Application of lycium ruthenicum polysaccharide extract in preparation of medicine for treating peripheral neuropathy

Abstract

The invention provides application of lycium ruthenicum polysaccharide extract in preparation of a medicine for treating peripheral neuropathy, and belongs to the technical field of lycium ruthenicum polysaccharide. The lycium ruthenicum polysaccharide disclosed by the invention comprises galacturonic acid, galactose, glucose, arabinose, rhamnose, xylose, mannose, glucuronic acid and glucosamine, wherein the molar ratio of the galacturonic acid to the galactose to the glucose to the arabinose to the rhamnose to the xylose to the mannose to the glucuronic acid to the glucosamine is 0.309 to 0.164 to 0.156 to 0.141 to 0.107 to 0.074 to 0.035 to 0.009 to 0.007. The lycium ruthenicum polysaccharide has good antioxidant activity, relieves hematopoietic toxicity and liver and kidney injury induced by OXA, resists mechanical hyperalgesia and cold stimulation, enhances an antioxidant defense mechanism to improve injury caused by oxidative stress, and improves neuromorphological characteristics of oxaliplatin-induced dorsal root ganglion.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of Lycium ruthenicum Murr polysaccharides, and particularly relates to application of Lycium ruthenicum Murr polysaccharide extract in preparation of a medicament for treating peripheral neuropathy. BACKGROUND

[0002] Oxaliplatin (OXA) as the third generation of platinum antitumor drugs and the component of FOLFOX chemotherapy regimen is used as the first-line treatment for advanced or metastatic colorectal cancer. However, the long-term neurotoxicity of platinum compounds exists, which is manifested as transient acute syndrome or dose-limiting cumulative sensory neuropathy. The incidence of oxaliplatin-induced peripheral neuropathy (OIPN) reaches 81.5-98%, and when the cumulative dose reaches 540 mg / m 2 The incidence is higher. At present, the clinical treatment means for peripheral neuropathy is limited to symptomatic drugs for relieving neuropathic pain, and no drug intervention has been found to affect the potential pathological mechanism of chemotherapy-induced peripheral neuropathy. Therefore, it is of great value to explore a new treatment strategy which can effectively cope with oxaliplatin-induced neuropathic pain without interfering with its anticancer properties.

[0003] It is crucial to study the mechanisms behind acute and chronic OIPN for future prevention and treatment, but these mechanisms are still unclear. Oxaliplatin induces neuropathic pain through multiple pathways, including sodium ion channel regulation, calcium ion homeostasis changes, reactive oxygen species generation, increased apoptosis, inflammatory response, and signal pathway disruption. Among them, oxidative stress is a key factor leading to neuropathic pain. There is no prevention or treatment drug for OIPN at present, and only symptomatic treatment methods such as antidepressants and anticonvulsants are used, but these methods mostly have poor effects or are not confirmed in clinical trials. In addition, a recent review found insufficient evidence for the effectiveness of central nervous system drugs in treating OIPN. At present, there is a lack of effective prevention strategies, and the evidence for drug treatment of diagnosed OIPN is also very limited, and only duloxetine has been confirmed to significantly relieve symptoms. Therefore, it is urgent to develop new treatment strategies.

[0004] Lycium ruthenicum Murr. (LR), as one of the plants of medicine and food, belongs to Solanaceae and is distributed in arid and saline-alkali regions of northwest China. The fruits of Lycium ruthenicum Murr. are commonly known as "grey side of the horse" and "black side of the horse", which are rich in polysaccharides, anthocyanins and other nutrients. Polysaccharides (PSs) are the main bioactive components of Lycium ruthenicum Murr., which have various health benefits such as anti-tumor, anti-oxidation, anti-inflammatory, and can enhance immune function and regulate intestinal flora. Lycium ruthenicum polysaccharides (LRPs) have various biological activities, but there is no relevant report on the treatment of oxaliplatin-induced peripheral neuropathy by LRPs. SUMMARY

[0005] Therefore, one of the purposes of the present application is to provide a Lycium ruthenicum polysaccharide.

[0006] The second purpose of the present application is to provide a preparation method of the Lycium ruthenicum polysaccharide.

[0007] The third purpose of the present application is to provide an application of the Lycium ruthenicum polysaccharide in the preparation of a drug for treating and / or preventing peripheral neuropathy.

[0008] The fourth purpose of the present application is to provide a drug for treating oxaliplatin-induced peripheral neuropathy.

[0009] In order to achieve the above-mentioned purposes of the present application, the present application provides the following technical solutions: A Lycium ruthenicum polysaccharide, which comprises galacturonic acid, galactose, glucose, arabinose, rhamnose, xylose, mannose, glucuronic acid and glucosamine, and the molar ratio of the galacturonic acid, galactose, glucose, arabinose, rhamnose, xylose, mannose, glucuronic acid and glucosamine is 0.309, 0.164, 0.156, 0.141, 0.107, 0.074, 0.035, 0.009 and 0.007.

[0010] Preferably, the particle size of the Lycium ruthenicum polysaccharide is 357.5±11.7 nm, and the weight average molecular weight is 5.686 kDa.

[0011] The present application also provides a preparation method of the Lycium ruthenicum polysaccharide, which comprises the following steps: The Lycium ruthenicum is crushed and mixed with water, and then boiled water extraction is performed for 1.5-3.5 h, which is repeated for 2-3 times; the extraction solutions are combined and concentrated to obtain a concentrated solution; Anhydrous ethanol is added to the concentrated solution to make the volume fraction of ethanol in the mixed solution 20%, and the mixture is placed at 4°C overnight to collect the precipitate.

[0012] Preferably, the Lycium ruthenicum is crushed and mixed with water at a solid-liquid ratio of 1 g:8-12 mL.

[0013] The application also provides application of the Lycium ruthenicum Murr polysaccharide or the preparation method in preparation of a medicine for treating and / or preventing peripheral neuropathy.

[0014] Preferably, the peripheral neuropathy is oxaliplatin-induced peripheral neuropathy.

[0015] Preferably, the Lycium ruthenicum Murr polysaccharide can relieve mechanical allodynia and cold / hot allodynia.

[0016] Preferably, the Lycium ruthenicum Murr polysaccharide relieves oxidative stress of oxaliplatin-induced peripheral neuropathy.

[0017] Preferably, the Lycium ruthenicum Murr polysaccharide improves the nerve morphological characteristics of dorsal root ganglion.

[0018] The application also provides a medicine for treating oxaliplatin-induced peripheral neuropathy, wherein the medicine comprises the Lycium ruthenicum Murr polysaccharide.

