Hericium erinaceus polysaccharide and application thereof

Abstract

The application belongs to the technical field of natural product extraction and preparation, and particularly relates to a Hericium erinaceus polysaccharide and application thereof. The Hericium erinaceus polysaccharide is prepared by regulating the mycelium fermentation of Hericium erinaceus through zinc elements, combining with ultrasonic-assisted water extraction and alcohol precipitation, and two-step column chromatography purification. The Hericium erinaceus polysaccharide has excellent antioxidant and anti-inflammatory activities, can significantly relieve intestinal inflammation, restore intestinal peristalsis and secretion function, and protect the intestinal mucosal barrier. The Hericium erinaceus polysaccharide can effectively promote the generation of red blood cells in an anemia model, improve the blood supplementing effect, inhibit the activities of amylase and lipase, and realize the effects of reducing blood sugar and blood fat in combination with bile salts. The preparation process of the Hericium erinaceus polysaccharide obtained by the application is simple, the extraction efficiency is high, the purity is high, the prepared polysaccharide is a natural active ingredient, the safety is high, and the Hericium erinaceus polysaccharide has a good market application prospect.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and preparation technology, specifically relating to a Hericium erinaceus polysaccharide and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Gastrointestinal diseases are common digestive system disorders in clinical practice, encompassing various types such as enteritis, gastrointestinal dysfunction, and damage to the intestinal mucosal barrier. Their pathogenesis is closely related to factors such as intestinal inflammatory responses, abnormal intestinal motility, and impaired secretory function of intestinal goblet cells. Long-term illness not only reduces intestinal nutrient absorption efficiency but also causes a series of uncomfortable symptoms such as abdominal pain, diarrhea, and indigestion, seriously affecting human health. Meanwhile, anemia, a global nutritional deficiency problem, can be caused by various factors such as insufficient red blood cell production and excessive red blood cell destruction, leading to insufficient oxygen supply to the body, causing problems such as fatigue, sallow complexion, and decreased immunity. Gastrointestinal malabsorption is also a significant contributing factor to anemia. The abnormal coordination between gastrointestinal function and hematopoietic function has become a major issue affecting human health. In addition, the incidence of metabolic diseases such as hyperlipidemia and hyperglycemia is rising year by year. These diseases are closely related to abnormal activity of intestinal digestive enzymes and lipid metabolism disorders, and often occur concurrently with gastrointestinal diseases, anemia and other conditions, further aggravating the metabolic burden on the body. Therefore, the development of natural active substances that can protect the stomach and intestines, nourish blood and improve complexion, and regulate glucose and lipid metabolism has become a research hotspot in the fields of biomedicine and functional foods.

[0004] Hericium erinaceus, a traditional edible and medicinal fungus, contains Hericium erinaceus polysaccharides as its core active ingredient. These polysaccharides have been proven to possess various biological activities, including antioxidant, anti-inflammatory, gastrointestinal mucosal protection, and immune regulation. Furthermore, natural polysaccharides are more suitable for long-term consumption and conditioning compared to chemically synthesized drugs due to their high safety and minimal side effects. However, current technologies primarily rely on the extraction and preparation of Hericium erinaceus polysaccharides from conventional mycelia or fruiting bodies. There is still room for improvement in the structure and biological activity of its active ingredients. Moreover, single Hericium erinaceus polysaccharides often cannot simultaneously provide multiple benefits such as gastrointestinal protection, blood replenishment, and regulation of glucose and lipid metabolism. Additionally, research on the modification and enhancement of Hericium erinaceus polysaccharides, especially techniques for regulating polysaccharide structure through trace elements to improve its overall biological activity, has not been fully developed and applied. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a Hericium erinaceus polysaccharide and its applications. Through experimental research, this invention has discovered that Hericium erinaceus polysaccharides, especially zinc-rich Hericium erinaceus polysaccharides, extracted and prepared from Hericium erinaceus exhibit significant anti-inflammatory, antioxidant, gastrointestinal protective, blood-nourishing, and blood sugar-lowering effects, making them suitable as active ingredient components in health foods, pharmaceuticals, and many other fields. Based on the above research findings, this invention has been completed.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides the application of Hericium erinaceus polysaccharide in the preparation of products with anti-inflammatory, antioxidant, gastrointestinal protective, blood-nourishing and skin-beautifying, and blood sugar and lipid-lowering effects.

[0007] The products include, but are not limited to, food, pharmaceuticals, and animal feed.

[0008] A second aspect of the present invention provides a method for preparing Hericium erinaceus polysaccharide, the method comprising: S1. Fermentation culture was used to obtain Hericium erinaceus mycelium, and crude polysaccharide of Hericium erinaceus was extracted by ultrasonic-assisted water extraction and alcohol precipitation. S2. The obtained crude polysaccharide of Hericium erinaceus was purified by DEAE-52 cellulose ion exchange column chromatography and Sephadex G-100 gel column chromatography.

[0009] The present invention has proven through experiments that the Hericium erinaceus polysaccharide components, especially HMZP, obtained by the above method have excellent anti-inflammatory, antioxidant, gastrointestinal protective, blood-nourishing and skin-beautifying, as well as blood sugar and lipid-lowering effects.

[0010] The anti-inflammatory effect is at least manifested in inhibiting the migration and aggregation of neutrophils.

[0011] The antioxidant effect is at least manifested in vitro, specifically by exhibiting good reducing power, total antioxidant capacity, and free radical scavenging ability. The free radicals include at least hydroxyl radicals, superoxide anion radicals, ABTS radicals, and DPPH radicals.

[0012] Specifically, the protection of the gastrointestinal tract manifests as relieving intestinal inflammation, restoring intestinal peristalsis and secretion function, and protecting the intestinal mucosal barrier. The specific effects of replenishing blood and nourishing the complexion are to promote red blood cell production, significantly increase the hemoglobin-related optical density value, and restore red blood cell levels in anemic states.

[0013] Specifically, the blood sugar and lipid-lowering effects are achieved by inhibiting the activity of α-amylase and pancreatic lipase, and by binding with sodium glycocholate and sodium taurocholate to regulate glucose and lipid metabolism.

[0014] Therefore, in a third aspect, the present invention provides a product having the above-mentioned anti-inflammatory, antioxidant, gastrointestinal protective, blood-nourishing and skin-beautifying, and blood sugar and lipid-lowering effects, wherein the product contains at least the Hericium erinaceus polysaccharide obtained by the above preparation method.

[0015] The beneficial technical effects of one or more of the above technical solutions are as follows: The above technical solution utilizes zinc to regulate the fermentation of Hericium erinaceus mycelium, combined with ultrasound-assisted water extraction and alcohol precipitation, and two-step column chromatography purification using DEAE-52 and Sephadex G-100 to obtain high-purity Hericium erinaceus polysaccharides HMP and HMZP. Experimental verification shows that these Hericium erinaceus polysaccharides possess excellent antioxidant and anti-inflammatory activities, with HMZP exhibiting significantly superior activity compared to HMP. They can significantly alleviate intestinal inflammation, restore intestinal peristalsis and secretory function, and protect the intestinal mucosal barrier. They can effectively promote erythrocyte production in anemia models, enhancing blood replenishment. Simultaneously, they can inhibit amylase and lipase activity, and by binding with bile salts, achieve hypoglycemic and hypolipidemic effects.

