Application of dryopteris fragrans polysaccharide in treatment of chronic lead poisoning

Abstract

The invention discloses application of dryopteris fragrans polysaccharide in treatment of chronic lead poisoning, and belongs to the technical field of biology. The invention aims at solving the technical blank of application of dryopteris fragrans polysaccharide in chronic lead poisoning and solving the technical problems of complicated operation, low polysaccharide retention rate and low polysaccharide purity of a polysaccharide extraction and purification method in the prior art. According to the method for extracting and purifying the dryopteris fragrans polysaccharide, pigments and residual protein in the dryopteris fragrans polysaccharide can be effectively removed, the effect of reducing the deproteinization treatment frequency is achieved, the decolorization rate reaches 78.76%, the polysaccharide retention rate is high, and the method can be used for industrially producing the dryopteris fragrans polysaccharide. The dryopteris fragrans acidic polysaccharide obtained through the extraction and purification method can be applied to treatment of chronic lead poisoning, so that the purpose of reducing growth delay, liver and kidney injury and intestinal villus lesion caused by chronic lead poisoning is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the application of Pteris vittata polysaccharide in the treatment of chronic lead poisoning. Background Technology

[0002] Chronic lead poisoning is a systemic disease caused by long-term, continuous exposure to lead or lead compounds, leading to the cumulative absorption of lead in the body. Symptoms include neurological disorders, digestive system diseases, and hematopoietic disorders. Studies have shown a strong link between the accumulation of heavy metals in the body and abnormal protein expression, as well as the pathogenesis of neurodegenerative diseases. Lead is a commonly used non-ferrous metal in industry and is also widely present in living and natural environments. Sources of environmental pollution include mining, smelting, manufacturing, and recycling activities, as well as metallic lead used in various products. Once accumulated in the environment, lead is difficult to degrade. Lead can enter the body through various routes, including the respiratory and digestive tracts. The body stores lead in teeth, bones, liver, kidneys, and brain, and it can accumulate over time. In conclusion, multi-organ dysfunction caused by chronic lead exposure has become a significant public health problem.

[0003] Fragrant scales ( Dryopteris fragrans (L.) Schott is a wild perennial fern belonging to the genus *Dryopteris* in the family Dryopterisceae. Its fresh leaves are fragrant when crushed, hence the name "fragrant-leaf dryopteris." Unlike most ferns that thrive in humid and hot environments, fragrant dryopteris grows in extremely unique conditions, such as on slippery slopes, lava crevices, and volcanic magma fissures formed after volcanic eruptions.

[0004] Pteris vittata polysaccharides possess various pharmacological activities, including anti-inflammatory and antioxidant effects; however, there are relatively few research reports on the isolation and purification of Pteris vittata polysaccharides. This is because plant polysaccharides are biological macromolecules with complex compositions and structures. Furthermore, the crude polysaccharides obtained from Pteris vittata contain a significant amount of pigments and proteins, which can interfere with the structural identification and bioactivity studies of the polysaccharides. Currently, the Sevage method is commonly used to remove proteins from polysaccharides, but this method is risky and cumbersome. A single treatment can only remove a small amount of protein, and repeated treatments result in substantial polysaccharide loss. Among commonly used pigment removal methods, activated carbon has weak adsorption for pigments such as flavonoids and polyphenols that readily bind to polysaccharides, and the carbon powder is difficult to remove. Hydrogen peroxide decolorization involves harsh reaction conditions that can easily damage the polysaccharides.

[0005] There are no reports on the therapeutic effects of Pteris vittata polysaccharide on chronic lead poisoning; therefore, those skilled in the art are eager to develop an easy-to-operate and highly efficient method for extracting and purifying Pteris vittata polysaccharide, and hope to provide a new approach for the treatment of chronic lead poisoning through Pteris vittata polysaccharide. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of *Dryopteris crassirhizoma* polysaccharide in the treatment of chronic lead poisoning.

[0007] One objective of this invention is to provide the application of acidic polysaccharide from Dryopteris crassirhizoma in the preparation of a drug for treating chronic lead poisoning, wherein the drug has at least one of the following functions (1)-(3): (1) Reduce growth retardation caused by chronic lead poisoning; (2) Reduce liver and kidney damage caused by chronic lead poisoning; (3) Reduce intestinal villus lesions caused by chronic lead poisoning.

