm petri dish were randomly transferred to NGM prepared in step 1 with black goji berry polysaccharide concentrations of 0 and 100 μg / mL, respectively. Nematodes were cultured in OP50 medium at 20°C. Changes in their motility, including body swaying frequency, swimming speed, and swimming duration, were assessed on days 4, 5, and 6. NGM inoculated with black goji berry polysaccharide at a concentration of 0 was used. OP50 medium was used as a blank control group, and NGM was inoculated with black goji berry polysaccharide at a concentration of 100 μg / mL. OP50 medium was used as the experimental group; Among them, the nematodes at the 4th, 5th and 6th day of age can correspond to different functional states in higher animals during the transition from youth to middle and old age, and can be used to evaluate the effect of black goji berry polysaccharide on maintaining motor ability.

[0107] The results are as follows Figure 10 And as shown in Table 2: Table 2. Determination of nematode locomotion ability

[0108] Note: Swimming speed and swimming duration are based on the Control group on day 4 as 100% baseline.

[0109] The results show: Figure 10 (A) shows that, compared with the control group, the body wriggling frequency of adult nematodes in the black goji berry polysaccharide treatment group was higher on days 4, 5, and 6. The body wriggling frequency in the control group decreased from approximately 87 times / min to 73 times / min, while that in the treatment group decreased from approximately 98 times / min to 88 times / min. Compared with the control group, the treatment group showed an increase of approximately 13% on day 4, approximately 18% on day 5, and approximately 21% on day 6.

[0110] Figure 10 (B) shows that, in terms of swimming speed, the control group nematodes decreased from about 100% on day 4 to about 79% on day 6, while the black goji berry polysaccharide treatment group decreased from about 119% to about 96%; compared with the control group, the treatment group increased by about 19% on day 4, about 12% on day 5, and about 22% on day 6.

[0111] Figure 10 (C) shows that, in terms of swimming duration, the percentage of nematodes in the control group decreased from about 100% on day 4 to about 76% on day 6, while the percentage in the black goji berry polysaccharide treatment group decreased from about 100% to about 90%; compared with the control group, the treatment group increased by about 12% on day 5 and by about 18% on day 6.

[0112] The above results indicate that, at different life stages, the body undulation frequency, swimming speed, and swimming duration of nematodes in the blank control group all showed a decreasing trend, suggesting that the nematode's motility gradually weakens with the progression of life. In contrast, the black goji berry polysaccharide treatment group maintained a high level of motility at all detection time points. Combined with the aforementioned lipid metabolism-related experimental results and omics analysis, this demonstrates that black goji berry polysaccharides can maintain high motility at different life stages and mitigate the declining trend in activity frequency, motility efficiency, and sustained motility by regulating lipid metabolism and promoting the rational mobilization and utilization of lipids. These results suggest that black goji berry polysaccharides can improve the motility of individuals at different life stages by regulating lipid metabolism and can be used to prepare products that maintain motor function.

[0113] 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 improving fat accumulation, weight loss, body shaping, and / or improving individual athletic ability, 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.

4. The application according to claim 3, characterized in that, The 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.

5. 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.

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

7. A drug for improving fat accumulation, reducing fat, shaping body and / or improving individual athletic ability, 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 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.

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 liquids.

9. The drug according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable excipients.

10. The medicament according to claim 9, characterized in that, The 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.

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