Dictyophora rubrovolvata volva polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses dictyophora rubrovolvata volva polysaccharide and a preparation method and application thereof.According to the dictyophora rubrovolvata volva polysaccharide and the preparation method and application thereof, the high-viscosity technical bottleneck of the dictyophora rubrovolvata volva polysaccharide is solved through H2O2, heating and ultrasonic-assisted multi-factor synergistic viscosity reduction operation under the alkaline condition, efficient separation is achieved, decomposition of H2O2 is promoted through the cavitation effect of ultrasonic waves, and the effect of reducing the viscosity of the dictyophora rubrovolvata volva polysaccharide is achieved. Hydroxyl free radicals with extremely high activity are efficiently generated under the alkaline condition, the free radicals can controllably attack glucosidic bonds on polysaccharide chains, polysaccharide macromolecular chains are broken, the molecular weight is reduced, the kinematic viscosity of a dictyophora rubrovolvata volva polysaccharide solution is remarkably reduced, and compared with independent use of H2O2, the activity of the dictyophora rubrovolvata volva polysaccharide solution is greatly improved. The H2O2 and ultrasonic-assisted synergistic method disclosed by the invention is milder in reaction condition, higher in efficiency and more remarkable in viscosity reduction effect, the subsequent chromatography is changed from infeasible to efficient and feasible, and the industrial problem in the background technology is solved.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, specifically to a polysaccharide from *Dictyophora indica*, its preparation method, and its application. Background Technology

[0002] Dictyophora rubrovolvata is a large fungus whose fruiting body (bamboo fungus) has high edible and medicinal value. Studies have shown that the bioactivity of bamboo fungus is closely related to polysaccharides, one of its main chemical components. Existing research has confirmed that bamboo fungus polysaccharides possess various biological activities, including immunomodulation, antitumor, antioxidant, and anti-aging effects, and have broad application prospects in functional foods, health products, and adjuvant drug therapy.

[0003] Currently, there are numerous reports on the extraction and purification of bamboo fungus polysaccharides. For example, Chinese patent CN104262500B discloses a method for preparing polysaccharides from *Dictyophora indicum*, which involves water extraction, Sevage protein removal, ethanol precipitation, and subsequent purification via ion exchange and gel filtration. However, this method, as well as the method disclosed in Chinese patent CN104277134B, involves relatively simple raw material processing and lacks detailed removal steps for pigments, lipid-soluble impurities, and bound proteins, resulting in relatively limited purity and extraction rate of the obtained polysaccharides. Chinese patent CN103044566B also discloses a method using a strong alkaline solution (5% NaOH solution) for extraction. While this may improve the dissolution of certain components, the strong alkaline environment easily leads to polysaccharide degradation or structural changes, and subsequent large-scale acid neutralization is required, making the operation complex and difficult to control.

[0004] More importantly, a common and challenging technical problem exists in the preparation of fungal polysaccharides, especially those from the genus *Dictyophora*: high viscosity. The inventors of this invention discovered that the polysaccharide extract obtained after preparing crude polysaccharides from the volva of *Dictyophora indusiata* using conventional methods (such as hot water extraction and alcohol precipitation) exhibits extremely high viscosity. This observation is not an isolated case, but rather a common characteristic of *Dictyophora* polysaccharides. Related academic research has confirmed that polysaccharides from *Dictyophora indusiata* possess high viscosity, a natural rheological property. This high viscosity has become a major obstacle to research and industrial production in this field, as it severely limits subsequent purification operations such as filtration, concentration, and chromatographic separation, and significantly increases production costs. High-viscosity solutions, when subjected to column chromatography (such as ion exchange or gel filtration), lead to low mass transfer efficiency, a sharp increase in column pressure, column blockage, and even damage, making the separation process difficult.

[0005] Although high viscosity is a key technical bottleneck in the purification of bamboo fungus polysaccharides, existing patent literature mostly focuses on the extraction steps or the activity of the final product, generally neglecting effective solutions to the intermediate problem of high viscosity in crude polysaccharides. Existing patents (such as Chinese patent CN104262500B) directly use alcohol precipitation for chromatography, which is acceptable in small-scale laboratory tests (with very small throughput), but in pilot-scale or industrial-scale production, high-viscosity solutions will lead to separation failure of chromatography columns (such as DEAE-52) or high costs. Existing technologies attempt to solve the viscosity problem of fungal polysaccharides, but they mostly use methods such as ultrafiltration, changing the extraction method, or radiation, lacking a mild, controllable, and compatible viscosity-reducing pretreatment process that is compatible with subsequent purification steps (such as ion exchange).

[0006] Therefore, there is an urgent need in the field for a new method for separating and purifying polysaccharides from *Dictyophora indicum*. This method can not only efficiently remove impurities, but also effectively solve the problem of high viscosity of crude polysaccharides in the purification process, thereby achieving efficient and high-purity separation. To this end, the present invention provides polysaccharides from *Dictyophora indicum*, its preparation method, and its application. Summary of the Invention

[0007] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a polysaccharide from *Dictyophora indica*, its preparation method, and its application.