[0016] The above-mentioned technical solution yields Hericium erinaceus polysaccharide with a simple preparation process, high extraction efficiency, and high purity. The resulting polysaccharide is a natural active ingredient with high safety. It can be widely used in the preparation of foods (including health foods), pharmaceuticals, or feeds that protect the stomach and intestines, nourish blood and improve complexion, and regulate glucose and lipid metabolism, and has good market application prospects. Attached Figure Description

[0017] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0018] Figure 1 Elution curves of DEAE-52 and Sephadex G-100 in Example 1 of this invention: (A) Elution curve of crude polysaccharide from Hericium erinaceus mycelium on a DEAE-52 cellulose ion exchange column; (B) Elution curve of crude polysaccharide from zinc-rich Hericium erinaceus mycelium on a DEAE-52 cellulose ion exchange column; (C) Elution curve of crude polysaccharide component 2 from Hericium erinaceus mycelium on a Sephadex G-100 dextran gel column; (D) Elution curve of crude polysaccharide component 2 from zinc-rich Hericium erinaceus mycelium on a Sephadex G-100 dextran gel column. Numbers indicate component numbers.

[0019] Figure 2 The above are the HPGPC spectra of HMP and HMZP in Embodiment 2 of the present invention; (A) HPGPC spectra of HMP; (B) HPGPC spectra of HMZP; the numbers are peak numbers.

[0020] Figure 3 The FT-IR spectra of HMP and HMZP in Embodiment 2 of the present invention are shown.

[0021] Figure 4 The following are HPLC chromatograms of HMP and HMZP in Example 2 of this invention: (A) HPLC chromatogram of standard monosaccharides; (B) HPLC chromatogram of HMP; (C) HPLC chromatogram of HMZP; The numbers in the figures represent: 1: Man; 2: GlcN; 3: Rib; 4: Rha; 5: GlcUA; 6: GalUA; 7: GalN; 8: Glc; 9: Gal; 10: Xyl; 11: Ara; 12: Fuc.

[0022] Figure 5 The antioxidant activities of HMP and HMZP in Example 3 of this invention are: (A) reducing power; (B) total antioxidant capacity; (C) hydroxyl radical scavenging capacity; (D) superoxide anion radical scavenging capacity; (E) ABTS radical scavenging capacity; and (F) DPPH radical scavenging capacity. Values ​​are expressed as mean ± SD (n=3).

[0023] Figure 6 The effects of HMP and HMZP on acute inflammation in zebrafish in Example 4 of this invention; ### p<0.001 compared to the Ctl group; p<0.001 compared to the CuSO4 group; aaa p<0.001 compared with the HMP-100 group; bbb p<0.001 compared with the HMP-200 group; c p<0.05 compared with the HMP-400 group.

[0024] Figure 7 The effects of HMP and HMZP on intestinal inflammatory cells in zebrafish, as shown in Example 5 of this invention; ### p<0.001 compared to the Ctl group; p<0.001 compared to the TNBS group; aaa p<0.001 compared with the HMP-100 group; bbb p<0.001 compared with the HMP-200 group; c p<0.05 compared with the HMP-400 group.

[0025] Figure 8 This illustrates the effects of HMP and HMZP on the intestinal excretion rate and intestinal peristalsis frequency in zebrafish in Example 5 of the present invention. ### p<0.001 compared to the Ctl group; p<0.05, p<0.01, p<0.001 compared to the TNBS group; b p<0.05 compared with the HMP-200 group.

[0026] Figure 9 The effect of HMP and HMZP on the secretion of zebrafish intestinal goblet cells in Example 5 of the present invention; ### p<0.001 compared to the Ctl group; p<0.01, p<0.001 compared to the TNBS group; cc p<0.01 compared with the HMP-400 group.

[0027] Figure 10 In Example 6 of this invention, HMP and HMZP enhance the ability of erythrocyte formation. ## p<0.01 compared with the NC group; p<0.01 compared with the MC group.

[0028] Figure 11 This refers to the inhibitory effects of HMP and HMZP on amylase in Example 7 of this invention. At the same sample concentration, different letters represent significant differences.

[0029] Figure 12 This refers to the lipase inhibitory capacity of HMP and HMZP in Example 8 of this invention. At the same sample concentration, different letters represent significant differences.

[0030] Figure 13 This refers to the sodium glycocholate binding capacity of HMP and HMZP in Example 8 of this invention. At the same sample concentration, different letters represent significant differences.

[0031] Figure 14 This refers to the sodium taurocholate binding capacity of HMP and HMZP in Example 8 of this invention. At the same sample concentration, different letters represent significant differences. Detailed Implementation

[0032] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0033] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for the purpose of describing specific embodiments and not for limiting the scope of protection of the present invention.

[0034] In a typical embodiment of the present invention, Hericium erinaceus polysaccharide is provided for use in the preparation of products with anti-inflammatory, antioxidant, gastrointestinal protective, blood-nourishing and skin-beautifying, blood sugar-lowering and lipid-lowering effects.

[0035] The products include, but are not limited to, food, pharmaceuticals, and animal feed.

[0036] It should be noted that the term "food" used in this invention should be interpreted broadly, and can be understood as any form that can be eaten. For example, food in this invention includes ordinary food and special food. The special food mentioned in this invention includes health food and food for special medical purposes. Ordinary food, in contrast to special food, is food suitable for everyone.

[0037] The food includes, but is not limited to, solid food and liquid food; the solid food includes, but is not limited to, baked goods, candy, and solid beverages; the liquid food includes, but is not limited to, liquid beverages.

[0038] The drug can be administered in unit doses, and the dosage form can be liquid or solid. Liquid dosage forms can be true solutions, colloids, microparticles, emulsions, or suspensions. Other dosage forms include tablets, capsules, pellets, aerosols, pills, powders, solutions, emulsions, granules, suppositories, lyophilized powder for injection, inclusion complexes, implants, patches, and liniments.

[0039] The feed refers to the food for animals raised in agriculture or animal husbandry. The rose waste liquid polysaccharide of the present invention can be added as a feed additive to any type of feed, including but not limited to complete compound feed, concentrated feed and premixed feed.

[0040] In another specific embodiment of the present invention, the Hericium erinaceus polysaccharide can be obtained by extracting and purifying Hericium erinaceus mycelium through ordinary fermentation culture or zinc-enriched fermentation culture; when the ordinary fermentation culture is used, the corresponding product is Hericium erinaceus mycelium polysaccharide HMP; when the zinc-enriched fermentation culture is used, the corresponding product is Hericium erinaceus zinc-enriched mycelium polysaccharide HMZP.

[0041] The molecular weight (Mw) of the hericium erinaceus polysaccharide is 1-10×10⁻⁶. 5 Da, further, is 4-7×10 5 In one specific embodiment of the present invention, the Hericium erinaceus polysaccharide is a pyranose linked by α- and β-glycosidic bonds, and mainly contains glucose, with the glucose content accounting for more than 90%.

[0042] In another specific embodiment of the present invention, the monosaccharide composition of the Hericium erinaceus mycelial polysaccharide HMP further includes mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, galactosamine, galactose and xylose. The monosaccharide composition of the zinc-rich mycelial polysaccharide HMZP from Hericium erinaceus also includes mannose, glucosamine, ribose, glucuronic acid, galacturonic acid, galactosamine, galactose, and arabinose.

[0043] In another specific embodiment of the present invention, a method for preparing Hericium erinaceus polysaccharide is provided, the method comprising: S1. Fermentation culture was used to obtain Hericium erinaceus mycelium, and crude polysaccharide of Hericium erinaceus was extracted by ultrasonic-assisted water extraction and alcohol precipitation. S2. The obtained crude polysaccharide of Hericium erinaceus was purified by DEAE-52 cellulose ion exchange column chromatography and Sephadex G-100 gel column chromatography.