[0008] In a preferred embodiment of the present invention, the extraction and purification method of the *Dryopteris crassirhizoma* polysaccharide is as follows: S1: Spread out the stems and leaves of the Dryopteris crassirhizoma, air dry them, then crush them and pass them through a 40-mesh sieve to obtain Dryopteris crassirhizoma powder; S2: The dried powder of *Dryopteris crassirhizoma* obtained in S1 was mixed with petroleum ether and soaked for 24 h for degreasing treatment. The degreasing treatment was repeated twice. The solid was then filtered to obtain solids. The solids were dried and mixed with water. The mixture was then placed in a water bath at 80-90℃ for 3 h for extraction treatment. The solids were filtered and the filtrate was collected. The extraction treatment was repeated three times. S3: After centrifuging the filtrate obtained in S2, concentrate it under reduced pressure, add anhydrous ethanol, mix well, and precipitate it at 4℃ for 24 h; centrifuge it at 4000 r / min for 10-15 min, add distilled water until the precipitate is completely dissolved, and concentrate it under reduced pressure for 1-2 h until the obtained sugar solution slightly clings to the wall. S4: Add papain to the sugar solution obtained in S3, stir well, and then enzymatically hydrolyze in a water bath at 55℃ for 4-6 hours to obtain the enzymatic hydrolysate; after cooling to room temperature, add Sevage reagent in proportion, shake vigorously for 10 min to mix well, and then centrifuge at 4000 r / min for 15-20 min. Take the supernatant after centrifugation and concentrate under reduced pressure to obtain the enzymatically hydrolyzed sugar solution. S5: Add 60-100 mesh polyamide to the sugar solution after enzymatic hydrolysis in S4, decolorize at 30-70℃ for 20-100 min, filter, dialyze, and freeze dry to obtain crude polysaccharide of Dryopteris crassirhizoma. S6: Mix the crude polysaccharide of Dryopteris crassirhizoma obtained in S5 with deionized water to obtain a crude polysaccharide solution of Dryopteris crassirhizoma. After passing through a 0.45 μm aqueous filter membrane, the solution is loaded onto the column. The loading volume is ≤ 3% of the column volume. The eluent after ultrasonic degassing is subjected to gradient elution. The eluents of different gradients are collected and dialyzed. After lyophilization, the polysaccharide component of Dryopteris crassirhizoma is obtained.

[0009] In a preferred embodiment of the present invention, the mass ratio of dried fern powder to petroleum ether in S2 is 1 g: 3 mL.

[0010] In a preferred embodiment of the present invention, the mixing mass ratio of solids to water in S2 is 1 g: 15 mL, and the extraction treatment temperature is 90°C.

[0011] In a preferred embodiment of the present invention, the volume ratio of the filtrate to anhydrous ethanol in S3 is 1:3.

[0012] In a preferred embodiment of the present invention, the amount of papain added in S4 is 8‰ by mass, and the enzymatic hydrolysis time is 6 h.

[0013] In a preferred embodiment of the present invention, the mixing volume ratio of the enzymatic hydrolysate to the Sevage reagent in S4 is 5:1, wherein the Sevage reagent is obtained by mixing dichloromethane and n-butanol in a 4:1 ratio.

[0014] In a preferred embodiment of the present invention, the mass ratio of the enzymatically hydrolyzed sugar solution to the polyamide in S5 is 1 mL: 40 mg, and the decolorization treatment is performed at a temperature of 50°C for a time of 60 min.

[0015] In a preferred embodiment of the present invention, the dialysis bag used in S5 has a molecular weight cutoff of 3500 Da, and the dialysis time is 3 days; wherein, first, running water is used for dialysis for 2 days, and then deionized water is used for dialysis for 1 day.

[0016] In a preferred embodiment of the present invention, the concentration of the crude polysaccharide solution of Dryopteris crassirhizoma in S6 is 25 mg / ml, and the eluent is a degassed NaCl solution with concentrations of 0, 0.1, 0.2, and 0.4 mol / L, respectively.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides the application of *Dryopteris crassirhizoma* polysaccharide in chronic lead poisoning, and to solve the problems of cumbersome operation, low conversion rate and purity in the existing methods for extracting and purifying polysaccharides, a method for extracting and purifying *Dryopteris crassirhizoma* polysaccharide is provided; the method involves pretreatment of *Dryopteris crassirhizoma* by drying, pulverizing, defatting and soaking, combined with ethanol precipitation and centrifugation to remove insoluble impurities; removing residual ethanol by vacuum concentration, followed by enzymatic hydrolysis using papain and removal of protein impurities using Sevage reagent; decolorization by adding polyamide in a specific ratio, and further purification of crude polysaccharide (DFP) by ion exchange chromatography to obtain polysaccharide components.