[0019] Compared with the prior art, the application has the following beneficial effects: The application provides a Lycium ruthenicum Murr polysaccharide, which has good antioxidant activity, can relieve hematopoietic toxicity and liver and kidney damage induced by OXA, can resist abnormal sensations such as mechanical allodynia and cold stimulation, can enhance the antioxidant defense mechanism to improve damage caused by oxidative stress, and can improve the nerve morphological characteristics of dorsal root ganglion (DRGs) induced by oxaliplatin. The Lycium ruthenicum Murr polysaccharide provided by the application can be applied to research and development of a medicine for treating oxaliplatin-induced peripheral neuropathy. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 To determine the molecular weight and purity of LRP-20, LRP-50 and LRP-80 by high-performance gel permeation chromatography-differential refractive detection; Figure 2 To determine the molar ratio (A) of monosaccharides and ion chromatogram (B) of LRP-20, LRP-50 and LRP-80; monosaccharide standards show peaks at 2.0 minutes (sodium hydroxide) and 40.5 minutes (sodium acetate); wherein the monosaccharides include fucose (Fuc), galactosamine (GalN), rhamnose (Rha), arabinose (Ara), glucosamine (GlcN), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (GalA), guluronic acid (GulA), glucuronic acid (GlcA), and mannuronic acid (ManA); Figure 3UV spectra (A) and Fourier transform infrared spectra (B) of LRP-20, LRP-50 and LRP-80; Figure 4 SEM images of LRP-20, LRP-50 and LRP-80 at different magnifications (500x, 2000x and 10000x); Figure 5 X-ray diffraction patterns, thermogravimetric (TG) and differential scanning calorimetry (DSC) analysis curves of LRPS-20, LRP-50 and LRP-80; wherein, A is the X-ray diffraction patterns of LRPS-20, LRP-50 and LRP-80; B is the thermogravimetric and differential scanning calorimetry analysis curves of LRPS-20; C is the thermogravimetric and differential scanning calorimetry analysis curves of LRPS-50; D is the thermogravimetric and differential scanning calorimetry analysis curves of LRPS-80; Figure 6 Antioxidant activities of the three LRPs with Vc; wherein, A is the DPPH free radical scavenging capacity; B is the hydroxyl radical scavenging rate determination; C is the reducing capacity determination; Figure 7 Effects of LRP-20 on OIPN rats; wherein, A is the execution process of using rat model for drug administration and behavior test; B is the time effect curve of LRP-20 on rat body weight; C is the behavior test: von Frey experiment, cold plate experiment and hot plate experiment are used to determine the mechanical withdrawal threshold, cold allodynia and hot withdrawal threshold, respectively; D is the effect of LRP-20 on the oxidative stress level of OIPN rats: by determining the malondialdehyde (MDA) level as a lipid peroxidation index, evaluating the superoxide dismutase (SOD) activity to reflect the antioxidant enzyme function, and detecting the glutathione (GSH) level to measure the antioxidant capacity; E is the observation of dorsal root ganglion (DRG) section after hematoxylin-eosin (H&E) and Nissl staining under 40x optical microscope (scale = 25 μm); F is the evaluation of the number of surviving neurons by analyzing the nucleolus area of Nissl-stained cells; G is the labeling of rat hind paw epidermal nerve fibers (IENF) using PGP9.5 staining (green) and DAPI (blue), and displaying a representative image (scale = 50 μm); H is the quantitative analysis of IENF density; I is the detection of serum nerve growth factor (NGF) level in each group; #P<0.05, ##P<0.01, ###P<0.001 indicate comparison with the control group, P<0.05, P<0.01, P<0.001 indicate comparison with the OIPN group (n=6 rats per group); Figure 8Safety evaluation of LRP-20 intervention on OIPN rats; wherein, A is to evaluate the influence of LRP-20 on the hematopoietic system of OIPN rats by blood routine test, and the number of white blood cells (WBC), red blood cells (RBC), platelets (PLT), neutrophils (Neu), monocytes (Mon), mean corpuscular volume (MCV), and the percentage of lymphocytes (Lym) and neutrophils are statistically analyzed to evaluate the hematotoxicity; B is the influence of LRP-20 on the liver function of OIPN rats, and the liver function is evaluated by detecting the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC) and triglyceride (TG) in serum; C is the influence of LRP-20 on the kidney function of OIPN rats, and the kidney function is evaluated by detecting the levels of albumin (ALB), creatinine (CRE) and blood urea nitrogen (BUN) in serum; D is the pathological influence of LRP-20 on the heart, liver, spleen, lung, kidney and brain tissue (scale = 100 μm); P<0.05, ##P<0.01, ###P<0.001 represent comparison with the control group, P<0.05, P<0.01, P<0.001 represent comparison with the OIPN group (n=6 rats per group). DETAILED DESCRIPTION

[0021] The present application provides a lycium ruthenicum polysaccharide, which comprises galacturonic acid, galactose, glucose, arabinose, rhamnose, xylose, mannose, glucuronic acid and glucosamine, and the molar ratio of the galacturonic acid, galactose, glucose, arabinose, rhamnose, xylose, mannose, glucuronic acid and glucosamine is 0.309, 0.164, 0.156, 0.141, 0.107, 0.074, 0.035, 0.009 and 0.007. The particle size of the lycium ruthenicum polysaccharide is 357.5±11.7 nm, and the weight average molecular weight is 5.686 kDa.

[0022] The present application also provides a preparation method of the lycium ruthenicum polysaccharide, which comprises the following steps: after the lycium ruthenicum is crushed, the lycium ruthenicum is mixed with water, boiled water extraction is carried out for 1.5-3.5 h, and the operation is repeated for 2-3 times; the extraction liquid is concentrated to obtain a concentrated liquid; anhydrous ethanol is added to the concentrated liquid, so that the volume fraction of ethanol in the mixed liquid is 20%, and the mixed liquid is placed at 4°C overnight to collect the precipitate.

[0023] In the preparation method, the Lycium ruthenicum Murr is preferably dried Lycium ruthenicum Murr; the Lycium ruthenicum Murr is preferably crushed and then passed through a 60-mesh sieve; the crushed Lycium ruthenicum Murr is mixed with water in a ratio of 1 g: 8-12 mL, which can be 1 g: 8 mL, 1 g: 9 mL, 1 g: 10 mL, 1 g: 11 mL or 1 g: 12 mL in some examples. The boiling water extraction time can be 1.5 h, 2 h, 2.5 h, 3 h or 3.5 h in some examples. After the extraction solutions are combined, they are concentrated to 1 / 10 of the total volume. The collected precipitate is preferably subjected to decolorization of the Lycium ruthenicum Murr extract using AB-8 macroporous adsorption resin, the solution after decolorization is subjected to deproteinization, and then freeze-dried into a powder.

[0024] The application also provides use of the Lycium ruthenicum Murr polysaccharide or the preparation method in preparation of a drug for treating and / or preventing peripheral neuropathy. The peripheral neuropathy preferably induced by oxaliplatin.

[0025] In the use, the Lycium ruthenicum Murr polysaccharide can relieve mechanical hyperalgesia and cold and heat hyperalgesia, relieve oxidative stress of oxaliplatin-induced peripheral neuropathy, and improve the nerve morphological characteristics of the dorsal root ganglion.

[0026] The application also provides a drug for treating oxaliplatin-induced peripheral neuropathy, which comprises the Lycium ruthenicum Murr polysaccharide.

[0027] The technical solutions provided by the application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of the application.