[0044] In step S1, the fermentation culture can be carried out using either a conventional fermentation culture method or a zinc-enriched fermentation culture method. Specifically, the conventional fermentation culture method involves inoculating the Hericium erinaceus seed liquid into a fungal culture medium (such as corn steep liquor powder medium) for liquid fermentation culture. The zinc-enriched fermentation culture method is the same as the conventional fermentation culture method, except that the culture medium used is a zinc-enriched fungal culture medium (such as corn steep liquor powder zinc-enriched medium). The corn steep liquor powder zinc-enriched medium is made by adding zinc element to the corn steep liquor powder medium. The zinc element is added in the form of zinc salt, specifically zinc sulfate, with a concentration of 10-500 mg / L, preferably 150 mg / L.

[0045] The specific method of the ultrasonic-assisted water extraction and alcohol precipitation method includes: after drying the mycelium of Hericium erinaceus, crushing and sieving it (such as through an 80-mesh sieve), adding water at a material-to-water ratio of 1:10-30 (preferably 1:20, g / mL), ultrasonically crushing and heating the mixture, centrifuging and collecting the supernatant. This step can be repeated 2-4 times (preferably 3 times); combining the obtained supernatants, concentrating them to 1 / 2 to 1 / 3 of the original supernatant volume, then adding 1-10 times the volume of the concentrated supernatant (preferably 3 times the volume) of ethanol, letting it stand overnight for alcohol precipitation, centrifuging to obtain the precipitate, and drying it to obtain the final product.

[0046] The specific conditions for the ultrasonic fragmentation treatment are as follows: treatment at 100-500 W (preferably 300 W) for 1-30 min (preferably 10 min); the heating treatment can be water bath heating treatment, and the specific heating conditions are as follows: water bath heating treatment at 80-100℃ (preferably 90℃) for 1-5 h (preferably 2 h).

[0047] The ethanol is high-concentration ethanol, and the ethanol can be 70% or higher concentration ethanol, such as 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ethanol. In one specific embodiment of the present invention, the ethanol is 95% ethanol.

[0048] In another specific embodiment of the present invention, the method for extracting crude polysaccharides from Hericium erinaceus using ultrasound-assisted water extraction and alcohol precipitation further includes resolubilizing the dried precipitate and removing proteins. Specifically, the method includes adding the precipitate to deionized water, wherein the mass-to-volume ratio of the precipitate to deionized water is 1:10-100 (preferably 1:50, g / mL). The protein removal method can employ any method known in the art, such as the Sevag method, and is not specifically limited herein.

[0049] The specific method of step S2 includes: purification using a DEAE-52 cellulose ion exchange column, eluting sequentially with water and 0.05 mol / L and 0.1 mol / L NaCl solutions at a flow rate of 0.5-2 mL / min (preferably 1 mL / min), collecting 30 tubes of eluent for each elution concentration, with each tube containing 0.5-4 mL (preferably 2 mL), and collecting tubes 1-30 (water elution fraction), tubes 31-60 (0.05 mol / L NaCl elution fraction), and tubes 61-90 (0.1 mol / L NaCl elution fraction). The 0.05 mol / L NaCl elution fraction is then purified using a Sephadex G-100 dextran gel column, specifically eluting with water at a flow rate of 0.05-0.5 mL / min (preferably 0.1 mL / min), with each tube containing 0.5-4 mL (preferably 2 mL), and collecting the fractions corresponding to a single absorption peak.

[0050] In another specific embodiment of the present invention, the elution process is detected tube by tube using the phenol-sulfuric acid method, and the elution curve is plotted with the number of tubes as the abscissa and the absorbance (A490 nm) as the ordinate.

[0051] In one specific embodiment of the present invention, when using Sephadex G-100 dextran gel column for purification, 9-23 tubes of crude polysaccharide from Hericium erinaceus mycelium were collected and freeze-dried to obtain HMP; 7-19 tubes of zinc-rich mycelium crude polysaccharide were collected and freeze-dried to obtain HMZP.

[0052] The present invention has proven through experiments that the Hericium erinaceus polysaccharide components, especially HMZP, obtained by the above method have excellent anti-inflammatory, antioxidant, gastrointestinal protective, blood-nourishing and skin-beautifying, as well as blood sugar and lipid-lowering effects.

[0053] The anti-inflammatory effect is at least manifested in inhibiting the migration and aggregation of neutrophils.

[0054] The antioxidant effect is at least manifested in vitro, specifically by exhibiting good reducing power, total antioxidant capacity, and free radical scavenging ability. The free radicals include at least hydroxyl radicals, superoxide anion radicals, ABTS radicals, and DPPH radicals.

[0055] Specifically, the protection of the gastrointestinal tract manifests as relieving intestinal inflammation, restoring intestinal peristalsis and secretion function, and protecting the intestinal mucosal barrier. The specific effects of replenishing blood and nourishing the complexion are to promote red blood cell production, significantly increase the hemoglobin-related optical density value, and restore red blood cell levels in anemic states.

[0056] Specifically, the blood sugar and lipid-lowering effects are achieved by inhibiting the activity of α-amylase and pancreatic lipase, and by binding with sodium glycocholate and sodium taurocholate to regulate glucose and lipid metabolism.

[0057] Therefore, in another specific embodiment of the present invention, the product provided above that has anti-inflammatory, antioxidant, gastrointestinal protective, blood-nourishing and beauty-enhancing, and blood sugar and lipid-lowering effects, the product at least contains Hericium erinaceus polysaccharide obtained by the above preparation method.

[0058] The products mentioned above include, but are not limited to, food, pharmaceuticals, and animal feed.

[0059] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0060] Example 1: Fermentation culture and polysaccharide extraction and purification Hericium erinaceus spawn (CICC 14080) was transferred from agar slant to PDA solid medium plates and incubated at 25°C for 14 days until robust, white mycelium covered the plate. A 1 cm sample was then inoculated using an inoculation spatula in a clean bench. 2 Large and small inoculum strains were inoculated into PDA liquid medium and cultured in shake flasks at 25℃ and 180 r / min for 10 days to obtain Hericium erinaceus seed culture solution. Then, the Hericium erinaceus seeds were inoculated into corn steep liquor medium (8 g / L corn steep liquor, 30 g / L glucose, 1 g / L KH2PO4, 0.25 g / L MgSO4·7H2O, 1 L water, natural pH), and 250 mL of the medium was placed in a 500 mL Erlenmeyer flask and cultured in shake flasks at 25℃ and 180 r / min for 14 days.

[0061] Hericium erinaceus mycelium and zinc-enriched mycelium were obtained by culturing with corn steep liquor at zinc concentrations of 0 and 150 mg / L (zinc sulfate, calculated as elemental zinc). Polysaccharides were extracted from Hericium erinaceus using an ultrasonic-assisted water extraction and alcohol precipitation method. The dried Hericium erinaceus mycelium was crushed into powder and passed through an 80-mesh sieve. The powder was accurately weighed and mixed with water at a ratio of 1:20 (g / mL). The mixture was ultrasonically crushed (300W, 10 min), heated in a water bath at 90℃ for 2 h, centrifuged (10000 r / min, 10 min), and the supernatant was collected. The residue was added to water, and the ultrasonic-assisted water extraction and alcohol precipitation method was repeated three times. The three polysaccharide extracts were mixed and concentrated to 1 / 3 volume using a rotary evaporator, followed by the addition of three volumes of 95% ethanol solution. After standing overnight for alcohol precipitation, the mixture was centrifuged at 10000 r / min for 10 min, the precipitate was retained, and dried at 55℃ to constant weight. The dried sample was ground and dissolved in an appropriate amount of deionized water (25 mg / mL) in a 55℃ constant temperature water bath for 2 h. The supernatant was collected by centrifugation. A reagent (chloroform:n-butanol = 5:1, v / v) was added at a volume ratio of 4:1, and the mixture was shaken thoroughly for 15 min. Then, it was centrifuged at 8000 r / min for 10 min to remove the organic phase and the white emulsion layer (denatured protein) at the interface between the organic and aqueous phases. This process was repeated until the emulsion layer at the interface between the organic and aqueous phases disappeared, yielding a crude polysaccharide aqueous solution of Hericium erinaceus. After freeze-drying, crude polysaccharide of Hericium erinaceus mycelium was obtained.