[0018] The extraction and purification method for Pteris vittata polysaccharides provided by this invention can effectively remove pigments, achieving a decolorization rate of 78.76% and a high polysaccharide retention rate. Furthermore, this method can further remove residual proteins after the Sevage deproteinization process, thereby reducing the number of deproteinization treatments. It can be used for the industrial production of Pteris vittata polysaccharides.

[0019] The polysaccharide obtained by the extraction and purification method of this invention can be used to treat chronic lead poisoning. Experiments have shown that the acidic polysaccharide component (DPF-2) obtained by the extraction and purification method provided by this invention can alleviate growth retardation in mice caused by chronic lead poisoning and alleviate liver and kidney damage in mice caused by chronic lead poisoning. It also has a certain restorative effect on the pathological changes in the small intestinal villi of the jejunum segment, such as villi shortening, breakage, and shedding. Furthermore, it can effectively reduce lead storage in liver and kidney tissues and increase the small intestinal tight junction protein gene. ZO-1, Occludin mRNA expression levels.

[0020] In summary, the polysaccharide obtained by the extraction and purification method of this invention can be used to treat chronic lead poisoning, with the aim of reducing growth retardation, liver and kidney damage and intestinal villus lesions caused by chronic lead poisoning. It has the advantages of fewer side effects and higher safety. Attached Figure Description

[0021] Figure 1 The standard curve for determining the purity of polysaccharides from Dryopteris crassirhizoma by the phenol-sulfuric acid method is shown; the vertical axis represents absorbance, and the horizontal axis represents glucose concentration. Figure 2 The graph shows the detection of decolorization rate and polysaccharide retention rate; A represents the change in polyamide dosage; B represents the change in decolorization treatment time; C represents the change in decolorization treatment temperature; the left vertical axis represents decolorization rate, the right vertical axis represents polysaccharide retention rate, the horizontal axis represents polyamide usage, the processing time represents decolorization time, and the processing temperature represents decolorization temperature. Figure 3 The images show the appearance color before and after the decolorization treatment; Dosage is the amount of polyamide used, Time is the time, and Temperature is the temperature. Figure 4 This is a full-wavelength UV scan; the vertical axis represents absorbance, the horizontal axis represents wavelength, Before Treatment represents before decolorization treatment, and After Treatment represents after decolorization treatment. Figure 5 The ion exchange chromatography elution curve of crude polysaccharide DFP from Dryopteris crassirhizoma is shown; the vertical axis represents absorbance and the horizontal axis represents the number of elution tubes. Figure 6 The graph shows the changes in body weight of mice in different experimental groups; the vertical axis represents body weight, and the horizontal axis represents 1 week, 2 weeks, 3 weeks, and 4 weeks of treatment, respectively. Figure 7 Liver index plots for mice in different experimental groups; the vertical axis represents the liver index. Figure 8 This is a graph showing the renal index of mice in different experimental groups; the vertical axis represents the renal index. Figure 9 HE staining images of the jejunum of mice in different experimental groups; Figure 10 The graphs show the lead content of mice in different experimental groups; A is the liver lead content graph; B is the kidney lead content graph; the vertical axis represents liver lead levels; the vertical axis represents kidney lead levels. Figure 11 Genes of tight junction protein in the small intestine of mice in different experimental groups ZO-1 The graph shows the relative expression levels of ZO-1 mRNA; the vertical axis represents the relative expression levels of ZO-1 mRNA. ZO-1 The relative mRNA expression level of the gene; Figure 12 Genes of tight junction protein in the small intestine of mice in different experimental groups Occludin The graph shows the relative expression levels of Occludin mRNA; the vertical axis represents the relative expression levels of Occludin mRNA. Occludin The relative mRNA expression level of the gene. Detailed Implementation

[0022] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0024] The reagents and experimental procedures involved in the following examples; 1. Determination of the purity of polysaccharides from Dryopteris crassirhizoma by phenol-sulfuric acid method (1) Reagent preparation Accurately weigh 600 mg of phenol using an analytical balance, and dissolve it in deionized water to a 10 mL volumetric flask to prepare a 6% phenol solution. Accurately weigh 10 mg of anhydrous glucose using an analytical balance, and dissolve it in deionized water to a 10 mL volumetric flask to prepare a 1 mg / mL glucose standard solution stock solution.