[0028] Example 1 A Lycium ruthenicum Murr polysaccharide is prepared by the following method: 1200 g of Lycium ruthenicum Murr (dried product) crushed to pass through a 60-mesh sieve is placed in a container, 10 times the volume of water is added, boiling water extraction is performed for 2 hours, the process is repeated twice, the two extraction solutions are combined, impurities are removed by filtration, and the solution is concentrated to 1 / 10 of the total volume.

[0029] Anhydrous ethanol is added to the concentrated solution to make the volume fraction of ethanol in the mixed solution 20%, and the mixture is placed at 4°C overnight. The precipitate LRP-20 is obtained by centrifugation at 6000 r / min for 5 min and stored in a 4°C refrigerator.

[0030] After the precipitate LRP-20 is dissolved in pure water, AB-8 macroporous adsorption resin is used for decolorization, and the eluate is concentrated to obtain a concentrated decolorized solution.

[0031] The concentrated decolorized solution was added with 1 / 5 volume of Sevage reagent (chloroform: n-butanol = 4:1) and stirred rapidly by a magnetic stirrer for 10 min. The solution was centrifuged in a centrifuge tube at 6000 r / 10 min to remove the protein. The solution was observed to be layered after centrifugation, and the white layer in the middle was the protein. The upper polysaccharide solution was sucked out, and the above steps were repeated several times until there was no precipitate, and the deproteinized solution was collected.

[0032] The deproteinized solution was observed, and if the color was clear and light yellow, it was concentrated to dark yellow. The dialysis bag was cut to about 30 cm and soaked in pure water in a beaker. The beaker was placed in a small pot containing water and boiled for 10 min to achieve activation. After activation, one end was clamped with a clamp, and the other end was added with the deproteinized solution using a dropper, and the solution was less than 1 / 2 of the dialysis bag. After the solution was added, the other end was clamped with a clamp, and placed in a container containing pure water, and dialyzed for 48 h, and the pure water was changed every 8 h. The solution in the dialysis bag was directly poured out after dialysis, and the dialysis was completed. The dialyzed solution was freeze-dried into a powder to obtain the purified LRP-20.

[0033] Comparative Example 1 A Lycium ruthenicum Murr polysaccharide was prepared by the following method: 1200 g of Lycium ruthenicum Murr (dry product) was ground to pass through a 60-mesh sieve and placed in a container, 10 times the volume of water was added, and boiled water was extracted for 2 hours, repeated 2 times, and the two extraction solutions were combined and filtered to remove impurities, and concentrated to 1 / 10 of the total volume.

[0034] Anhydrous ethanol was added to the concentrated solution to make the volume fraction of ethanol in the mixed solution 20%, and it was placed at 4°C overnight. Centrifuged at 6000 r / min for 5 min, and the supernatant was taken, and evaporated at 50°C to remove ethanol, then an equal volume of anhydrous ethanol was added to make the volume fraction of ethanol in the mixed solution 50%, and stirred uniformly, and placed at 4°C overnight. Centrifuged at 6000 r / min for 5 min to obtain the precipitate LRP-50, which was stored in a 4°C refrigerator.

[0035] The precipitate LRP-50 was dissolved in pure water, and AB-8 macroporous adsorption resin was used for decolorization, and the eluate was concentrated to obtain the concentrated decolorized solution.

[0036] The concentrated decolorized solution was added with 1 / 5 volume of Sevage reagent (chloroform: n-butanol = 4:1) and stirred rapidly by a magnetic stirrer for 10 min. The solution was centrifuged in a centrifuge tube at 6000 r / 10 min to remove the protein. The solution was observed to be layered after centrifugation, and the white layer in the middle was the protein. The upper polysaccharide solution was sucked out, and the above steps were repeated several times until there was no precipitate, and the deproteinized solution was collected.

[0037] The deproteinized solution was observed. If the color was clear and light yellow, it was concentrated to dark yellow. The dialysis bag was cut about 30 cm and soaked in pure water in a beaker. The beaker was placed in a small pot filled with water and boiled for 10 min to achieve activation. After activation, one end was clamped with a clamp and the other end was added with the deproteinized solution using a dropper. The added solution was less than 1 / 2 of the dialysis bag. After the addition of the solution was completed, the other end was clamped with a clamp and placed in a container containing pure water for dialysis for 48 h, and the pure water was changed every 8 h. After dialysis, the solution in the dialysis bag was directly poured out to complete the dialysis. The dialyzed solution was freeze-dried into a powder to obtain purified LRP-50.

[0038] Comparative Example 2 A Lycium ruthenicum Murr. polysaccharide was prepared by the following method: Lycium ruthenicum Murr. (dry product) 1200 g (ground to pass through a 60 mesh sieve) was placed in a container, 10 times the volume of water was added, and boiling water extraction was performed for 2 hours, repeated 2 times, and the two extraction solutions were combined and filtered to remove impurities, and concentrated to 1 / 10 of the total volume.

[0039] Anhydrous ethanol was added to the concentrated solution to make the volume fraction of ethanol in the mixed solution 20%, and it was placed at 4°C overnight. Centrifugation was performed at 6000 r / min for 5 min, and the supernatant was taken. Evaporation was performed at 50°C to remove ethanol, and then an equal volume of anhydrous ethanol was added to make the volume fraction of ethanol in the mixed solution 50%, and it was stirred uniformly and placed at 4°C overnight. Centrifugation was performed at 6000 r / min for 5 min, and the supernatant was taken. Evaporation was performed at 50°C to remove ethanol, and then 4 times the volume of anhydrous ethanol was added to make the volume fraction of ethanol in the mixed solution 80%, and it was stirred uniformly and placed at 4°C overnight. Centrifugation was performed at 6000 r / min for 5 min to obtain the precipitate LRP-80, which was stored in a 4°C refrigerator.

[0040] After the precipitate LRP-80 was dissolved in pure water, AB-8 macroporous adsorption resin was used for decolorization, and the eluate was concentrated to obtain a concentrated decolorized solution.

[0041] Sevage reagent (chloroform:n-butanol=4:1) was added to the concentrated decolorized solution at 1 / 5 volume, and the solution was rapidly stirred with a magnetic stirrer for 10 min. The solution was centrifuged in a centrifuge tube at 6000 r / 10 min to remove protein. The solution was observed after centrifugation, and the middle white layer was protein. The upper polysaccharide solution was sucked out, and the above steps were repeated several times until there was no precipitate, and the deproteinized solution was collected.

[0042] The deproteinized solution was observed. If the color was clear and light yellow, it was concentrated to dark yellow. The dialysis bag was cut about 30 cm and soaked in pure water in a beaker. The beaker was placed in a small pot filled with water and boiled for 10 min to achieve activation. After activation, one end was first clamped with a clamp, and the other end was added with a dropper to add the deproteinized solution, which was less than 1 / 2 of the dialysis bag. After adding the solution, the other end was clamped with a clamp, and placed in a container containing pure water, and dialyzed for 48 h, with the pure water changed every 8 h. After dialysis, the solution in the dialysis bag was directly poured out to complete the dialysis. The dialyzed solution was freeze-dried into a powder to obtain purified LRP-80.