[0008] The present invention achieves the above objectives through the following technical solutions: This invention provides a method for preparing polysaccharide from *Dictyophora indica*, comprising the following steps: (1) Fresh red-capped bamboo fungus volva are dried, crushed and sieved to obtain volva powder; (2) The volva powder was extracted with hot water, the extracts were combined, the supernatant was collected by centrifugation and concentrated to obtain concentrated supernatant; (3) The concentrated supernatant was subjected to alcohol precipitation, the precipitate was collected by centrifugation and dried to obtain the precipitate; (4) The precipitate is reconstituted to obtain a liquid, which is then subjected to enzymatic hydrolysis, enzyme inactivation, and deproteinization using Sevage reagent to obtain the deproteinized supernatant. (5) The supernatant after protein removal is treated with macroporous adsorption resin, the effluent is collected, and after dialysis and drying, a high-viscosity crude polysaccharide is obtained. (6) Dissolve the high-viscosity crude polysaccharide in water, adjust the pH to alkaline, add hydrogen peroxide solution, and carry out a synergistic viscosity reduction reaction under heating and ultrasonic-assisted conditions to obtain the viscosity-reduced reaction solution; (7) Adjust the pH of the reaction solution after viscosity reduction to neutral, centrifuge to collect the supernatant, concentrate and precipitate again with alcohol, collect the precipitate and redissolve to obtain the crude polysaccharide solution after viscosity reduction. (8) Centrifuge the viscosity-reducing crude polysaccharide solution to obtain the supernatant, pass it through a DEAE-52 anion exchange column, and elute it with NaCl solutions of different concentration gradients. Collect the eluent in separate tubes. (9) Combine the eluents corresponding to the target elution peak in step (8), concentrate and desalt by dialysis, purify by Superdex200 gel filtration chromatography column, collect the single component, freeze dry to obtain purified red-topped bamboo fungus polysaccharide.

[0009] As a further optimization of the present invention, the compound ginkgo kernel extract comprises ginkgo kernel extract, chitosan, and coconut shell activated carbon mixed in a mass ratio of 3-5:1:1.

[0010] As a further optimization of the present invention, in step (1), the sieving is performed through a 60-mesh sieve; In step (2), the hot water temperature for extraction is 75-85℃, the material-to-liquid ratio is 1:36-42 (g / mL), the extraction time is 3-5h, and the extraction is repeated 2-4 times. In step (3), the alcohol precipitation uses 95% (v / v) ethanol, the volume ratio of ethanol to concentrated supernatant is 3.5-4.8:1, the alcohol precipitation is allowed to stand at 4℃ for 20-25h, the centrifugation speed is 8000rpm, and the centrifugation time is 10min.

[0011] As a further optimization of the present invention, in step (4), the enzymatic hydrolysis uses at least one of papain, complex protease, pectinase, and cellulase; the volume ratio of Sevage reagent to liquid is 1:3-5.

[0012] As a further optimization of the present invention, in step (5), the macroporous adsorption resin is AB-8 type; the dialysis uses a dialysis bag with a molecular weight cutoff of 3000 Da.

[0013] As a further optimization of the present invention, in step (6), the heating temperature of the viscosity reduction treatment is 58-65℃ and the reaction time is 0.5-1.5h; in step (6), the viscosity is observed after cooling to room temperature. If the viscosity does not change significantly, step (6) is repeated once or multiple times.

[0014] As a further optimization of the present invention, in step (7), the second alcohol precipitation uses 95% (v / v) ethanol at 4 times the volume of the concentrated liquid after supernatant concentration. In step (8), the DEAE-52 anion exchange column is sequentially eluted with pure water, 0.1M NaCl solution, 0.2M NaCl solution and 0.3M NaCl solution; In step (9), the target elution peak is the second component obtained by elution with 0.1M NaCl solution; the Superdex 200 gel filtration chromatography column uses deionized water as the eluent and the flow rate is 1 mL / min.

[0015] The present invention also provides a polysaccharide from *Dictyophora indica*, prepared by the method described above.

[0016] This invention also provides the application of polysaccharide from *Dictyophora indicum* in the preparation of drugs for treating or preventing radiation, frostbite, and mechanical damage.

[0017] As a further optimization of the present invention, the medicinal dosage of purified *Dictyophora indicum* polysaccharide is 100-800 mg / kg.