[0025] (2) Draw the standard curve Accurately transfer 1000, 800, 600, 400, and 200 μL of the stock solution and dilute to 10 mL with deionized water in volumetric flasks to prepare glucose standard solutions of 0.1, 0.08, 0.06, 0.04, and 0.02 mg / mL, respectively. Accurately transfer 2 mL of the prepared glucose standard solution into a glass test tube, replacing the blank tube with deionized water. Then, add 1 mL of 6% phenol solution and 5 mL of concentrated sulfuric acid, shake rapidly to mix, and let stand for 15 min. Transfer the reaction solution into a quartz cuvette and measure the absorbance at 490 nm using a spectrophotometer. Perform the measurements in triplicate. Finally, plot a standard curve with glucose concentration on the x-axis and absorbance on the y-axis, as shown below. Figure 1 As shown.

[0026] The regression equation obtained is: y = 7.7243x - 0.0087, R² = 0.9986.

[0027] (3) Determination of the purity of polysaccharides from Dryopteris crassirhizoma. Accurately weigh 10 mg of Pteris vittata polysaccharide and dilute to 10 mL in a volumetric flask with deionized water to prepare a 1 mg / mL polysaccharide stock solution. Transfer 1 mL of the stock solution and dilute to 10 mL in a volumetric flask with deionized water to obtain a 0.1 mg / mL polysaccharide solution. Accurately transfer 2 mL of the polysaccharide solution into a glass test tube, add 1 mL of 6% phenol solution, shake to mix, then add 5 mL of concentrated sulfuric acid and shake rapidly to mix. After standing for 15 min, transfer the reaction solution into a quartz cuvette and measure the absorbance at 490 nm using a spectrophotometer. Perform three parallel measurements. Substitute the absorbance values ​​into the regression equation to obtain the polysaccharide concentration C1. The polysaccharide purity is:

[0028] 2. Decolorization rate The decolorization rate was calculated by performing a full-wavelength ultraviolet scan on the polysaccharide before decolorization, with the wavelength of 220 nm (where the maximum absorbance value is located) as the detection wavelength:

[0029] In the formula, A1 is the absorbance value of the polysaccharide solution before decolorization at 220 nm, and A2 is the absorbance value of the polysaccharide solution after decolorization at 220 nm.

[0030] 3. Polysaccharide retention rate

[0031] In the formula, C3 is the polysaccharide concentration before decolorization, and C4 is the polysaccharide concentration after decolorization.

[0032] 4. Comprehensive weighted scoring method To determine the optimal conditions, a comprehensive assessment of decolorization rate and polysaccharide retention rate is necessary. A weighted scoring method is employed, using decolorization rate and polysaccharide retention rate as scoring indicators, and the maximum value of each indicator is used as a reference to normalize the data. The weighting coefficients for decolorization rate and polysaccharide retention rate are each 0.5.

[0033]

[0034] In the formula, X is the polysaccharide decolorization rate, X Max Y represents the maximum polysaccharide decolorization rate; Y represents the polysaccharide retention rate. Max This represents the maximum polysaccharide retention rate.