[0043] Example 2 Characterization of the three Lycium ruthenicum polysaccharides obtained in Example 1 and Comparative Examples 1-2.

[0044] 1. Yield, chemical composition, molecular weight, particle size, PDI and zeta potential of LRP: 1.1 As shown in Table 1, the yield of LRP decreases in the following order: LRP-50 > LRP-80 > LRP-20. Overall, the polysaccharide yield increases with increasing ethanol volume fraction. Therefore, the yield of LRP-50 is higher than that of LRP-20. However, the yield of LRP-50 is higher than that of LRP-80 due to the precipitation of low molecular weight polysaccharides in high concentration ethanol with lower polarity. The reason for the lower yield of LRP-80 is that there is less low molecular weight polysaccharide. Studies have shown that higher ethanol concentration can promote the separation of polysaccharides and lipid-soluble impurities, while reducing the solubility of polysaccharides and the extraction rate. The total sugar content of LRP-50 (54.225 ± 4.713%) is higher than that of ASLP-80.

[0045] 1.2 Determination of molecular weight: The peak molecular weight (Mp), weight average molecular weight (Mw) and number average molecular weight (Mn) of the three LRP obtained in Example 1 and Comparative Examples 1-2 were evaluated using high performance gel permeation chromatography (HPGPC) equipped with RID-20A detector and BRT105-103-101 series gel column. The experimental parameters were: injection volume 25 μL, column temperature 40℃, flow rate 0.7 mL / min, mobile phase 0.05 M NaCl, LRP concentration 5 mg / mL.

[0046] The molecular weight of polysaccharides has a significant impact on their physical properties (solubility, viscosity, thermal stability, etc.) and biological activities (immunity, antioxidant, anti-inflammatory, etc.). Therefore, the molecular weight of polysaccharides is crucial for their application in functional foods, drugs and materials. The peak molecular weight (Mp), weight average molecular weight (Mw) and number average molecular weight (Mn) of LRP were determined by HPGPC-RI, and the standard curves were y = -0.2034x + 11.911 (R 2=0.9948), y=-0.2052x+11.971 (R 2 =0.9952) and y=-0.2048x+11.949 (R = ... 2 =0.9948). The molecular weights of LRP-20, LRP-50, and LRP-80 are 5.686 kDa, 7.140 kDa, and 9.753 kDa, respectively. Figure 1 (See Table 1). LRPs have a wide molecular weight distribution, typically ranging from 17.0 to 2650 kDa. The molecular weight difference of LRPs depends on the specific polysaccharide type present within them. The black goji berry polysaccharide extracted in this invention has a relatively small molecular weight.

[0047] 1.3 Particle size and zeta potential of LRPs: In polysaccharide research, the interaction of charged particles significantly affects the stability of these systems, as well as their sensory and structural properties. Zeta potential is a key parameter characterizing the electrical interactions or dispersion systems within polysaccharide matrices. In food science, zeta potential is primarily used to characterize the surface charge and stability of mixed systems. Generally, the higher the absolute value of the zeta potential, the stronger the stability of the polysaccharide system. Systems with an absolute zeta potential below 10 mV are generally considered unstable, while values ​​in the 10–20 mV range indicate relative stability. As shown in Table 1, the zeta potentials of LRP-20, LRP-50, and LRP-80 solutions are -18.6 ± 2.2 mV, -16.4 ± 1.0 mV, and -14.6 ± 0.5 mV, respectively, corresponding to particle sizes of 357.5 ± 11.7 nm, 748.3 ± 14.3 nm, and 637.8 ± 12.9 nm. The negative charge value indicates that the polysaccharide component is primarily acidic, while the larger potential values ​​indicate that the three different LRP solutions exhibit relative stability.

[0048] The zeta potential of polysaccharides is a key parameter for assessing their aggregation tendency. Regardless of whether they are negatively or positively charged, particles with higher absolute zeta potentials exhibit stronger electrostatic repulsion, thus reducing aggregation and increasing particle stability. Therefore, LRP molecules exhibit extremely weak aggregation and maintain a small average particle size, stemming from the large absolute zeta potential in LRP solutions. This significant electrostatic repulsion between particles effectively inhibits aggregation. As indicated by the zeta potential, this repulsive force is sufficient to ensure the stability of the LRP system.

[0049] Table 1. Yield, chemical composition, molecular weight, particle size, PDI and ζ-potential of different black goji berry polysaccharides

[0050] 2. Determination of monosaccharide composition: Accurately weigh 5 mg sample into ampoule, add 3M TFA 2 mL, hydrolyze at 120 °C for 3 h. Accurately pipette the acid hydrolysis solution into a tube and dry under nitrogen, add 5 mL water and vortex to mix. Pipette 100 µL of 50%, 80% fractions into 900 µL DI water, pipette 200 µL of 2% fraction into 800 µL DI water, centrifuge at 12000 rpm for 5 min. Take supernatant for IC analysis.

[0051] The results of monosaccharide composition analysis of Lycium ruthenicum polysaccharides provided by Example 1, Comparative Example 1 and Comparative Example 2 are shown in Table 1. Figure 2 LRP-20 was mainly composed of galacturonic acid (GalA), galactose (Gal), glucose (Glc), arabinose (Ara), rhamnose (Rha), xylose (Xyl), mannose (Man), glucuronic acid (GlcA), and glucosamine (GlcN) with molar ratios of 0.309, 0.164, 0.156, 0.141, 0.107, 0.074, 0.035, 0.009, and 0.007, respectively. Similarly, LRP-50 mainly contained GalA, Gal, Ara, Xyl, Rha, Glc, GlcA, and GlcN with molar ratios of 0.329, 0.255, 0.146, 0.108, 0.078, 0.064, 0.017, and 0.003, respectively. In contrast, LRP-80 was mainly composed of Gal, Ara, Glc, Rha, GalA, Man, Xyl, GlcA, and GlcN with ratios of 0.377, 0.296, 0.141, 0.090, 0.025, 0.024, 0.020, 0.019, and 0.008, respectively. These findings indicated that LRP-20 and LRP-80 had similar polysaccharide compositions, while LRP-50 lacked mannose. Both LRP-20 and LRP-50 exhibited higher GalA content, while LRP-80 had lower GalA content, which might affect its biological activity. This was attributed to the elevated levels of GalA being associated with enhanced ABTS free radical scavenging, nitric oxide free radical scavenging, and iron-reducing activity. Furthermore, LRP-80 contained significantly higher concentrations of Ara and Gal compared to LRP-20 and LRP-50, which might contribute to its enhanced anti-inflammatory activity. For example, the major monosaccharides in LRP-20 included five types: GalA, Gal, Glc, Ara, and Rha; while LRP-50 mainly contained four: GalA, Gal, Ara, and Xyl; and LRP-80 was mainly composed of three monosaccharides: Gal, Ara, and Glc. The composition and ratios of these monosaccharides significantly influenced the biological activity of the polysaccharides.

[0052] 3. Ultraviolet (UV) spectral analysis: The black goji berry polysaccharide samples provided in Example 1, Comparative Example 1, and Comparative Example 2 were prepared into 1 mg / mL solutions and analyzed using a UV-Vis spectrophotometer in the wavelength range of 200–400 nm.