[0062] DEAE-52 cellulose ion exchange column chromatography Accurately weigh 50 mg of crude polysaccharide from Hericium erinaceus mycelium or zinc-enriched crude polysaccharide, dissolve in 6 mL of deionized water, incubate at 55℃ with shaking for 2 h, then centrifuge at 10000 r / min for 10 min at 4℃. Filter the supernatant through a 0.45 μm microporous membrane. Load the filtrate onto a DEAE-52 cellulose column (2.6 cm × 30 cm), and elute sequentially with a gradient of deionized water, 0.05 mol / L, and 0.1 mol / L NaCl solution (flow rate 1 mL / min). Collect 2 mL samples per tube, changing the elution concentration gradient every 30 tubes. Analyze each tube using the phenol-sulfuric acid method, measuring absorbance at 490 nm using a microplate reader, and plot the elution curve. Combine the solutions from the tubes corresponding to the main peak, and lyophilize to collect the polysaccharide sample.

[0063] Sephadex G-100 gel column purification The fractions obtained from DEAE-52 chromatography were loaded onto a Sephadex G-100 gel column (1.6 cm × 50 cm) and eluted with deionized water at a flow rate of 0.1 mL / min. One tube was collected for every 2 mL of elution, for a total of 30 tubes. The phenol-sulfuric acid method was used for tube-by-tube detection, and absorbance was measured at 490 nm using a microplate reader to plot elution curves. Fractions corresponding to single absorption peaks were combined, lyophilized, and HMP and HMZP were obtained.

[0064] Experimental results The DEAE-52 elution curves of crude polysaccharide from Hericium erinaceus mycelium and zinc-enriched crude polysaccharide from Hericium erinaceus, and the Sephadex G-100 elution curves of crude polysaccharide fraction 2 from Hericium erinaceus mycelium and zinc-enriched crude polysaccharide fraction 2 from Hericium erinaceus mycelium are shown below. Figure 1 As shown.

[0065] HMP and HMZP were obtained by separation and purification using a DEAE-52 cellulose ion exchange column and a Sephadex G-100 dextran gel column. Crude polysaccharides from Hericium erinaceus mycelium and zinc-enriched Hericium erinaceus mycelium were purified using a DEAE-52 cellulose ion exchange column, eluting with deionized water and 0.05 mol / L NaCl solution to obtain two fractions. Fraction 2 (crude polysaccharide from Hericium erinaceus mycelium, collected in tubes 38-53; crude polysaccharide from zinc-enriched mycelium, collected in tubes 39-56) was further purified using a Sephadex G-100 dextran gel column. Figure 1 It can be seen that both crude polysaccharide component 2 of Hericium erinaceus mycelium and crude polysaccharide component 2 of zinc-rich Hericium erinaceus mycelium exhibit a single symmetrical peak. After collecting (collecting crude polysaccharide from tubes 9-23 of Hericium erinaceus mycelium; collecting crude polysaccharide from tubes 7-19 of zinc-rich mycelium), HMP and HMZP were obtained by freeze drying.

[0066] Example 2: Structural Characterization of HMP and HMZP (1) Determination of molecular weight of HMP and HMZP 10 mg of HMP and HMZP were dissolved in 1 mL of 0.1 mol / L NaNO3. Molecular weight was determined using the HPGPC method. Instrumentation: Waters high-performance liquid chromatograph; Waters differential refractive index detector; column: aqueous gel filtration column (8 × 300 mm, TSKgel GMPWxl); mobile phase: 0.1 mol / L NaNO3; flow rate: 0.6 mL / min; column temperature: 30℃; injection volume: 30 μL; processing software: Empower3. A standard curve was plotted using a series of narrow-distribution pullulan polysaccharides as standards, with retention time on the x-axis and the reciprocal of weight-average molecular weight (Mw) on the y-axis, and the regression equation was derived. The corresponding Mw was calculated by substituting the sample data into the curve.

[0067] (2) FT-IR analysis of HMP and HMZP Dry HMP or HMZP samples and dry KBr particles were placed in a clean agate mortar and ground thoroughly. The ground powder was then pressed into a thin sample slide. KBr was prepared into a slide using the same method and placed in the sample chamber of a Fourier transform infrared spectrometer as a blank background. The sample slide was then placed in the optical path, and the infrared spectrum of the polysaccharide sample was measured at room temperature.

[0068] (3) Determination of monosaccharide composition of HMP and HMZP The monosaccharide composition of the samples was determined by high-performance liquid chromatography (HPLC). 0.1 g of HMP or HMZP sample was accurately weighed into a chromatographic vial, 5 mL of 2 mol / L trifluoroacetic acid was added, and the vial was sealed with nitrogen (10 mL / min, 1 min), then hydrolyzed in a 120 ℃ oven for 2 h. 1 mL of the hydrolysis residue was taken, 1 mL of methanol was added, and the mixture was dried under nitrogen in a 70 ℃ water bath. This process was repeated twice to remove trifluoroacetic acid. 1 mL of 0.3 mol / L NaOH solution was added to fully dissolve the residue, yielding the polysaccharide hydrolysate. Take 400 μL of monosaccharide standard solution or hydrolysate of the test sample and mix thoroughly with 400 μL of 1-phenyl-3-methyl-5-pyrazolone methanol solution (0.5 mol / L). Incubate at 70℃ for 120 min, cool to room temperature, add 400 μL of 0.3 mol / L HCl to neutralize to pH 6-8, and add 1200 μL of water. Add an equal volume of chloroform to the mixture, vortex to mix, then let stand, discard the chloroform phase, and repeat the extraction twice. Filter the supernatant through a 0.45 μm microporous membrane and analyze by HPLC.

[0069] Instrumentation: High-performance liquid chromatography (HPLC); Column: C18 column (NanoChrom, 250 mm × 4.6 mm, 5 μm); Mobile phase A: PBS (0.1 M, pH 6.4); Mobile phase B: Acetonitrile; Detection wavelength: 245 nm; Column temperature: 30℃; Flow rate: 1 ml / min; Injection volume: 20 μL. Linear gradient elution: 0–30 min, 95%–80% A; 30.1–45 min, 60% A; 45.1–60 min, 95% A. The monosaccharide composition of each sample was calculated by comparing it with monosaccharide standards. The standard monosaccharides are as follows: mannose (Man), glucosamine (Glc-N), ribose (Rib), rhamnose (Rha), glucuronic acid (Glc-UA), galacturonic acid (Gal-UA), galactoseamino (Gal-N), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara), and fucose (Fuc).

[0070] Experimental results (1) Molecular weight results The HPGPC spectra of HMP and HMZP are as follows: Figure 2 As shown.

[0071] Depend on Figure 2 As observed, the Mw of HMP is 6.42 × 10⁻⁶. 5 The Mw of Da,HMZP is 5.27×10 5 The results showed that the molecular weight of HMZP decreased compared to HMP after the addition of zinc.