[0035] Example 1: Extraction of polysaccharides from Dryopteris crassirhizoma. S1: Spread out the stems and leaves of the Dryopteris crassirhizoma, air dry them, then pulverize them and pass them through a 40-mesh sieve to obtain 400 g of Dryopteris crassirhizoma powder. S2: The dried powder of *Dryopteris crassirhizoma* obtained in S1 was mixed with petroleum ether at a mass ratio of 1 g: 3 mL and soaked for 24 h for defatting treatment. The defatting treatment was repeated twice. The solid was then filtered to obtain solids. The solids were dried and mixed with water at a mass ratio of 1 g: 15 mL. The mixture was then placed in a 90℃ water bath for extraction treatment for 3 h. The filtrate was collected and the extraction treatment was repeated 3 times. S3: Centrifuge the filtrate obtained in S2 at 4000 r / min for 15 min to remove insoluble impurities, then concentrate under reduced pressure. Add anhydrous ethanol at a volume ratio of 1:3 and stir thoroughly. Then precipitate at 4℃ for 24 h. After the precipitation is complete, centrifuge at 4000 r / min for 15 min, collect the precipitate, add distilled water until completely dissolved, and concentrate under reduced pressure for 1-2 h until the sugar solution slightly clings to the wall. S4: Papain was added at a ratio of 8‰ of the volume of the sugar solution obtained in S3. After stirring evenly, the mixture was enzymatically hydrolyzed in a water bath at 55℃ for 6 h to obtain the hydrolysate. After cooling to room temperature, Sevage reagent (obtained by mixing dichloromethane and n-butanol in a 4:1 ratio) was added at a volume ratio of 5:1. The mixture was vigorously shaken for 10 min to mix evenly and then centrifuged at 4000 r / min for 15 min. The centrifuged solution was divided into three layers, with the middle layer being the protein layer. The uppermost clear liquid was taken and concentrated under reduced pressure to remove the residual Sevage reagent, thus obtaining the enzymatically hydrolyzed sugar solution. S5: Add 60-100 mesh polyamide to the sugar solution after enzymatic hydrolysis in S4 at a mass-volume ratio of 1 mL: 40 mg, decolorize at 50℃ for 60 min, filter, and dialyze using a dialysis bag with a molecular weight cutoff of 3500 Da for 3 days; first dialyze with running water for 2 days, then dialyze with deionized water for 1 day, and obtain crude polysaccharide DFP from Dryopteris crassirhizoma after freeze-drying.

[0036] Effect Experiment: (1) Single-factor experiment This embodiment analyzes the decolorization effect of polyamide by changing three dimensions: polyamide dosage, decolorization treatment time, and decolorization treatment temperature. A 2 mg / mL solution of crude polysaccharide DFP was prepared, and 60-100 mesh polyamide was added to the enzymatically hydrolyzed sugar solution at ratios of 1:8, 1:16, 1:24, 1:32, and 1:40 (v / m). Single-factor experiments were conducted with treatment temperature gradients of 30, 40, 50, 60, and 70°C, and treatment time gradients of 20, 40, 60, 80, and 100 min. The crude sugar solution was scanned across the entire wavelength range (200-800 nm) to determine the position of the pigment absorption peak. After filtration and separation of the filtrate, the absorbance value was measured at this wavelength to calculate the decolorization rate. The polysaccharide retention rate was determined using the phenol-sulfuric acid method.

[0037] like Figure 2 As shown, when the ratio of enzymatically hydrolyzed sugar solution to polyamide exceeds 1:32 (polysaccharide retention rate is 87.82%), the decolorization treatment time exceeds 60 min (polysaccharide retention rate is 88.32%), and the decolorization treatment temperature exceeds 40℃ (polysaccharide retention rate is 91.88%), the polysaccharide retention rate decreases sharply.

[0038] (2) Orthogonal experiment Based on the results of the single-factor experiments, a three-factor, three-level orthogonal experiment was designed as shown in Table 1. The polysaccharide decolorization rate, polysaccharide retention rate, and polysaccharide purity were comprehensively evaluated. Each group was tested in three parallel experiments, and the results are shown in Table 2. The result with the largest mean (K) was taken as the optimal parameter level. It was found that the optimal decolorization treatment conditions were A3B2C3, that is, the mixing ratio of crude sugar to polyamide was 1 mL: 40 mg, the decolorization treatment time was 60 min, and the decolorization treatment temperature was 50℃.

[0039] At the same time, the appearance color before and after the decolorization treatment is compared, such as Figure 3 As shown in Tables 3-4, and through orthogonal experimental visualization and variance analysis, it can be found that the factors affecting the polysaccharide decolorization rate from high to low are: polyamide dosage > decolorization temperature > decolorization time.

[0040] Table 1

[0041] Table 2

[0042] Table 3

[0043] Table 4

[0044] Note: * This indicates a significant difference ( P <0.05) The optimal conditions obtained by orthogonal experiment were used for decolorization treatment, namely: the mixing ratio of crude sugar to polyamide was 1 mL: 40 mg, the decolorization treatment time was 60 min, and the decolorization treatment temperature was 50℃. Under the above conditions, the decolorization rate of polysaccharide was 78.76%, the polysaccharide retention rate was 69.85%, and the polysaccharide purity was 66.5%.