[0053] The results are as follows Figure 3 As shown in Figure A, no absorption peaks were observed at wavelengths of 260 nm and 280 nm, indicating that the protein and nucleic acid content was negligible or completely absent. This observation result is highly consistent with the data from the total protein content analysis.

[0054] 4. Infrared spectral analysis of polysaccharides: Accurately weigh 2 mg of sample and 200 mg of potassium bromide, compress them into tablets, and use potassium bromide powder tablets as blank control. Scan and record the results using a Fourier transform infrared spectrometer (FT-IR650).

[0055] like Figure 3 As shown in Figure B, the infrared spectra of LRP-20, LRP-50, and LRP-80 exhibit typical polysaccharide characteristic absorption peaks. (3399 cm⁻¹) -1 3394 cm -1 and 3365 cm -1 The broad peak at 2927 cm⁻¹ corresponds to the stretching vibration of the OH bond. Furthermore, LRP-20 shows a peak at 2927 cm⁻¹. -1 and 2854 cm -1 The peak of LRP-50 is at 2921 cm⁻¹. -1 and 2852 cm -1 The peak at 2931 cm⁻¹, and LRP-80 at 2931 cm⁻¹ -1 The peaks at this location all characterize CH bond stretching vibrations. The C=O stretching vibration peaks of LRP-20 and LRP-50 are located at 1741 cm⁻¹. -1 Asymmetric stretching vibrations lead to 1619 cm -1 A peak appears at 1650 cm⁻¹. LRP-80 peaks at 1650 cm⁻¹. -1 The peak at 1550 cm⁻¹ is attributed to the C=O stretching vibration. -1 The absorption peak at that point may originate from the C=O asymmetric stretching vibration.

[0056] LRP-20 at 1415 cm -1 1147 cm -1 and 1081 cm -1 The peak of LRP-50 is at 1419 cm⁻¹. -1 and 1141 cm -1 The peak at 1405 cm⁻¹, and LRP-80 at 1405 cm⁻¹ -1 and 1074 cm -1The peak at 1317 cm -1 and 1240 cm -1 for LRP-20, at 1315 cm -1 , 1243 cm -1 , 1072 cm -1 and 1020 cm -1 for LRP-50, and at 1405 cm -1 and 1074 cm -1 for LRP-80 can be attributed to C-O stretching vibration. The peaks at 1069.7 cm -1 and 1042.3 cm -1 for LRP-20 are related to C-O-H or C-O stretching vibration in C-O-C (pyranoside) structure.

[0057] The peak at 889 cm -1 for LRP-20 indicates C-H out-of-plane bending vibration due to anomeric effect of pyranose ring, and the peak at 777 cm -1 shows D-pyranose ring symmetric stretching vibration, suggesting the presence of pyranose ring structure in the molecule. The peaks at 1419 cm -1 and 1141 cm -1 for LRP-50 also originate from C-O stretching vibration. The peaks at 1315 cm -1 , 1243 cm -1 , 1072 cm -1 and 1020 cm -1 for LRP-80 belong to O-H out-of-plane bending vibration. The absorption peaks at 1405 cm -1 and 1074 cm -1 can be attributed to C-O stretching vibration, while the peaks at 1243 cm -1 and 1033 cm -1 may be related to O-H out-of-plane bending vibration. The absorption peak at 896 cm -1 for LRP-80 may originate from C-H deformation vibration due to β-anomeric effect of pyranose ring.

[0058] 5. Scanning Electron Microscope (SEM) observation analysis: About 5 mg of dried sample was adhered to a conductive carbon film containing double-sided adhesive, and placed in the sample chamber of an ion sputtering instrument for about 40 s of gold spraying. After the sample was taken out, it was placed in the observation chamber of the scanning electron microscope, and the acceleration voltage was 5 KV for observation.

[0059] The surface morphology of polysaccharides was observed by scanning electron microscopy (SEM). The morphology of polysaccharides under electron microscopy reflects their inherent macroscopic structural characteristics, which differ with changes in their physicochemical properties. Figure 4 SEM images of three LRPs at different magnifications (x500, x2000, and x10000) are shown. The scanning electron micrographs of LRP-20, LRP-50, and LRP-80 after 500x, 2000x, and 10000x magnification, respectively. LRP-20 exhibits an irregularly curved lamellar structure with visible hillock-like protrusions on the surface. LRP-50 and LRP-80 exhibit a continuous lamellar structure with visible fragments on the surface. As the ethanol concentration increases, the lamellar structure becomes more pronounced, and the surface appears smoother. This may be because LRP is purified by ethanol precipitation, and changes in ethanol concentration can change the structure of the polysaccharide sample.

[0060] 6. X-ray diffraction analysis: X-ray diffraction patterns of samples were measured using an X-ray diffractometer with a copper target, Kα radiation, a working voltage of 45 kilovolts, a current of 40 milliamperes, and a scanning speed of 1.2 degrees per minute in the range of 5-90 degrees (2θ).

[0061] X-ray diffraction (XRD) is a key technology for characterizing the crystal structure of polymers, which can be used to further analyze the structure of polysaccharides. As shown in A of Figure 5 LRP-20 and LRP-50 have two weak signal peaks in the diffraction angle 2θ range of 5°-90°, mainly at 19.79° and 45.98°, indicating that they are mainly amorphous and contain a small amount of crystalline form. Similarly, LRP-80 has a signal peak in the diffraction angle 2θ range of 5°-90°, mainly at 45.98°, which is believed to be amorphous. By comparing the X-ray patterns of the three different levels of LRPs, it was found that they have similar X-ray diffraction curves, peak positions, and peak intensities, indicating that their aggregate structures are similar.

[0062] 7. Thermogravimetric analysis: Precisely weigh 3 mg of sample and place it in a crucible. After pressing, use a thermogravimetric analyzer (Netzsch STA 449C, USA) under the protection of nitrogen gas, with a temperature increase from 25°C to 800°C at a rate of 10°C / min.

[0063] Thermal stability is a key attribute in the study of the physical properties of polysaccharides. The present invention uses thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) to evaluate the thermal stability of LRPs. DSC observes the thermal transition of polysaccharide structure deformation or crystalline polysaccharide melting process, and becomes an important tool for exploring the interaction between macromolecules. As shown in Figure 5As shown in Figs. B-D, the weight loss curves of the LRPs can be divided into three distinct stages. For LRP-20, the second stage of mass loss occurred between 263.3 °C and 390.9 °C, resulting in a 26.1% weight reduction, with a residual mass of 27.20% at 805.5 °C. The second stage of mass loss for LRP-50 occurred between 237.3 °C and 365.3 °C, with a 26.1% weight reduction, and a residual mass of 24.7% at 805.5 °C. The second stage for LRP-80 occurred between 257.9 °C and 351.9 °C, with a 21.68% weight reduction, and a residual mass of 32.94% at 805.5 °C. These results indicate that all three LRP variants exhibit significant thermal stability. Of particular note, LRP-80 exhibits superior thermal stability, which can be attributed to differences in molecular conformation, monosaccharide composition, and structural features of its polysaccharide components.