[0072] (2) FT-IR analysis of HMP and HMZP The FT-IR spectra of HMP and HMZP are as follows: Figure 3 As shown in the infrared spectra, both HMP and HMZP exhibit typical polysaccharide characteristic peaks. Both HMP and HMZP are pyranoses linked by α- and β-glycosidic bonds.

[0073] (3) Determination of monosaccharide composition of HMP and HMZP The HPGPC spectra of HMP and HMZP are as follows: Figure 4As shown in Table 1, the molar percentage of monosaccharide composition of HMP and HMZP are presented. HMP mainly contains Glc residues, and also contains Man, GlcN, Rib, Rha, GlcUA, GalUA, GalN, Gal, and Xyl residues. HMZP mainly contains Glc residues, and also contains Man, GlcN, Rib, GlcUA, GalUA, GalN, Gal, and Ara residues.

[0074] Table 1. Monosaccharide composition (molar percentage) of HMP and HMZP

[0075] Note: " / " indicates not detected.

[0076] Example 3: Analysis of the in vitro antioxidant activity of HMP and HMZP (1) Determination of the reducing power of HMP and HMZP The reducing power of HFCP and HMCP was determined using the Prussian blue method. After preparing a series of polysaccharide sample aqueous solutions, 1 mL of the sample, 2.5 mL of phosphate buffer (0.2 mol / L, pH 6.6), and 1 mL of potassium ferricyanide solution (10 g / L) were mixed thoroughly in a test tube and placed in a 50℃ water bath for 20 min. Then, 2 mL of trichloroacetic acid solution (100 g / L) and 1.2 mL of ferric chloride solution (1 g / L) were added, mixed well, and the absorbance was measured at 700 nm. Vitamin C was used as a positive control.

[0077] (2) Determination of the total antioxidant capacity of HMP and HMZP Transfer 0.2 mL of the sample to be tested or the vitamin C standard solution to a clean test tube, and add 2 mL of the P reaction solution prepared by trisodium phosphate (28 mmol / L), ammonium molybdate (4 mmol / L), and sulfuric acid (0.6 mol / L) to each tube. Heat the mixed solution in a 95℃ constant temperature water bath for 90 min, cool to room temperature, and then use an ELISA reader to detect the absorbance of the sample group and the vitamin C group at a wavelength of 695 nm.

[0078] (3) Determination of the hydroxyl radical scavenging ability of HMP and HMZP A composite detection method was constructed based on the Fenton reaction and salicylic acid colorimetric system. The experimental procedure was as follows: Ferrous sulfate solution (9 mmol / L, 1 mL), polysaccharide test solution (1 mL), salicylic acid ethanol solution (9 mmol / L, 1 mL), and hydrogen peroxide solution (8.8 mmol / L, 1 mL) were sequentially added to a clean test tube and vortexed to mix. The mixture was then incubated at 37℃ for 30 min, centrifuged (6000 r / min, 10 min), and the supernatant was collected for absorbance detection at a wavelength of 510 nm. The hydroxyl radical scavenging rate was calculated according to Formula 1.

[0079]

[0080] In the formula, A0 means the absorbance of the blank control (deionized water replaces the sample), and A means the absorbance of the sample after the reaction.

[0081] (4) Determination of the superoxide anion radical scavenging capacity of HMP and HMZP Take 1 mL of the test sample and add 2 mL of pre-prepared Tris-HCl buffer (50 mmol / L, pH 8.2) to a test tube, and vortex to mix. Incubate the mixture in a 25°C water bath for 20 min, then quickly add 0.4 mL of preheated pyrogallol solution (5 mmol / L), and immediately vortex to ensure complete reaction. After mixing, immediately monitor the absorbance at 325 nm using a UV spectrophotometer, collecting absorbance data every 20 s for a total recording time of 3 min. A blank control group was set up in the experiment, using an equal volume of deionized water instead of the sample solution and performing the same procedure. Calculate the superoxide anion scavenging rate according to Formula 2.

[0082]

[0083] In the formula, S0 means the slope of the absorbance of the blank control (deionized water instead of the sample), and S means the slope of the absorbance of the sample after the reaction.

[0084] (5) Determination of ABTS radical scavenging ability of HMP and HMZP A stock solution of 7 mmol / L ABTS was mixed with a 4.9 mmol / L potassium persulfate solution at a volume ratio of 1:1 and incubated at room temperature for 20 h in the dark to obtain an ABTS stock solution. This stock solution was then serially diluted with phosphate buffer (pH 7.4, 0.1 mol / L) and adjusted to an absorbance of 0.70 ± 0.02 (734 nm) using spectrophotometry to obtain the standard ABTS working solution. 3 mL of the working solution and 1 mL of the test sample were mixed thoroughly in a light-protected test tube and reacted at room temperature in the dark for 6 min. The absorbance was immediately measured at 734 nm. The ABTS radical scavenging rate was calculated according to Formula 3.

[0085]

[0086] In the formula, A means the absorbance of the sample solution after reacting with ABTS working solution, Apair means the absorbance of the sample solution after reacting with phosphate buffer, and Ablank means the absorbance of deionized water after reacting with ABTS working solution.

[0087] (6) Determination of DPPH radical scavenging capacity of HMP and HMZP Take 2 mL of the polysaccharide test solution into a light-protected centrifuge tube, add an equal volume of DPPH-ethanol solution (0.2 mmol / L), and vortex to mix. Incubate the mixture in a dark chamber for 30 min, and then measure the absorbance at 517 nm using a UV spectrophotometer. Calculate the DPPH free radical scavenging rate according to Formula 4.

[0088]

[0089] In the formula, A means the absorbance of the sample solution and the DPPH-ethanol solution mixture, A0 means the absorbance of the sample solution and the ethanol solution mixture, and A1 means the absorbance of the deionized water and the DPPH-ethanol solution mixture.

[0090] Experimental results The in vitro antioxidant test results of HMP and HMZP are as follows: Figure 5 As shown in Table 3.

[0091] Table 3 Antioxidant capabilities of HMP and HMZP

[0092] Note: Absorbance at 4000 mg / L is used as the reducing power and total antioxidant capacity. Data from different letter labels in the same industry show significant differences. p <0.05).

[0093] In vitro antioxidant experiments revealed that HMZP exhibited significantly superior antioxidant activity compared to HMP. In reducing power tests, the absorbance of HMZP (1.05±0.05) was significantly higher than that of HMP (0.91±0.04). p <0.05 indicates a stronger ability to donate electrons or hydrogen atoms. Simultaneously, HMZP significantly enhances the scavenging efficiency for various free radicals: its scavenging efficiency for hydroxyl radicals (-OH) and superoxide anions (O₂) is significantly improved. 2- EC of ABTS and DPPH free radicals 50 The values ​​decreased from 826.76 mg / L, 151.66 mg / L, 947.63 mg / L, and 505.12 mg / L of HMP to 470.78 mg / L, 94.64 mg / L, 788.71 mg / L, and 324.84 mg / L, respectively, representing a decrease of 35.7% to 43%. The most significant improvement was observed in the scavenging ability against hydroxyl radicals (EC50). 50 (Reduced by 43%).