[0045] The polysaccharide solutions before and after the above-mentioned optimal decolorization treatment were subjected to a full-wavelength UV scan (200-800 nm). Figure 4 As shown, the absorbance of the decolorized polysaccharide solution at 220 nm decreased significantly, indicating that the decolorization treatment provided by the present invention has a good effect. At the same time, it was found that the peak at 280 nm tended to be flat, which is the characteristic absorption peak of protein, indicating that the decolorization treatment using polyamide can further remove residual protein in the crude polysaccharide of Dryopteris crassirhizoma.

[0046] Example 2: Purification of polysaccharides from Dryopteris crassirhizoma. Column packing: Wash the DEAE celluLose-52 anion exchange packing material with deionized water to remove ethanol, and activate it by swelling with ultrasonically degassed deionized water for 24 h; when packing the column, first add 1 / 4 column volume of degassed deionized water into the column, and then slowly guide the activated packing material through a glass rod to perform wet packing (Φ 5 cm × 60 cm) to prevent the formation of discontinuities and air bubbles. After packing, elute with degassed deionized water at a flow rate of 1 mL / min for 4-6 column volumes.

[0047] Ion exchange chromatography purification of crude polysaccharide from Dryopteris crassirhizoma: The crude polysaccharide of *Dryopteris crassirhizoma* obtained in Example 1 was mixed with deionized water to obtain a crude polysaccharide solution of *Dryopteris crassirhizoma* (concentration 25 mg / ml). After passing through a 0.45 μm aqueous filter membrane, the solution was loaded with the solution, with the loading volume ≤ 3% of the column volume. The eluent (NaCl solutions with concentrations of 0, 0.1, 0.2, and 0.4 mol / L, respectively) after ultrasonic degassing was used for gradient elution. The absorbance of each eluent tube (10 mL per tube) was measured and recorded after color development by phenol-sulfuric acid method.

[0048] Eluents of the same concentration were collected and combined, dialyzed against deionized water for one day, and then lyophilized to obtain four polysaccharide fractions from *Dryopteris crassirhizoma*, named DFP-1, DFP-2, DFP-3, and DFP-4, respectively. Elution curves were plotted with the number of eluent tubes on the x-axis and absorbance at 490 nm on the y-axis, as shown below. Figure 5 As shown.

[0049] Furthermore, among the four polysaccharide components obtained by the extraction and purification method of Pteris vittata polysaccharide provided by this invention, the DPF-2 component with the highest acidic polysaccharide content of Pteris vittata was selected to verify the application of Pteris vittata polysaccharide in chronic lead poisoning drugs.

[0050] Example 3: Application of acidic polysaccharides from Dryopteris crassirhizoma in drugs for chronic lead poisoning 1. Experimental model construction and grouping Thirty four-week-old male Kunming mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were selected and weighed after one week of acclimatization. They were randomly divided into 5 groups of 6 mice each, and the following parallel treatments were performed: (1) Lead poisoning group (Pb group): 40 mg / kg lead acetate solution was administered by gavage at 9:00 am every day, and an equal amount of physiological saline was administered by gavage at 11:00 am. (2) Low, medium and high dose treatment groups (LD, MD and HD groups): 40 mg / kg lead acetate aqueous solution was administered by gavage at 9:00 am every day, and 50, 100 and 200 mg / kg DFP-2 aqueous solution (DFP-2 polysaccharide obtained in Example 2) was administered by gavage at 11:00 am. (3) Control group: The same amount of physiological saline was administered by gavage at 9:00 am and 11:00 am every day; The gavage volume was 0.1 mL / 10 g; During the experiment, mice were allowed free access to food and water. The mice were observed and their condition was recorded. The weight of mice in different groups was measured and recorded weekly. The experiment lasted for 4 weeks, and the weight was recorded at the end of the experiment. Mice were fasted for 24 hours but allowed free access to water before being sacrificed. Blood from the eyeballs, small intestine, liver, and kidneys were collected, weighed, and aliquoted. They were then fixed with fixative or frozen at -80°C.

[0051] 2. Observation of histopathological sections of mouse jejunum and liver under different conditions The jejunum of mice in the above different experimental groups was fixed with 4% paraformaldehyde fixative for 24 h, then embedded in paraffin and sectioned, and hematoxylin-eosin staining (HE staining) was performed to observe the effects of chronic lead poisoning on mouse tissues and organs and the therapeutic effect of DFP-2.