[0018] The present invention has the following beneficial effects: 1) This invention solves the high viscosity bottleneck of *Dictyophora indicum* polysaccharide by using synergistic viscosity reduction under alkaline conditions (H2O2), heating, and ultrasonic assistance, achieving efficient separation. The invention utilizes the "cavitation effect" of ultrasound to promote the decomposition of H2O2, efficiently generating highly reactive hydroxyl radicals under alkaline conditions. These radicals can controllably attack the glycosidic bonds on the polysaccharide chains, causing the polysaccharide macromolecular chains to break and the molecular weight to decrease. This significantly reduces the kinematic viscosity of the *Dictyophora indicum* polysaccharide solution. Compared to using H2O2 alone, the synergistic method of this invention (H2O2 + heating + ultrasonic assistance) significantly reduces the viscosity of the solution. 2和 The ultrasound-assisted reaction conditions are milder (60°C), more efficient, and have a more significant viscosity reduction effect, which transforms subsequent chromatography from "infeasible" to "highly feasible," solving the industry problems described in the background technology. 2) Before viscosity reduction, this invention uses a combination of "enzymatic hydrolysis and Sevage reagent" to remove free and bound proteins; then, overnight adsorption with AB-8 macroporous adsorption resin effectively removes pigments and small molecule phenolic impurities. This multi-purification combination ensures that the sample entering the viscosity reduction and chromatography steps has high purity, the purification path is reasonably designed, and the target components are uniform. 3) After solving the viscosity problem, this invention uses the classic "two-step chromatography method" for fine purification, and finally obtains the target polysaccharide with uniform molecular weight. This purification path (DEAE-52 in series with Superdex 200 / Sephadex G-100) is a proven high-purity preparation method in the field, which ensures the uniformity of the final product. 4) The purified polysaccharide of *Dictyophora indica* prepared in this invention has the function of treating or preventing radiation, frostbite, and mechanical damage. Attached Figure Description

[0019] Figure 1The chromatogram (elution curve) of the viscosity-reducing crude polysaccharide solution of Example 1 of the present invention after gradient elution on a DEAE-52 anion exchange column. Figure 2 As in Embodiment 1 of the present invention Figure 1 The second elution peak (0.1M NaCl fraction) was collected, concentrated, and purified by Superdex 200 gel filtration chromatography column. Figure 3 The image shows the CCK8 cytotoxicity test results of purified DRP-F2 polysaccharide from *Dictyophora indicum* in Example 1 of this invention. Detailed Implementation

[0020] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0021] I. Materials 1. Papain and compound protease were both purchased from Beijing Solarbio Science & Technology Co., Ltd. The activity of compound protease was 100,000 U / g, and the activity of papain was 100,000 U / g. The amount added accounted for 0.1-0.3% of the dry weight of the reconstituted product, w / w. 2. Pectinase: Purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g. The amount added is 0.1-0.2% of the dry weight of the reconstituted product, w / w. 3. Cellulase: Purchased from Shaanxi Runfeng Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g. The amount added is 0.1-0.2% of the dry weight of the reconstituted product, w / w. Unless otherwise specified, the methods used in this embodiment are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products.

[0022] II. Methods Example 1 In this embodiment, the present invention provides a method for preparing polysaccharide from *Dictyophora indica*, the specific steps of which are as follows: (1) Raw material preparation Weigh fresh red-topped bamboo fungus volva and dry them in a 60℃ forced-air drying oven until constant weight. Use a high-speed pulverizer to grind the dried volva into powder, sieve the powder through a 60-mesh sieve, collect the powder that passes through the sieve, and obtain dry volva powder.

[0023] (2) Extraction and concentration Weigh 100g of the strobila powder obtained in step (1) above, add 4000mL of pure water at a material-to-liquid ratio of 1:40 (g / mL), place in an 80℃ constant temperature water bath, and stir and extract for 4h; filter with gauze, collect the filtrate, and repeat the extraction twice under the same conditions (80℃, 1:40, 4h); combine the three extracts (total volume about 12000mL), centrifuge at 4℃ and 8000rpm for 15min using a high-speed refrigerated centrifuge, and take the supernatant; concentrate the supernatant to 1 / 5 of the original volume (about 2400mL) using a rotary evaporator under reduced pressure at 60℃ to obtain concentrated supernatant.

[0024] (3) Alcohol precipitation and crude extraction Cool the concentrated supernatant to room temperature, and slowly add 95% (v / v) ethanol while stirring, so that the final volume ratio of ethanol to concentrated supernatant is 4:1 (i.e., add 9600 mL of ethanol); place the mixture in a refrigerator at 4℃ and let it stand for ethanol precipitation for 24 h; centrifuge the precipitate at 4℃ and 8000 rpm for 10 min, discard the supernatant, and collect the precipitate; dry the precipitate in an oven at 60℃ until there is no obvious ethanol odor, and obtain the coarse precipitate.