[0094] Example 4: Determination of the activity of HMP and HMZP in alleviating acute inflammation A Tg(ELE:EGFP) transgenic zebrafish inflammation model was used, and the anti-inflammatory effects of HMP and HMZP were evaluated using fluorescence tracing technology. The experiment included a blank control group, a model group, a positive control group, and a sample group. The positive control group was supplemented with 20 μM indomethacin, while the sample groups were supplemented with HMP or HMZP (100, 200, and 400 μg / mL). All groups were brought to a final volume of 2.0 mL with E3 culture medium. Except for the blank control group, the other groups were pre-incubated in the dark for 2 h, followed by 1 h of exposure to 20 μM CuSO4 to stimulate acute inflammation. Subsequently, the zebrafish juveniles were placed laterally under a fluorescence microscope with both eyes aligned to observe the neutrophil inflammatory response, and the migration of neutrophils to the lateral line region was counted. An Olympus SZX16 fluorescence microscope equipped with a CellSens Standard imaging system was used to capture fluorescence signals and statistically analyze the number of inflammatory cell aggregates.

[0095] Experimental results The effects of HMP and HMZP on acute inflammation in zebrafish, such as Figure 6 As shown.

[0096] In a zebrafish acute inflammation model, the copper sulfate model group exhibited significant neutrophil aggregation near the thalamus and above the lateral line, indicating successful induction of the inflammatory response. The positive control group (Indometacin) showed significantly reduced neutrophil aggregation compared to the model group, validating the reliability of the experimental system. Analysis of the intervention effects of HMP and HMZP showed that both drugs exhibited a dose-dependent anti-inflammatory trend; with increasing concentration, the amount of neutrophils above the lateral line gradually decreased. HMZP showed a more pronounced inhibitory effect at the highest concentration, with the number of neutrophils above the lateral line in its fluorescence image approaching the level of the normal control group. Furthermore, the number of neutrophils above the lateral line in 400 μg / mL HMZP was reduced by 82.66% compared to the model group, a statistically significant difference (p<0.001). Low to medium concentrations of HMZP demonstrated superior anti-inflammatory activity compared to the corresponding concentrations of HMP. These results indicate that HMZP is significantly more effective than HMP in regulating neutrophil migration or inhibiting inflammation.

[0097] Example 5: Verification of intestinal protective efficacy (1) Model construction and grouping The experiment selected healthy developing children. AB Wild-type zebrafish and genetically modified strains Tg ( ELE:EGFP Neutrophil-labeled zebrafish were used as the experimental subjects. Juvenile fish from the same batch that reached 72 hpf were randomly assigned to 24-well culture plates, with 10 juvenile fish (2 mL) loaded into each well, and three independent biological replicates were set up. The experimental groups included a blank control group, a model group, a positive control group, and groups treated with different concentrations of samples.

[0098] The control group maintained an E3 culture medium environment throughout the entire process. The model group, positive control group, and sample treatment group were treated with 50 μg / mL 2,4,6-trinitrobenzenesulfonic acid solution (TNBS) added to the E3 culture medium to establish the pathological model. After 24 h of light-protected incubation, each group was replaced with freshly prepared treatment medium. After 48 h of light-protected incubation, TNBS was thoroughly removed by rinsing three times with E3 culture medium. The model group was then replaced with E3 culture medium, the positive control group with 2 μmol / L 5-aminosalicylic acid (5-ASA) culture medium, and the sample treatment group with culture medium containing 2 μmol / L 5-ASA and HMP (100, 200, 400 μg / mL) or HMZP (100, 200, 400 μg / mL), and the standard culture conditions were maintained for another 24 h. Independent ventilation systems were used in all procedures to ensure the stability of the culture environment.

[0099] (2) Determination of intestinal inflammatory cells in zebrafish Select 72 hpf Tg ( ELE:EGFP ) Neutrophil fluorescent labeling was used to label juvenile zebrafish for experiments. The experimental groups and operations were carried out as described in (1) above. Phenotypic observation was performed using an Olympus SZX16 fluorescence microscope equipped with a CellSensStandard imaging system. Fluorescence signals in the gastrointestinal region of the juvenile fish were captured in lateral focusing mode, and the number of inflammatory cell aggregations was statistically analyzed.

[0100] (3) Determination of zebrafish intestinal extravasation rate Select 72 hpf AB Wild-type zebrafish juveniles were used in the experiment. The experimental groups were as described in (1) above. After TNBS treatment for 48 h, the TNBS residue was washed away, and each group was stained with 0.2% calcein solution for 1 h. After washing three times with E3 culture medium, the blank group and the model group were photographed under a fluorescence microscope, and the mean integrated optical density (IOD) of the zebrafish intestine was counted and named as IOD. C0 and IOD T0 The model group was replaced with E3 culture medium, the positive control group with 2 μmol / L 5-ASA culture medium, and the sample treatment groups with culture medium containing 2 μmol / L 5-ASA and HMP (100, 200, 400 μg / mL) or HMZP (100, 200, 400 μg / mL). Standard culture conditions were maintained for 16 h. Images were acquired again using an Olympus SZX16 fluorescence microscope and CellSens Standard software. The mean IOD values ​​of the blank group, model group, positive control group, and different sample treatment groups were statistically analyzed and named as IOD. C IOD T IOD A and IOD H Calculate the intestinal excretion rate according to formulas 1 and 2.

[0101]

[0102]

[0103] (4) Determination of the number of intestinal peristalsis in zebrafish Select 72 hpf ABWild-type zebrafish juveniles were used in the experiment. The experimental groups and procedures were as described in (1) above. After the drug treatment, the juveniles in each group were transferred to 0.2% calcein solution for staining in the dark for 1 h. Then, the juveniles were washed three times with E3 culture medium to remove residual dye. Eight juveniles from each treatment group were randomly selected. After anesthetizing the zebrafish, they were horizontally fixed in a methylcellulose matrix. The mid-intestinal region was dynamically imaged using an inverted fluorescence microscope, and the fluctuation of calcein fluorescence signal was continuously recorded within 1 min. The number of intestinal peristalsis per minute was used as the evaluation index. Each group of experiments was independently repeated three times to ensure data reliability.

[0104] (5) Determination of secretion from goblet cells in zebrafish intestine Select 72 hpf AB Wild-type zebrafish juveniles were used in the experiment. The experimental groups and operations were performed as described in (1) above. After drug treatment, the juvenile fish were washed three times with E3 culture medium. The zebrafish were fixed with 4% paraformaldehyde solution at 4℃ for 12 h to achieve tissue solidification. After removing the fixative, they were washed three times with a mixture of 1% hydrochloric acid and ethanol. Freshly prepared 2% Alcian blue staining working solution (glacial acetic acid-anhydrous ethanol volume ratio 2:8) was transferred in, and the fish were stained at room temperature in the dark for 2 h before being stained at 4℃ for 12 h to enhance specific binding. After staining was terminated, the fish were washed three times with acidic decolorizing solution (1% hydrochloric acid-ethanol) to thoroughly remove non-specific adsorbed dyes. Images were acquired using a Zeiss fluorescence microscope, focusing on capturing the distribution characteristics of goblet cells in the intestine. For quantitative analysis, ImagePro Plus V6.0 software was used to perform statistical analysis of the stained area in the intestinal region.

[0105] Experimental results (1) Effects of HMP and HMZP on intestinal inflammatory cells in zebrafish The effects of HMP and HMZP on intestinal inflammatory cells in zebrafish, such as Figure 7 As shown.

[0106] According to the experimental results, the fluorescence signal of neutrophils in the gastrointestinal tract of zebrafish in the TNBS model group was significantly enhanced compared with that in the control group (p<0.001), indicating that the inflammatory response was successfully induced and accompanied by abnormal aggregation of immune cells. Under the intervention of the positive control drug 5-ASA, neutrophil infiltration was significantly alleviated, verifying the reliability of the experimental system. HMP and HMZP showed strong dose-dependent effects. However, the number of neutrophil fluorescence signals in the gastrointestinal tract of 400 μg / mL HMZP was significantly lower than that of 400 μg / mL HMP (p<0.05), indicating that HMZP has better activity in alleviating enteritis than HMP.