[0052] like Figure 6 As shown, the weight gain of mice in the Pb model group was inhibited under chronic lead poisoning conditions. However, after treatment with low, medium, and high doses of DPF-2, the average weight of mice in each treatment group was higher than that in the Pb group. This indicates that the acidic polysaccharide DFP-2 from Dryopteris crassirhizoma provided by this invention can alleviate the growth retardation in mice caused by chronic lead poisoning.

[0053] like Figure 7-8 As shown in the organ index graphs of the liver and kidneys of mice in each experimental group, it can be seen that the organ indices of both the liver and kidneys increased under lead poisoning conditions, with the liver index showing a particularly significant increase. P <0.05%, indicating that lead accumulation caused varying degrees of damage to the liver and kidneys of mice; after treatment with low, medium, and high doses of DPF-2, the liver and kidney indices of mice in all treatment groups recovered, especially the liver index of mice in the HD group (high-dose treatment group) showed the most significant recovery. P <0.05); indicating that the acidic polysaccharide DFP-2 from *Dryopteris crassirhizoma* provided by this invention has the effect of alleviating liver and kidney damage in mice caused by chronic lead poisoning.

[0054] Results of HE staining of mouse jejunum as follows Figure 9 As shown, compared with the blank group, HE staining sections of the Pb group showed obvious pathological changes in the villi of the jejunum segment, with the villi becoming shorter and exhibiting breakage and shedding. After treatment with low, medium, and high doses of DPF-2, the pathological changes in the villi of the jejunum segment of mice in each treatment group were reversed to a certain extent, the villi arrangement returned to normal, and the spacing between the villi shortened. This indicates that the acidic polysaccharide DFP-2 from *Dryopteris crassirhizoma* provided in this invention can reverse the pathological changes in the villi of the jejunum segment caused by chronic lead poisoning.

[0055] 3. Detection of lead content After being absorbed by the body, heavy metal lead accumulates in organs such as the liver and kidneys, which can have adverse effects on health. To assess the accumulation of lead under chronic lead poisoning and the mechanism of action of DFP-2, concentrated nitric acid was used to digest the liver and kidney tissues of mice in each experimental group, and the lead content in the tissues was detected by inductively coupled plasma mass spectrometry (ICP-MS).

[0056] like Figure 10 As shown, compared with the control group, the lead content in the liver and kidney tissues of mice in the chronic lead poisoning Pb group was significantly increased, indicating that the lead ingested into the mice was accumulated in the liver and kidneys. This accumulation can cause serious damage to the organs and affect their normal function. After treatment with low, medium and high doses of DPF-2, the lead content in the liver and kidneys of mice in each treatment group was significantly reduced. P <0.05); At the same time, it was found that more lead was accumulated in the liver and the downward trend was more significant, which indicates that the protective effect of DFP-2 acidic polysaccharide of Dryopteris crassirhizoma is better on liver tissue than on kidney tissue.

[0057] 4. Detection of expression levels of small intestinal barrier-related genes Total RNA was extracted from the small intestine of mice in each experimental group using the Trizol method, and cDNA was obtained by reverse transcription (kit purchased from Nanjing Novizan Biotechnology Co., Ltd.). The small intestinal tight junction protein gene ZO was analyzed by qPCR. -1, Occludin The relative quantification of mRNA expression levels was performed, and the primer sequences used are shown in Table 5.

[0058] Table 5

[0059] like Figure 11-12 As shown, the small intestinal tight junction protein gene in mice with chronic lead poisoning (Pb) ZO-1, Occludin The mRNA expression level of lead was significantly reduced, demonstrating that lead intake disrupts the small intestinal barrier; and after treatment with low, medium, and high doses of DPF-2, the expression level of the small intestinal tight junction protein gene was significantly reduced in mice in each treatment group. ZO-1, Occludin The mRNA expression levels were restored to some extent.

[0060] Therefore, it can be seen that the acidic polysaccharide DFP-2 of *Dryopteris crassirhizoma* obtained by the polysaccharide extraction and purification method provided by this invention can alleviate / treat the damage caused by chronic lead poisoning by reducing the accumulation of lead in organs such as the liver and kidneys and repairing the intestinal barrier.