[0064] Example 3 Evaluation of antioxidant capacity in vitro of different Lycium ruthenicum polysaccharides.

[0065] The antioxidant capacity of LRP-20 provided in Example 1, LRP-50 provided in Comparative Example 1, and LRP-80 provided in Comparative Example 2 was evaluated using the following experiment.

[0066] DPPH radical scavenging test procedure: 50 mg of LRP-20, LRP-50, and LRP-80 samples were each extracted with 800 μL of nitrogen radical extract in a constant temperature water bath at 40 °C for 60 minutes. The mixture was centrifuged at 10,000 rpm for 10 minutes in a refrigerated centrifuge, and the supernatant was collected for analysis.

[0067] Four experimental groups (n = 6) were set up in parallel: the blank control tube contained 50 μL of nitrogen radical extract, 450 μL of absolute ethanol, and DPPH solution; the sample test tube contained 50 μL of sample solution, 225 μL of absolute ethanol, and 225 μL of DPPH solution; the sample control tube contained 50 μL of sample solution and 450 μL of absolute ethanol; and the positive control contained 50 μL of vitamin C solution (concentration of 60 μg / mL), 225 μL of ethanol, and 225 μL of DPPH solution. After vortex mixing, the mixture was incubated at room temperature for 30 minutes in the dark, and the absorbance was measured at 517 nm using a Multiskan GO microplate reader (Thermo Scientific, USA). The formula for calculating the percentage of DPPH scavenging activity is: scavenging rate% = A0- (A1-A2) / A0 x 100%, A0 is the absorbance value of the blank control; A1 is the absorbance value of the test sample; and A2 is the absorbance value of the control sample.

[0068] Hydroxyl radical scavenging experiment procedure: Take 3 samples, each sample is LRP-20, LRP-50 and LRP-80 with a weight of 50 mg, and each sample is extracted with 1 mL of 80% ethanol. Ultrasonic extraction is carried out at 50°C for 30 minutes, and the mixture is shaken every 5 minutes. Then the sample is centrifuged at room temperature at 12,000 rpm for 10 minutes, and the supernatant is collected for analysis.

[0069] The detection is carried out using a kit provided by Shanghai Yuan Ye Biotechnology Co., Ltd., and the reagents 1, 2 and 3 are provided in the kit. Four experimental groups (n=6) are set in parallel, the blank control detection sample contains reagents 1, 2 and 3 without adding sample; the sample test detection sample is LRP-20, LRP-50 and LRP-80 respectively adding reagents 1, 2 and 3; the sample control detection sample is LRP-20, LRP-50 and LRP-80 respectively adding reagents 1 and 2; the positive control detection sample is vitamin C solution (concentration of 60 μg / mL), and the volume is made up with distilled water.

[0070] According to the kit instructions, add the sample and reagent in turn, mix well, incubate at 37°C for 20 minutes, then centrifuge at room temperature at 8,000 rpm for 5 minutes. Transfer 200 μL of clear liquid to a 96-well plate, containing 6 parallel samples per group. The Multiskan GO microplate reader (Thermo Scientific, USA) is used to measure the absorbance value at 510 nm. The hydroxyl radical scavenging rate calculation formula is: scavenging rate%=A0-(A1-A2) / A0x100%, A0 is the absorbance value of the blank control; A1 is the absorbance value of the test sample; A2 is the absorbance value of the control sample.

[0071] Reducing power: In this determination, the total antioxidant capacity is quantified in terms of ferrous ion concentration (Fe 2+ Concentration, expressed in mM). For the blank sample, add 30 μL of distilled water and 264 μL of FRAP solution to the test tube. Similarly, add 30 μL of distilled water and 264 μL of FRAP solution to the sample tube. Both groups of test tubes are incubated in a water bath at 37°C for 30 minutes. The sample relative reducing power calculation formula is as follows: A x =A1-A0, y=1.7473x+0.1014 (R 2 =0.998), A0 is the absorbance value of the blank control; A1 is the absorbance value of the test sample.

[0072] Anticancer drugs trigger cancer cell apoptosis by generating reactive oxygen species (ROS), but these reactive oxygen species also damage normal cells, leading to side effects such as cardiotoxicity, nephrotoxicity, and neurotoxicity. Since the discovery of chemotherapy-induced peripheral neuropathy (CIPN), its clear pathological mechanism has been rarely studied. Recent studies have confirmed that oxidative stress and neuroinflammation play an important role in this process. Understanding oxidative agent-induced neuronal damage may provide ideas for developing alternative therapies based on natural antioxidants or synthetic free radical scavengers. Antioxidants with multiple effects on CIPN-related pathways are expected to improve treatment outcomes. The present invention uses three oxidation evaluation experiments to detect the antioxidant properties of LRP-20, LRP-50 and LRP-80 provided by Lycium ruthenicum polysaccharides of Example 1, Comparative Example 1 and Comparative Example 2.

[0073] DPPH assay evaluated the scavenging activity of these compounds by measuring the reduction of DPPH by a proton donor, with vitamin C as a positive control. As shown in A of FIG. 1, the DPPH scavenging ability of LRP-20 and LRP-50 increased with increasing concentration. At a concentration of 2.5 mg / mL, the DPPH scavenging ability of LRP-80 decreased, possibly due to inhibition at high concentrations. Overall, LRPS showed excellent scavenging ability, with LRP-20 being the strongest, followed by LRP-50, and then LRP-80. Figure 6

[0074] As shown in B of FIG. 1, LRP-20 showed stronger hydroxyl radical scavenging activity than LRP-50 and LRP-80 in the concentration range of 1-5 mg / mL, indicating a significant effect on neutralizing hydroxyl radicals. FRAP assay measures the reducing power of antioxidants in samples through a redox-related colorimetric reaction and is commonly used to analyze individual and overall antioxidant activity in plant extracts. Figure 6

[0075] As shown in C of FIG. 1, the antioxidant capacity of LRP-20, LRP-50 and LRP-80 increased with increasing concentration. LRP-80 showed the highest total antioxidant capacity, while the radical scavenging activity of LRP-20 was stronger than that of LRP-50. This is because radical scavenging is only one of several antioxidant mechanisms, other mechanisms include chain initiation prevention, metal ion binding, peroxide decomposition, hydrogen extraction prevention, and reducing power. The results show that LRPs have antioxidant properties, with LRP-20 showing the strongest DPPH and hydroxyl radical scavenging ability and moderate reducing power. Figure 6 Example 4

[0076] Study on prevention and treatment of Lycium ruthenicum polysaccharide against oxaliplatin-induced peripheral neuropathy.

[0077] ​​​1. Eight-week-old male Sprague-Dawley rats were selected from the Animal Experimental Center of Xinjiang Medical University and were raised in a specific pathogen-free level experimental animal facility. The experimental environment was constant at a temperature of 22±2°C and a humidity of 45±10%, with a 12-hour light-dark cycle starting at 8 am every day, and food was freely available. All rats underwent a one-week environmental adaptation period before the experiment. The animal feeding and experimental operation of this study were approved by the University Experimental Animal Ethics Committee (Approval No.: K202507-65).