[0025] (4) Enzymatic hydrolysis and deproteinization The crude precipitate obtained in step (3) was fully reconstituted with 1000 mL of pure water. Papain and complex protease were added to the solution (each added at 1% of the dry weight of the reconstituted product, w / w). The pH was adjusted to 6.5, and the solution was enzymatically hydrolyzed overnight (12 h) in a 50 °C water bath. After the enzymatic hydrolysis was completed, the solution was heated in a 100 °C water bath for 15 min to inactivate the enzyme. After cooling, Sevage reagent (chloroform: n-butanol = 4:1, v / v) was added so that the volume ratio of Sevage reagent to the enzymatic hydrolysate was 1:4. After vigorous shaking for 30 min, the solution was centrifuged at 4000 rpm for 10 min. The supernatant was collected, and the Sevage reagent deproteinization operation was repeated 3-5 times until there was no obvious protein precipitation at the interface between the aqueous phase and the organic phase. The deproteinized supernatant was obtained.

[0026] (5) Adsorption and dialysis (step f, core step): The supernatant after deproteinization was passed through a pretreated AB-8 macroporous adsorption resin column (column volume 500 mL) at a controlled flow rate (2 BV / h) to allow it to adsorb overnight in the column to remove pigments and small molecule impurities. The eluent was collected and placed into a dialysis bag with a molecular weight cutoff (MWCO) of 3000 Da. Dialysis was performed in flowing deionized water for 48 h, with the dialysis water changed every 6 h. The dialysis liquid was collected and dried in a 65 °C oven (or freeze-dried) to obtain high-viscosity crude polysaccharide (denoted as Crude-DRP-H), which was then weighed.

[0027] (6) Viscosity reduction treatment Add all the high-viscosity crude polysaccharide solid (Crude-DRP-H) obtained in step (5) to 1000 mL of pure water to dissolve it completely; adjust the pH to 9.0 using 1 M NaOH solution; add 30% (w / v) H2O2 solution to make the final concentration in the mixture 1% (v / v); place the mixture in a 60°C water bath and heat and stir for 1 h, while simultaneously turning on ultrasonic assistance (frequency 40 kHz, power 200 W); after 1 h, stop heating and ultrasonication, and cool to room temperature; use a rotational viscometer to sample and measure the viscosity; in this embodiment, the viscosity has decreased significantly after 1 h of treatment. (Optional) If the viscosity does not decrease significantly, add H2O2 again (e.g., to make the final concentration in the mixture 0.5%, v / v) and repeat this step once.

[0028] (7) Neutralization and recycling After confirming a significant reduction in viscosity (e.g., viscosity reduced to <20 mPa·s), the pH of the reaction solution was adjusted to neutral (pH 7.0) using 1M HCl solution. The neutralized solution was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The supernatant was concentrated to 200 mL by rotary evaporation. Four times the volume (800 mL) of 95% ethanol was added, and the solution was precipitated overnight at 4°C. The precipitate was collected by centrifugation (8000 rpm for 10 min) and reconstituted with 50 mL of pure water to obtain the viscosity-reducing crude polysaccharide solution (denoted as Crude-DRP-L).

[0029] (8) Ion exchange chromatography The Crude-DRP-L solution obtained in step (7) was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected. The supernatant was loaded onto a DEAE-52 anion exchange column (2.6 × 40 cm) equilibrated with pure water. After loading, the column was first eluted with 1000 mL of pure water (to remove unadsorbed neutral components, denoted as Frac-1). Subsequently, gradient elution was performed with 1000 mL of 0.1 M NaCl solution (denoted as Frac-2), 1000 mL of 0.2 M NaCl solution (denoted as Frac-3), and 1000 mL of 0.3 M NaCl solution (denoted as Frac-4). The flow rate was controlled at 4 mL / min. The eluent was collected in separate tubes (10 mL / tube) using an automatic fraction collector. The total sugar content of the eluent in each collection tube was determined using the sulfuric acid-phenol method. An ion purification elution curve was plotted with the number of collection tubes on the x-axis and absorbance (OD value) on the y-axis. The experimental results are as follows: Figure 1 As shown, Figure 1 The results show that Frac-1, Frac-2, Frac-3, and Frac-4 are all elution peaks containing polysaccharides.

[0030] (9) Gel filtration chromatography according to Figure 1 The elution curves were analyzed, and the eluents from each collection tube corresponding to the second elution peak (Frac-2, i.e., the 0.1M NaCl eluent) were combined. The Frac-2 collection solution was concentrated to approximately 50 mL by rotary evaporation under reduced pressure at 60 °C. The concentrated product was placed in a 3000 Da dialysis bag and dialyzed in deionized water for 24 h to desalt it. The desalted product was then freeze-dried. The freeze-dried powder was reconstituted with 10 mL of deionized water and loaded onto a pre-equilibrated Superdex 200 gel filtration column (1.5 × 100 cm). Deionized water was used as the eluent, and the flow rate was controlled at 1 mL / min. The sulfuric acid-phenol method was also used to determine the sugar content of the effluent, and the elution curve was plotted as follows: Figure 2 As shown, Figure 2 The peak appears as a sharp, symmetrical single elution peak, indicating that the component is a homogeneous polysaccharide component. collect Figure 2 The component corresponding to the single symmetrical peak in the middle was collected and freeze-dried to obtain purified red-topped bamboo fungus polysaccharide (denoted as Purified-DRP-F2), weighed, and the yield was calculated (mass of Purified-DRP-F2 / mass of dried fungus powder from step 1).