[0107] (2) Effects of HMP and HMZP on intestinal extravasation rate and number of intestinal motility in zebrafish The effects of HMP and HMZP on intestinal extravasation rate and intestinal motility in zebrafish, such as Figure 8 As shown.

[0108] Analysis of zebrafish intestinal function experiments showed that the TNBS model group had significantly lower intestinal efflux rate and peristaltic frequency compared to the normal control group (p<0.001), indicating impaired intestinal function. After intervention with the positive control drug 5-ASA, both indicators showed a significant recovery trend. Both HMP and HMZP treatment groups exhibited dose-dependent improvement effects; with increasing concentration, intestinal efflux rate and peristaltic frequency gradually increased, reaching the optimal effect at 400 μg / mL. Data showed that the intestinal efflux rate of 200 μg / mL HMZP (60.04%) was significantly higher than that of 200 μg / mL HMP (49.97%, p<0.05). Therefore, HMZP has better activity than HMP in alleviating impaired intestinal motility in zebrafish.

[0109] (3) Effects of HMP and HMZP on secretion by zebrafish intestinal goblet cells The effects of HMP and HMZP on the secretion of zebrafish intestinal goblet cells, such as Figure 9 As shown.

[0110] In zebrafish, goblet cells in the intestine primarily secrete mucin, forming a protective barrier to safeguard epithelial cells. When the intestine is damaged, the number of goblet cells significantly decreases. This experiment used Alcian blue dye to specifically label acidic mucin and statistically analyzed the staining area of ​​intestinal goblet cells to assess their secretory function. Experimental data showed that the TNBS-induced inflammation model group had a significantly reduced goblet cell staining area compared to the normal control group (p<0.001), indicating that inflammation severely impaired goblet cell function. The number of goblet cells significantly increased after intervention with the positive control drug 5-ASA, validating the model's effectiveness. Both HMP and HMZP intervention drugs showed concentration-dependent ameliorative effects, but their effects differed in intensity. The staining area of ​​intestinal goblet cells with 400 μg / mL HMZP was 2.78 × 10⁻⁶. 4 μm 2 The intestinal goblet cell staining area was significantly higher than that of 400 μg / mL HMP (2.33 × 10⁻⁶). 4 μm 2 (p<0.01). Therefore, the HMZP group showed better recovery of goblet cell secretory function than the HMP group.

[0111] Example 6 Evaluation of activity in improving anemia Healthy wild-type AB zebrafish that had reached 56 hpf (hour post-fertilization) were placed in six-well plates and divided into 5 groups of 10 fish each. The blank control group (NC) was given culture water; the anemia model group (MC) was supplemented with 0.1 μg / mL phenylhydrazine; the HMP control group (HMP) was supplemented with 0.1 μg / mL phenylhydrazine and HMP (25 μg / mL); and the low, medium, and high-dose HMZP intervention groups (HMZP-I, HMZP-II, and III) were supplemented with 0.1 μg / mL phenylhydrazine and different concentrations of HMZP (5 μg / mL, 15 μg / mL, and 25 μg / mL). The zebrafish in each group were incubated for 3 dpf (day post-fertilization). The samples were treated with an anesthetic (cocaine) for 1 min, and the embryos were stained with o-anisidine dye in the dark for 20 min. Images were acquired under a microscope. The optical density (IOD) values ​​of the stained areas were quantified using ImageJ (1.0, USA) software. A model of anemia was created by phenylhydrazine destroying red blood cells. The hemoglobin in mature red blood cells catalyzes the oxidation of o-anisidine by hydrogen peroxide, resulting in a rust-colored (reddish) appearance. When the number of mature red blood cells decreases (anemia), the average optical density value of red blood cell staining at the heart of juvenile fish will decrease.

[0112] Depend on Figure 10 It was found that after phenylhydrazine-induced modeling, the IOD value of the heart in juvenile zebrafish in the MC group was significantly lower than that in the NC group (p<0.01). Phenylhydrazine effectively inhibited erythropoiesis, indicating that the zebrafish anemia model was successfully established. Under low, medium, and high doses of HMZP intervention, the IOD value of the heart in juvenile zebrafish gradually increased, showing a clear dose-response relationship. The IOD values ​​of zebrafish in HMZP-I, -II, and -III groups were 0.82 × 10⁻⁶. 6 1.25×10 6 and 1.48×10 6 These values ​​represent increases of 70.83%, 160.42%, and 208.83% respectively compared to the MC group (IOD value). Furthermore, the IOD value of the HMP group zebrafish was 1.27 × 10⁻⁶. 6 The IOD value of HMZP was 164.58% higher than that of the MC group. Therefore, HMZP showed a significant hematopoietic effect in the zebrafish anemia model, and its intervention ability was significantly stronger than that of HMP. Thus, both HMZP and HMP can significantly restore red blood cell levels in anemic states and have the potential to serve as natural hematopoietic agents.

[0113] Example 7 Blood Sugar Lowering Ability Inhibition rate of polysaccharides against α-amylase was determined. Different concentrations of polysaccharides (0.2, 0.6, 1.2, 1.4, 1.8, 2 mg / mL) and 1% soluble starch were prepared using PBS buffer (0.1 mol / L, pH 6.9); 1 U / mL α-amylase was prepared using 6.7 mmol / L NaCl solution. 50 μL of polysaccharide solution was mixed with 50 μL of α-amylase and incubated at 37℃ for 10 min. Then, 50 μL of 1% soluble starch was added, and incubation continued for another 10 min. The reaction was then terminated with 100 μL of DNS reagent, and the amylase was inactivated by boiling in water for 5 min. After cooling to room temperature, 1 mL of distilled water was added for dilution, and the absorbance was measured at 520 nm. The inhibition rate of α-amylase was calculated using the formula. Acarbose was used as a positive control.

[0114]

[0115] In the formula: A1 is the absorbance of the sample group; A2 is the background absorption of PBS buffer instead of α-glucosidase solution; A0 is the absorbance of the blank control group with PBS buffer instead of the sample.

[0116] Polysaccharides that inhibit amylase activity can slow down the hydrolysis of starch into glucose, thus possessing a certain ability to lower blood sugar. For example... Figure 11 As shown, at 2 mg / mL, the amylase inhibition rates of HMP and HMZP were 72.22% and 82.06%, respectively. Therefore, both HMP and HMZP have the ability to inhibit amylase (hypoglycemic capacity), and HMZP has a stronger hypoglycemic capacity than HMP.

[0117] Example 8: Lipid-lowering ability (1) Determination of the inhibition rate of polysaccharides on lipase. Polysaccharide solutions of different concentrations (1, 5, 10, 15, 20 mg / mL) were prepared using PBS buffer (0.1 mol / L, pH 6.9). 50 μL of each polysaccharide sample solution was accurately pipetted into test tubes, and 150 μL of porcine pancreatic lipase solution (1250 U / mL) and 350 μL of PBS buffer (0.1 mol / L, pH 7.4) were added respectively. The mixture was preheated at 37℃ for 10 min, followed by the addition of 450 μL of substrate solution. After thorough mixing, the mixture was placed in a 37℃ water bath for 2 h. The mixture was then centrifuged at 11000 r / min for 10 min, and the absorbance of the supernatant was measured at 405 nm. The inhibition rate of pancreatic lipase was calculated using the formula.