[0078] 2. Establishment of an oxaliplatin-induced OIPN model in rats and dosing regimen: After completing the baseline behavioral tests, the rats were randomly divided into six groups (n=6 per group): OIPN group: The OIPN model was established by intraperitoneal injection of oxaliplatin (4 mg / kg dissolved in 5% glucose solution) on specific dates (injected on days 1, 2, 8, 9, 15, 16, 22, and 23, respectively) and daily gavage with sterilized water; Control group: Intraperitoneal injection of 0.15 mL of 5% glucose solution was performed according to the dates of oxaliplatin injection within 28 days, and daily gavage with sterilized water was performed; OIPN+LRP-20 low-dose group (30 mg / kg): From the first oxaliplatin injection, LRP-20 (30 mg / kg) in Example 1 was administered daily for 4 weeks, denoted as OIPN+LRP-20-L; OIPN+LRP-20 high-dose group (60 mg / kg): From the first oxaliplatin injection, LRP-20 (60 mg / kg) in Example 1 was administered daily for 4 weeks, denoted as OIPN+LRP-20-H.

[0079] Mechanical pain threshold, cold hyperalgesia, and thermal sensory function were evaluated at baseline, during treatment for 4 weeks, and at the end of treatment. At the time of sacrifice, all rats in each group were anesthetized with Zoletil-50, and whole blood, dorsal root ganglion (DRG), and hind paw skin samples were collected.

[0080] After successfully establishing the OIPN rat model (A in Figure 7 , the in vivo safety of LRP-20 was verified (B in Figure 8 ).

[0081] Routine hematology analysis (C in Figure 8A) showed that, compared with the control group, the number of white blood cells (WBC) and neutrophils (Neu) was significantly decreased (P<0.05), and the percentage of lymphocytes (Lym) was also reduced. Notably, LRP-20-H administration improved OXA-induced Neu reduction (P<0.05). These results indicate that in OIPN model rats, LRP-20 not only did not produce toxic effects on the hematopoietic system, but also alleviated OXA-induced hematopoietic toxicity. Liver function ( Figure 8 B) and kidney function ( Figure 8 The C) test showed that when OXA was used as a chemotherapy drug, it caused liver and kidney damage in OIPN model rats. LRP-20 not only did not aggravate these damages, but also showed a protective effect. Hematoxylin-eosin (HE) staining histopathological examination showed that LRP-20 did not cause pathological changes in the heart, liver, spleen, lungs, and kidneys. Figure 8 (D in the text). We assessed the effect of LRP-20 on body weight in OIPN rats ( Figure 7 (B) Compared with the control group, the body weight of OIPN rats was significantly reduced (P<0.001). Compared with the OIPN group, LRP-20 had no significant effect on body weight (P>0.05).

[0082] 3. Behavioral tests: Von Frey mechanical pain threshold test: Oxaliplatin-induced mechanical pain threshold was determined using the Von Frey filament test on days 0, 7, 14, 21, and 28. Before the experiment, animals were acclimatized for 15 minutes in an acrylic box with a grid bottom. Using Von Frey filaments of varying sizes (0.008 to 300 grams), the skin in the middle of the sole of each hind paw was gently touched at 3-4 second intervals. The minimum pressure value that elicited a claw withdrawal response was recorded. A positive response was defined as rapid claw withdrawal (involuntary movement) occurring in 5 out of 10 tests.

[0083] The cold plate test is used to assess cold hyperalgesia: This test uses a cold plate apparatus to assess the response of mice to non-noxious cold stimuli. Mice are placed on a cold plate set at 4±1°C, with their movement restricted to a cylindrical plexiglass chamber 10 cm in diameter and 15 cm high. A foot pedal-controlled timer records the latency from when the mouse is placed on the cold plate until the onset of pain-related behaviors such as hind paw licking. The maximum allowed time for paw lifting or licking is 30 seconds.

[0084] Hot plate test for thermal hyperalgesia: This test assesses the thermal pain threshold by placing rats on a metal surface at 50–55°C and recording the time it takes for them to exhibit noxious behavior. Thermal hyperalgesia assessment was performed at 52°C with a 30-second cutoff time to prevent tissue damage.

[0085] An OIPN rat model was established by intraperitoneal injection of oxaliplatin (OXA), successfully mimicking clinical symptoms. Rats received a cumulative dose of 32 mg / kg over 4 weeks, with regular behavioral tests performed to validate the model and assess the efficacy of LRP-20. Figure 7 (C) Mechanical pain thresholds were measured using Von Frey fibers on days 0, 7, 14, 21, and 28. By day 7, the pain threshold in the hind paw of the OIPN group was significantly reduced to 6.74 ± 2.08 g (P < 0.001), showing a significant difference from the control group. The high-dose LRP-20 administration group significantly alleviated OXA-induced hyperalgesia (P < 0.01), with pain thresholds maintained at 11.09 ± 1.89 g and 9.38 ± 1.25 g, respectively, and this protective effect persisted throughout the experimental period.

[0086] The sensitivity of rats to temperature stimulation was measured using a hot / cold plate apparatus. The latency of paw withdrawal under cold stimulation showed no significant difference among the groups within 60 seconds. By day 7, the latency in the OIPN group was significantly shortened to 20.03±6.26 seconds, which was statistically significant compared to the control group (P<0.001). The latency in the LRP-20-L and LRP-20-H groups was prolonged to 30.36±0.93 seconds and 35.04±4.69 seconds, respectively (P<0.01). On days 21 and 28, the latency in the LRP-20-H group slowly prolonged (P<0.001), while the latency in the OIPN group continued to decrease, reaching 6.47±1.30 seconds (P<0.001).

[0087] The results of heat-induced paw withdrawal latency showed that no response was observed in any rat group within 30 seconds, thus establishing baseline levels. No significant changes were observed in the control group. However, the oxaliplatin-induced peripheral neuropathy (OIPN) group showed a significantly shortened latency on day 7 (P<0.001), which continued to decrease to 8.22±1.35 seconds on day 28. In contrast, the LRP-20-H group exhibited a higher threshold (17.90±1.43 seconds, P<0.05), and its latency was significantly longer than that of the OIPN group by day 28 (P<0.001). LRP-20-H effectively alleviated oxaliplatin-induced thermosensitive reactions, indicating that this drug can combat symptoms of mechanical hyperalgesia and sensory abnormalities such as cold stimulation.

[0088] 4. LRP-20 alleviated oxidative stress in OIPN rats: Oxidative stress is a key pathogenic factor in oxaliplatin-induced peripheral neuropathy (OIPN), and reducing oxidative stress in vivo can bring multiple benefits. The main biomarkers for assessing oxidative stress include glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD). Notably, previous studies have reported that in a mouse model of oxaliplatin-induced neuropathic pain, SOD and GSH activities were significantly reduced after oxaliplatin (OXA) administration, while MDA levels were significantly increased.