[0031] Example 2 To verify the effect of the viscosity reduction process in steps (6) and (7) of Example 1 of the present invention on the final product, in this example, the present invention provides a method for preparing polysaccharide from *Dictyophora indicum*. Based on Example 1, steps (1)-(5) are strictly performed to obtain high-viscosity crude polysaccharide (Crude-DRP-H). The difference is that the viscosity reduction process in steps (6) and (7) is omitted. That is, the high-viscosity crude polysaccharide (Crude-DRP-H) obtained in step (5) is reconstituted with 50 mL of pure water to obtain a crude polysaccharide solution with extremely high viscosity (Crude-DRP-H-Sol), and then the ion exchange chromatography in step (8) is directly performed.

[0032] Experimental results: During the sample loading process in step (8), the crude polysaccharide solution (Crude-DRP-H-Sol) with extremely high viscosity (measured viscosity >1000mPa·s) was loaded onto a DEAE-52 column (2.6×40cm) at a set flow rate of 4mL / min (using a peristaltic pump). The column inlet pressure quickly (<5min) exceeded the upper limit of the column material (DEAE-52) pressure (>3bar), causing the column bed to be severely compressed and blocked, and the eluent could not flow out.

[0033] Experimental conclusion: This shows that without the viscosity reduction process of steps (6) and (7) of the present invention, the crude polysaccharide of *Dictyophora indicum* is too viscous to be separated by subsequent DEAE-52 ion exchange chromatography. This proves that the viscosity reduction step of the present invention is the prerequisite and key to achieving subsequent fine purification, and solves the major technical obstacles existing in the background art.

[0034] Example 3 To verify the effect of the "ultrasonic-assisted" process in step (6) of Example 1 of the present invention on the final product, in this example, the present invention provides a method for preparing polysaccharide from *Dictyophora indicum*. Based on Example 1, steps (1)-(5) are strictly performed to obtain high-viscosity crude polysaccharide (Crude-DRP-H). The difference is that when performing step (6), only 60°C water bath heating and stirring is performed for 1 hour, and ultrasonic assistance is not turned on. After 1 hour of reaction, the viscosity is measured by sampling. Although the viscosity has decreased, it is still significantly higher than that of Example 1. The viscosity of the sample at the same time point (H2O2 + ultrasonic assistance) is as shown in Table 1.

[0035] To achieve the same target viscosity as in Example 1 (<20 mPa·s, for easy column loading), the reaction time in this example needs to be extended to 8 hours. Although the viscosity is reached after 8 hours and subsequent purification can be carried out, in the subsequent purification steps (8) and (9), the final yield of the target component purified from *Dictyophora indica* polysaccharide (Purified-DRP-F2) is only 45% of that in Example 1.

[0036] Experimental conclusions: Ultrasonic assistance can significantly accelerate the viscosity reduction reaction of H2O2, shortening the reaction time from 8h to 1h, and greatly improving production efficiency. More importantly, prolonged heating degradation may lead to excessive hydrolysis of polysaccharides or breakage of non-target bonds, resulting in a significant decrease in the recovery rate (yield) of the target components. Therefore, the "H2O2 + ultrasonic assistance" synergistic method in Example 1 of this invention achieves rapid (1h), mild, and efficient controllable viscosity reduction, which is a significant improvement over the existing technology (H2O2 degradation alone).

[0037] Based on the preparation methods and experimental results of Examples 1-3 above, the key indicators were summarized and compared, as shown in Table 1 below: Table 1. Effects of different viscosity-reducing treatments on the purification efficiency of polysaccharides from *Dictyophora indicum*. ; Note: Viscosity was measured at 25°C; Total purification time refers to the total time from the start of viscosity reduction in step (6) to obtaining the lyophilized powder in step (9); Polysaccharide yield = (final purified polysaccharide mass / mass of thallium spore powder in step (1)) × 100%; The purity of purified polysaccharide was determined by the phenol-sulfuric acid method. Experimental conclusions: Based on the data analysis in Table 1, it can be seen that the viscosity of Example 2 (1120.4 mPa·s) is extremely high, while that of Example 1 drops to 11.5 mPa·s within 1 hour, which is beneficial for chromatography. The viscosity of Example 3 is still as high as 485.3 mPa·s within the same time (1 hour), which proves that the lack of ultrasonic assistance results in extremely low viscosity reduction efficiency and makes it impossible to load onto the column. When the H2O2 treatment time of Example 3 is extended to 8 hours, although purification is completed, the yield of purified polysaccharide is only 2.3%, which is much lower than the 5.2% yield of purified polysaccharide in Example 1. This indicates that long-term treatment leads to a large loss of target polysaccharide, which shows that the treatment process in steps (6) and (7) of this invention is necessary. Moreover, the "H2O2 + ultrasonic assistance" synergistic viscosity reduction treatment process can significantly improve the efficiency and yield of purified polysaccharide. In practical applications, the beneficial effects are significant.