[0061] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. The application of *Dryopteris crassirhizoma* polysaccharide in the preparation of drugs for treating chronic lead poisoning, characterized in that... The drug has at least one of the following functions (1)-(3): (1) Reduce growth retardation caused by chronic lead poisoning; (2) Reduce liver and kidney damage caused by chronic lead poisoning; (3) Reduce intestinal villus lesions caused by chronic lead poisoning.

2. The application according to claim 1, characterized in that, The extraction and purification method of the polysaccharide from Dryopteris crassirhizoma is as follows: S1: Spread out the stems and leaves of the Dryopteris crassirhizoma, air dry them, then crush them and pass them through a 40-mesh sieve to obtain Dryopteris crassirhizoma powder; S2: The dried powder of *Dryopteris crassirhizoma* obtained in S1 was mixed with petroleum ether and soaked for 24 h for degreasing treatment. The degreasing treatment was repeated twice. The solid was then filtered to obtain solids. The solids were dried and mixed with water. The solids were then placed in a water bath at 80-90℃ for 3 h for extraction treatment. The solids were filtered and the filtrate was collected. The extraction treatment was repeated three times. S3: After centrifuging the filtrate obtained in S2, concentrate it under reduced pressure, add anhydrous ethanol, mix well, and precipitate it at 4℃ for 24h; centrifuge it at 4000 r / min for 10-15 min, add distilled water until the precipitate is completely dissolved, and concentrate it under reduced pressure for 1-2 h until the obtained sugar solution slightly clings to the wall. S4: Add papain to the sugar solution obtained in S3, stir well, and then enzymatically hydrolyze in a water bath at 55℃ for 4-6 h to obtain the enzymatic hydrolysate; after cooling to room temperature, add Sevage reagent in proportion, shake vigorously for 10 min to mix well, and then centrifuge at 4000 r / min for 15-20 min. Take the supernatant after centrifugation and concentrate under reduced pressure to obtain the enzymatically hydrolyzed sugar solution. S5: Add 60-100 mesh polyamide to the sugar solution after enzymatic hydrolysis in S4, decolorize at 30-70℃ for 20-100 min, filter, dialyze, and freeze dry to obtain crude polysaccharide of Dryopteris crassirhizoma. S6: Mix the crude polysaccharide of Dryopteris crassirhizoma obtained in S5 with deionized water to obtain a crude polysaccharide solution of Dryopteris crassirhizoma. After passing through a 0.45 μm aqueous filter membrane, the solution is loaded onto the column. The loading volume is ≤ 3% of the column volume. The eluent after ultrasonic degassing is subjected to gradient elution. The eluents of different gradients are collected and dialyzed. After lyophilization, the polysaccharide component of Dryopteris crassirhizoma is obtained.

3. The application according to claim 2, characterized in that, The mass ratio of dried fern powder to petroleum ether in S2 is 1 g: 3 mL.

4. The application according to claim 2, characterized in that, The mixing mass ratio of solids to water in S2 is 1 g: 15 mL, and the extraction treatment temperature is 90 °C.

5. The application according to claim 2, characterized in that, The volume ratio of the filtrate to anhydrous ethanol in S3 is 1:

3.

6. The application according to claim 2, characterized in that, In S4, the amount of papain added is 8‰ of the mass fraction, and the enzymatic hydrolysis time is 6 h.

7. The application according to claim 2, characterized in that, The volume ratio of the enzymatic hydrolysate to Sevage reagent in S4 is 5:

1. Sevage reagent is obtained by mixing dichloromethane and n-butanol in a 4:1 ratio.

8. The application according to claim 2, characterized in that, The mass ratio of the enzymatically hydrolyzed sugar solution to polyamide in S5 is 1 mL: 40 mg, and the decolorization treatment is carried out at a temperature of 50°C for 60 min.

9. The application according to claim 2, characterized in that, The dialysis bag used in S5 has a molecular weight cutoff of 3500 Da, and the dialysis time is 3 days; first, dialysis is performed with running water for 2 days, followed by dialysis with deionized water for 1 day.

10. The application according to claim 2, characterized in that, The concentration of the crude polysaccharide solution of Dryopteris crassirhizoma S6 is 25 mg / ml, and the eluent is a degassed NaCl solution with concentrations of 0, 0.1, 0.2, and 0.4 mol / L.

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