[0118]

[0119] In the formula: A1 is the absorbance of the blank group, with PBS buffer replacing the polysaccharide solution; A2 is the absorbance of the blank control group, with PBS buffer replacing the polysaccharide and pancreatic lipase solution; A3 is the absorbance of the sample group; A4 is the absorbance of the control group, with PBS buffer replacing the pancreatic lipase solution.

[0120] Polysaccharides with lipase-inhibiting activity can reduce the hydrolysis and absorption of dietary fat, thus having a lipid-lowering effect. For example... Figure 12 As shown, at a concentration of 20 mg / mL, the lipase inhibition rates of HMP and HMZP were 67.79% and 72.34%, respectively. Therefore, both HMP and HMZP have the ability to inhibit lipase (lipid-lowering ability). Moreover, the lipase-inhibiting ability of HMZP is stronger than that of HMP.

[0121] (2) Determination of the polysaccharide binding capacity to bile salts. Bile salt standard curve experiment: Take 1 mL of sodium glycocholate (0.04, 0.10, 0.20, 0.30, 0.40 mmol / L) or sodium taurocholate (0.05, 0.10, 0.20, 0.30, 0.40, 0.50 mmol) standard solutions of different molar concentrations and place them in stoppered test tubes. Add 3 mL of 60% concentrated H2SO4 solution, incubate at 70 ℃ for 20 min, then in an ice bath for 5 min, and measure the absorbance at a wavelength of 387 nm. Plot a standard curve of bile salt content with bile salt content as the abscissa and absorbance as the ordinate.

[0122] 10 mg / mL pepsin and trypsin were prepared using PBS buffer (0.1 mol / L, pH 6.3). 1.5 mL of polysaccharide solutions of different concentrations were added to 10 mL centrifuge tubes, along with 1.5 mL of pepsin and 0.5 mL of 0.01 mol / L HCl. The mixture was incubated at 37 °C with shaking for 1 h to simulate gastric digestion. The pH was then adjusted to 6.3 with 0.1 mol / L NaOH solution, followed by the addition of 2 mL of trypsin. The mixture was then incubated at 37 °C with shaking for 1 h to simulate intestinal digestion. After digestion, 2 mL of 0.4 mmol / L sodium glycocholate or 0.5 mmol / L sodium taurocholate was added to the samples, and the mixture was incubated at 37 °C with shaking for 1 h. The samples were then transferred to centrifuge tubes, centrifuged at 4000 r / min for 20 min, and 1 mL of the supernatant was collected. The contents of sodium glycocholate and sodium taurocholate were determined according to the standard curve method. Each sample was measured in triplicate. Calculate the binding rates of sodium glycocholate and sodium taurocholate according to the formula.

[0123]

[0124] In the formula: m1 is the mass of bile salt before binding, mg; m2 is the mass of bile salt after binding, mg.

[0125] Some polysaccharides can bind to sodium glycocholate and sodium taurocholate, promoting bile acid excretion, thereby accelerating the conversion of cholesterol into bile acids, lowering serum cholesterol levels, and exerting a lipid-lowering effect. For example... Figure 13 As shown, at a concentration of 20 mg / mL, the binding rates of HMP and HMZP to sodium glycocholate were 33.55% and 38.87%, respectively. Therefore, both HMP and HMZP possess sodium glycocholate binding capacity (lipid-lowering capacity), and HMZP has a stronger lipid-lowering capacity than HMP (sodium glycocholate binding capacity). Figure 14 As shown, at a concentration of 20 mg / mL, the binding rates of HMP and HMZP to sodium taurocholate were 37.39% and 42.09%, respectively. Therefore, both HMP and HMZP have the ability to bind sodium taurocholate (lipid-lowering ability), and HMZP has a stronger lipid-lowering ability than HMP (sodium taurocholate binding ability).

[0126] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of Hericium erinaceus polysaccharide in the preparation of products with anti-inflammatory, antioxidant, gastrointestinal protective, blood-nourishing and skin-beautifying, blood sugar-lowering and lipid-lowering effects.

2. The application as described in claim 1, characterized in that, The products include food, pharmaceuticals, and animal feed.

3. A method for preparing Hericium erinaceus polysaccharide, characterized in that, The preparation method includes: S1. Fermentation culture was used to obtain Hericium erinaceus mycelium, and crude polysaccharide of Hericium erinaceus was extracted by ultrasonic-assisted water extraction and alcohol precipitation. S2. The obtained crude polysaccharide of Hericium erinaceus was purified by DEAE-52 cellulose ion exchange column chromatography and Sephadex G-100 gel column chromatography.

4. The preparation method according to claim 3, characterized in that, In step S1, the fermentation culture adopts a conventional fermentation culture method or a zinc-enriched fermentation culture method. The specific method of the ultrasonic-assisted water extraction and alcohol precipitation method includes: after drying the mycelium of Hericium erinaceus, crushing and sieving it, adding water at a material-to-water ratio of 1:10-30, ultrasonically crushing and heating it, centrifuging it and taking the supernatant, repeating this step 2-4 times; combining the obtained supernatants, concentrating it to 1 / 2 to 1 / 3 of the original supernatant volume, then adding 1-10 times the volume of the concentrated supernatant ethanol, letting it stand overnight for alcohol precipitation, centrifuging to obtain the precipitate, and drying it to obtain the final product.

5. The preparation method according to claim 4, characterized in that, The specific conditions for the ultrasonic fragmentation treatment are: treatment at 100-500 W for 1-30 min; the heating treatment is a water bath heating treatment, and the specific heating conditions are: water bath heating treatment at 80-100℃ for 1-5 h.

6. The preparation method according to claim 4, characterized in that, The ethanol is ethanol with a concentration of 70% or higher.

7. The preparation method according to claim 3, characterized in that, The method for extracting crude polysaccharides from Hericium erinaceus using ultrasound-assisted water extraction and alcohol precipitation further includes resolubilizing the dried precipitate and removing proteins. Specifically, the method includes adding the precipitate to deionized water, wherein the mass-to-volume ratio of the precipitate to deionized water is 1:10-100; and the method for removing proteins is the Sevag method.

8. The preparation method according to claim 3, characterized in that, The specific method of step S2 includes: purification using a DEAE-52 cellulose ion exchange column, eluting sequentially with water and 0.05 mol / L and 0.1 mol / L NaCl solutions at a flow rate of 0.5-2 mL / min, collecting 30 tubes of eluent for each elution concentration, with each tube containing 0.5-4 mL, and collecting tubes 1-30 (water elution fraction), tubes 31-60 (0.05 mol / L NaCl elution fraction), and tubes 61-90 (0.1 mol / L NaCl elution fraction). The 0.05 mol / L NaCl elution fraction is then purified using a Sephadex G-100 dextran gel column, specifically eluting with water at a flow rate of 0.05-0.5 mL / min, with each tube containing 0.5-4 mL, and collecting the fractions corresponding to single absorption peaks. Preferably, when using Sephadex G-100 dextran gel column for purification, 9-23 tubes of crude polysaccharide from Hericium erinaceus mycelium are collected and freeze-dried to obtain HMP; 7-19 tubes of zinc-rich mycelium crude polysaccharide are collected and freeze-dried to obtain HMZP.

9. A product with anti-inflammatory, antioxidant, gastrointestinal protective, blood-nourishing, skin-beautifying, blood sugar-lowering, and lipid-lowering effects, characterized in that, The product contains at least the Hericium erinaceus polysaccharide obtained by the preparation method according to any one of claims 3-8.

10. The product as described in claim 9, characterized in that, The products include food, pharmaceuticals, and animal feed.

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