[0038] This invention employs DEAE-52 anion exchange: utilizing the differences in binding capacity of different polysaccharide components (neutral or acidic) under different ionic strengths (0.1M, 0.2M, 0.3M NaCl concentrations), preliminary separation is achieved (e.g., ...). Figure 1 (As shown); Superdex 200 gel filtration: Superdex 200 is a highly efficient molecular sieve medium (a composite matrix of dextran and agarose) that utilizes differences in molecular size for separation. It is used for the separation of components obtained from DEAE-52 (such as... Figure 2 As shown, the present invention selects the second component) for desalting and purification to finally obtain the target polysaccharide with uniform molecular weight. This purification route (DEAE-52 in series with Superdex200 / Sephadex G-100) is a proven high-purity preparation method in the art, which ensures the uniformity of the final product.

[0039] Example 4 To verify the effect of papain, complex protease, cellulase, and pectinase on the final product in step (4) of Example 1 of the present invention, in this example, the present invention provides a method for preparing polysaccharide from *Dictyophora indicum*, based on Example 1, and also adds cellulase (the amount added is 0.8% of the dry weight of the reconstituted product, w / w); the rest are consistent with Example 1.

[0040] Example 5 To verify the effect of papain, complex protease, cellulase and pectinase combined on the final product in step (4) of Example 1 of the present invention, in this example, the present invention provides a method for preparing polysaccharide from *Dictyophora indicum*, based on Example 1, and also adds pectinase (the amount added is 0.5% of the dry weight of the reconstituted product, w / w); the rest are consistent with Example 1.

[0041] Example 6 To verify the effect of papain, complex protease, cellulase, and pectinase on the final product in step (4) of Example 1 of the present invention, in this example, the present invention provides a method for preparing polysaccharide from *Dictyophora indicum*, based on Example 1, by adding cellulase and pectinase (the amount of cellulase added is 0.8% of the dry weight of the reconstituted product, and the amount of pectinase added is 0.5% of the dry weight of the reconstituted product, w / w); the rest are consistent with Example 1.

[0042] Example 7 To verify the effect of papain, complex protease, cellulase, and pectinase on the final product in step (4) of Example 1 of the present invention, in this example, the present invention provides a method for preparing polysaccharide from *Dictyophora indicum*, based on Example 1, by replacing papain in step (4) with an equal amount of neutral protease; the rest are consistent with Example 1.

[0043] The same test methods as those used in Examples 1-3 were applied to Examples 4-7. Key indicators of the test results were summarized and compared, as shown in Table 2 below. Table 2. Effects of different enzymatic hydrolysis reagents on the purification efficiency of polysaccharides from *Dictyophora indicum*. ; Experimental conclusions: As shown in Table 2, the specific combination of papain, complex protease, cellulase, and pectinase for enzymatic hydrolysis can improve the purity and yield of purified polysaccharides while increasing the flow rate on the DEAE-52 column, resulting in the best effect. There is a synergistic effect among them.

[0044] III. Experimental Verification of the Effect of Purified DRP-F2 from *Dictyophora indicum* (1) CCK8 cytotoxicity assay 1) Remove the cells from the incubator and observe them under an inverted microscope. Take C2C12 cells in good condition with about 80% cell confluence and prepare a cell suspension. Count the cells and seed them evenly in a 96-well plate at 5000 cells per well. Incubate the plate in an incubator for 24 hours. 2) The original culture medium was removed the next day, and polysaccharides of different concentrations (purified polysaccharides of *Dictyophora indicum* from Example 6) were added and cultured for another 96 hours. The concentrations of polysaccharides were set to 0 (control), 0.5, 1.0, 2.0 and 4.0, which were prepared by diluting 8 mg / mL polysaccharide lyophilized powder stock solution (8 mg of polysaccharide lyophilized powder dissolved in 1 mL of DMEM high-glucose medium). The medium was changed daily. 3) At 0h, 24h, 48h, 72h, and 96h, respectively, the old culture medium in the 96-well plate was aspirated, and 100μL of CCK-8 detection reagent (CCK-8: complete culture medium = 1:10) was added to each well. Then, the plate was incubated at 37°C in the dark for 1h. 4) After 1 hour, remove the 96-well plate and first observe the color intensity with the naked eye to make a preliminary judgment on the experimental results. Then, use an ELISA reader to detect the effect of polysaccharides on cell activity. The detection wavelength is set to 450nm to obtain the OD value of each well. Calculate the survival rate of C2C12 cells after treatment with different concentrations of polysaccharides. Cell proliferation activity (%) = (OD experiment - OD blank) / (OD control - OD blank). Each experiment is performed 3 times.

[0045] The experimental data are shown in Table 3, which demonstrates that the purified DRP-F2 polysaccharide from *Dictyophora indicum* of this application promotes cell proliferation at a certain concentration.