[0089] In this embodiment, serum GSH, MDA, and SOD levels were quantitatively detected, and the results are as follows: Figure 7 As shown in D in the figure. MDA level detection showed that the OIPN group had a significantly higher MDA level than the control group (P<0.001); and the low / high dose LRP-20 group had a lower MDA level than the OIPN group (P<0.05). SOD and GSH level analysis showed that the OIPN group had a significantly lower SOD and GSH level than the control group (P<0.001); while the low / high dose LRP-20 group had a significantly higher MDA level than the OIPN group (P<0.01). These results suggest that LRP-20 may improve oxidative stress-induced damage by enhancing antioxidant defense mechanisms.

[0090] 5. Histopathological analysis of the dorsal root ganglion (DRG): DRGs were fixed in 10% paraformaldehyde solution, washed, dehydrated with ethanol, rinsed with xylene, and then embedded in paraffin. Sections were 5 μm thick, stained with H&E, and observed under an optical microscope. After dewaxing and rehydration, Nissl staining was performed for 1 min with 0.5% cresol violet. The stained sections were analyzed under the same microscope, and the number of surviving neurons in the DRGs was counted.

[0091] Structural changes in the dorsal root ganglion (DRG) were observed using hematoxylin-eosin (H&E) staining and Nissl staining. Figure 7 In oxaliplatin-induced peripheral neuropathy (OIPN) rats, H&E staining showed pyknosis of neuronal nuclei, enlarged intercellular spaces, and numerous vacuolar formation, in stark contrast to the control group. Administration of LRP-20 (30, 60 mg / kg) dose-dependently alleviated oxaliplatin (OXA)-induced histopathological changes, manifested as an increase in the number of normal neurons and a reduction in intercellular spaces. Nissl staining showed severe neuronal damage in the DRG tissue of the OIPN group, characterized by significant nucleolar atrophy, cytoplasmic vacuolation, and a reduction in Nissl bodies. The low-dose and high-dose LRP-20 groups showed milder neuronal damage. Analysis of the nucleolar area, a neuronal activity indicator (… Figure 7 The results showed that the OIPN group had a significantly lower dose than the control group (P<0.001), while the high-dose LRP-20 group had a significantly higher dose than the OIPN group (P<0.001).

[0092] 6. Skin biopsy analysis of intraepidermal nerve fibers (IENF): After euthanasia of each animal, a 3 mm borehole was drilled in one hind paw to obtain a live tissue sample, which was fixed in paraformaldehyde-lysine-periodate solution and sectioned to a thickness of 20 μm. Three discontinuous sections were selected from each paw pad and immunostained with rabbit polyclonal PGP9.5 antibody. Under a 40x magnified optical microscope, PGP-positive fibers penetrating the dermal-epidermal junction were counted. The linear density of IENF / mm was calculated after measuring the epidermal length.

[0093] Intradermal nerve fibers (IENFs) of the dorsal root ganglion or trigeminal nerve are crucial for sensing and transmitting pain signals. This study investigated whether oxaliplatin (OXA) reduces the density of IENFs in the distal skin of the foot, a common site of damage in chemotherapy-induced neuropathy. Figure 7 G in the image shows the results of PGP9.5 immunostaining on the hind paw, while Figure 7 The H in the figure represents the statistical analysis of intradermal nerve fiber (IENF) density in each group. Compared with the control group, the IENFs in the oxaliplatin-induced peripheral neuropathy (OIPN) group were significantly reduced (P<0.01). However, LRP-20 administration inhibited this oxaliplatin-induced nerve fiber retraction phenomenon.

[0094] 7. Detection of Nerve Growth Factor (NGF) in Serum: Whole blood was allowed to stand at room temperature for 1 hour, then centrifuged at 3000 rpm for 10 minutes to collect serum. Serum NGF levels were detected by ELISA using a rat NGF ELISA kit according to the manufacturer's instructions. All assays were performed at least three times.

[0095] Nerve growth factor (NGF) is mainly secreted by neurons or glial cells, and its level is correlated with the severity of oxaliplatin-induced peripheral neuropathy (OIPN). Decreased NGF expression and transport function are key mechanisms in the development of chemotherapy-induced peripheral neuropathy (CIPN). Figure 7 The results showed that, compared with the control group, serum NGF levels were significantly lower in the oxaliplatin (OXA) treatment group (5.98±1.78 vs. 11.01±2.74 pg / ml; P<0.05). However, after intervention with low-dose (LRP-20-L) and high-dose (LRP-20-H) LRP-20, NGF levels increased to 11.65±1.47 pg / ml and 12.19±2.08 pg / ml, respectively, significantly higher than those in the OIPN model group (P<0.01). This suggests that LRP-20 may promote neuronal repair by increasing serum NGF levels.

[0096] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A Lycium ruthenicum polysaccharide, characterized in that, The Lycium ruthenicum Murr. polysaccharides comprise galacturonic acid, galactose, glucose, arabinose, rhamnose, xylose, mannose, glucuronic acid and glucosamine, and the molar ratio of the galacturonic acid, galactose, glucose, arabinose, rhamnose, xylose, mannose, glucuronic acid and glucosamine is 0.309, 0.164, 0.156, 0.141, 0.107, 0.074, 0.035, 0.009 and 0.

007.

2. The Lycium ruthenicum polysaccharide according to claim 1, characterized in that, The particle size of the Lycium ruthenicum Murr. polysaccharides is 357.5±11.7 nm, and the weight average molecular weight is 5.686 kDa.

3. The method for preparing Lycium ruthenicum polysaccharide according to claim 1 or 2, characterized in that, The method comprises the following steps: The Lycium ruthenicum Murr. is crushed and mixed with water, and boiled water extraction is performed for 1.5-3.5 h, and the extraction is repeated for 2-3 times; the extraction liquid is concentrated to obtain a concentrated liquid; Anhydrous ethanol is added to the concentrated liquid to make the volume fraction of ethanol in the mixed liquid 20%, and the mixture is placed at 4 ℃ overnight to collect the precipitate.

4. The production method according to claim 3, characterized by, The Lycium ruthenicum Murr. is crushed and mixed with water at a solid-liquid ratio of 1 g:8-12 mL.

5. The use of the Lycium ruthenicum Murr. polysaccharides of claim 1 or 2 or the preparation method of claim 3 or 4 in the preparation of a drug for treating and / or preventing peripheral neuropathy.

6. Use according to claim 5, characterized in that, The peripheral neuropathy is oxaliplatin-induced peripheral neuropathy.

7. Use according to claim 5, characterized in that, The Lycium ruthenicum Murr. polysaccharides can relieve mechanical hyperalgesia and cold / heat hyperalgesia.

8. Use according to claim 5, characterized in that, The Lycium ruthenicum Murr. polysaccharides relieve the oxidative stress response of oxaliplatin-induced peripheral neuropathy.

9. Use according to claim 5, characterized in that, The Lycium ruthenicum Murr. polysaccharides improve the nerve morphological characteristics of the dorsal root ganglion.

10. A medicament for treating oxaliplatin-induced peripheral neuropathy, characterized in that, The drug comprises the Lycium ruthenicum Murr. polysaccharides of claim 1 or 2.

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