[0046] (2) Construction of a mouse model of radiation injury Ninety healthy male Kunming mice of SPF grade were selected and divided into three groups: a blank control group, an irradiation model group, and experimental groups (Examples 1-7), with 10 mice in each group (half male and half female). Mice were given prophylactic medication before irradiation. The experimental groups were administered purified DRP-F2 (50-800 mg / kg, 180 mg / kg was used in this invention) by gavage, with a total gavage volume of 0.5 mL. The blank control group and the irradiation model group were administered an equal volume of physiological saline by gavage. Gavage was performed at fixed times daily for 10 consecutive days. On day 11, mice in the irradiation model group and experimental groups (Examples 1-7) were tested using... 60 A single whole-body irradiation of 5 Gy with CO-γ rays at a dose rate of 218.63 cGy / min and an irradiation time of 2 min 17 s with a source-to-skin distance of 80 cm was administered; the blank control group received no irradiation.

[0047] After radiation irradiation, the mice were sacrificed, and their spleens were harvested and homogenized. The homogenate was centrifuged at 3000 rpm for 15 min, and the supernatant was collected. The serum IL-6 and TNF-α levels of each group of mice were measured according to the ELISA kit instructions, as shown in Table 3. Table 3. Record of IL-6 and TNF-α levels in the serum of mice in each group. ; Experimental Conclusions: Analysis of the data in Table 3 shows that the serum IL-6 (145 pg / mL) and TNF-α (698 ng / mL) levels in the irradiation model group mice were significantly higher than those in the blank control group (IL-6 was 68 pg / mL and TNF-α was 513 ng / mL). This indicates that radiation irradiation successfully induced an increase in the level of inflammatory factors in mice, establishing a radiation damage model. The IL-6 and TNF-α levels in all example groups (1-7) were between those in the blank control group and the irradiation model group, indicating that each treatment regimen had a certain inhibitory effect on radiation-induced inflammatory response. Among the example groups, Example 6 had the most significant anti-inflammatory effect, with the lowest IL-6 (81 pg / mL) and TNF-α (542 ng / mL) levels, closest to the blank control group. This suggests that the purified *Dictyophora indica* polysaccharide from Example 6 had the best effect.

[0048] (3) Construction of mouse models of frostbite and mechanical injury Frostbite mouse model construction: A total of 80 healthy male SPF-grade Kunming mice were selected and divided into a frostbite model group and experimental groups (Examples 1-7), with 10 mice in each group and half male and half female. The frostbite mouse model was prepared by liquid nitrogen method. After anesthetizing the mice, hair was removed from a 0.6 cm area on the back. An iron sheet was immersed in liquid nitrogen for about 15 minutes to cool it to -196°C. The iron sheet was then immediately pressed tightly against the mouse skin for 90 seconds to form deep frostbite (degree III-IV). Once the local skin frostbite was established in the mice, the model was successfully established.

[0049] Mechanical injury mouse model construction: A total of 80 healthy male SPF Kunming mice were selected and divided into mechanical injury model group and experimental groups of Examples 1-7, with 10 mice in each group, half male and half female. After anesthetizing the mice, a circular full-thickness skin with a diameter of 0.6 cm was cut off with surgical scissors to establish local full-thickness skin injury in the mice, and the model was successfully established.

[0050] Administration method: The purified DRP-F2 polysaccharide obtained in Examples 1-7 was slowly added to deionized water (concentration 2-5%, w / v; 3.5%, w / v was used in this invention), stirred at room temperature for 30 min, heated in an 80°C water bath for 1.5 h, and poured into a mold while still hot after complete dissolution. The gel dressing was then refrigerated at 4°C for 18 h. The amount of gel dressing used for each mouse with a 0.6 cm lesion area was 65 μL (this invention). In the experiment, the amount of purified *Dictyophora indicum* polysaccharide used was approximately 114 mg / kg. Finally, a 3M transparent dressing was applied to the wound surface to fix the gel dressing. The gel dressing was changed every two days post-surgery, and on days 3, 5, 7, 9, 11, and 13. EGF levels in the mouse wound tissue on days 5 and 10 were quantitatively analyzed using ELISA, and the healing rate on day 14 was calculated. The healing rate was calculated using the formula: Healing Rate = {(A0 - A...} t ) / A0}×100%, where A0 is the initial wound area (t=0), A t This is the wound area on day t (t=14) after treatment. The experimental data are shown in Table 4. Table 4 EGF Quantitative Analysis Data Record Table ; Experimental conclusions: In both the frostbite and mechanical injury models, the EGF content and wound healing rate of all example groups (1-7) were significantly higher than those of the corresponding model groups, indicating that each treatment regimen could effectively promote wound healing. In the frostbite model, the healing rate reached 95.8% after 14 days; in the mechanical injury model, the healing rate reached 92.1%, which was much higher than other groups. Among them, the EGF level in the frostbite group of Example 6 reached 175 pg / mL on day 10, which was significantly higher than that in the mechanical injury group, which reached 154 pg / mL on day 10. This indicates that the purified *Dictyophora indica* polysaccharide of Example 6 of this application has the specific ability to activate the repair potential of frostbite tissue. It is speculated that its advantage in treating deep frostbite lies in the breakthrough therapeutic effect of shortening the healing cycle and increasing the healing rate by upregulating EGF expression early, continuously and efficiently, which has specific therapeutic value for complex traumas such as deep frostbite.

[0051] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing polysaccharide from *Dictyophora indicum*, characterized in that, Includes the following steps: (1) Fresh red-capped bamboo fungus volva are dried, crushed and sieved to obtain volva powder; (2) The volva powder was extracted with hot water, the extracts were combined, the supernatant was collected by centrifugation and concentrated to obtain concentrated supernatant; (3) The concentrated supernatant was subjected to alcohol precipitation, the precipitate was collected by centrifugation and dried to obtain the precipitate; (4) The precipitate is reconstituted to obtain a liquid, which is then subjected to enzymatic hydrolysis, enzyme inactivation, and deproteinization using Sevage reagent to obtain the deproteinized supernatant. (5) The supernatant after protein removal is treated with macroporous adsorption resin, the effluent is collected, and after dialysis and drying, a high-viscosity crude polysaccharide is obtained. (6) Dissolve the high-viscosity crude polysaccharide in water, adjust the pH to alkaline, add hydrogen peroxide solution, and carry out a synergistic viscosity reduction reaction under heating and ultrasonic-assisted conditions to obtain the viscosity-reduced reaction solution; (7) Adjust the pH of the reaction solution after viscosity reduction to neutral, centrifuge to collect the supernatant, concentrate and precipitate again with alcohol, collect the precipitate and redissolve to obtain the crude polysaccharide solution after viscosity reduction. (8) Centrifuge the viscosity-reducing crude polysaccharide solution to obtain the supernatant, pass it through a DEAE-52 anion exchange column, and elute it with NaCl solutions of different concentration gradients. Collect the eluent in separate tubes. (9) Combine the eluents corresponding to the target elution peak in step (8), concentrate and desalt by dialysis, purify by Superdex 200 gel filtration chromatography column, collect the single component, freeze dry to obtain purified red-topped bamboo fungus polysaccharide.

2. The method for preparing polysaccharide from *Dictyophora indica* according to claim 1, characterized in that, In step (1), the sieve is a 60-mesh sieve; In step (2), the hot water temperature for extraction is 75-85℃, the material-to-liquid ratio is 1:36-42 (g / mL), the extraction time is 3-5h, and the extraction is repeated 2-4 times. In step (3), the alcohol precipitation uses 95% (v / v) ethanol, the volume ratio of ethanol to concentrated supernatant is 3.5-4.8:1, the alcohol precipitation is allowed to stand at 4℃ for 20-25h, the centrifugation speed is 8000rpm, and the centrifugation time is 10min.

3. The method for preparing polysaccharide from *Dictyophora indica* according to claim 1, characterized in that, In step (4), the enzymatic hydrolysis uses at least one of papain, complex protease, pectinase, and cellulase; the volume ratio of Sevage reagent to liquid is 1:3-5.

4. The method for preparing polysaccharide from *Dictyophora indica* according to claim 1, characterized in that, In step (5), the macroporous adsorption resin is AB-8 type; the dialysis uses a dialysis bag with a molecular weight cutoff of 3000 Da.

5. The method for preparing polysaccharide from *Dictyophora indica* according to claim 1, characterized in that, In step (6), the heating temperature for the viscosity reduction treatment is 58-65℃ and the reaction time is 0.5-1.5h. In step (6), the viscosity is observed after cooling to room temperature. If the viscosity does not change significantly, step (6) is repeated once or multiple times.

6. The method for preparing polysaccharide from *Dictyophora indica* according to claim 1, characterized in that, In step (7), the second alcohol precipitation uses 95% (v / v) ethanol at 4 times the volume of the concentrated supernatant liquid; In step (8), the DEAE-52 anion exchange column is sequentially eluted with pure water, 0.1M NaCl solution, 0.2M NaCl solution and 0.3M NaCl solution; In step (9), the target elution peak is the second component obtained by elution with 0.1M NaCl solution; the Superdex 200 gel filtration chromatography column uses deionized water as the eluent and the flow rate is 1 mL / min.

7. A polysaccharide from *Dictyophora indicum*, characterized in that, It is prepared by any one of the preparation methods described in claims 1-6.

8. The use of the polysaccharide from *Dictyophora indicum* as described in claim 7 in the preparation of drugs for treating or preventing radiation, frostbite, and mechanical damage.

9. The application according to claim 8, characterized in that, The medicinal dosage of purified polysaccharide from *Dictyophora indicum* is 100-800 mg / kg.

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