阅读说明：本技术 一种黄柄黄肉牛肝菌粗多糖的制备方法及其应用 (Preparation method and application of boletus flavomarginatus crude polysaccharide ) 是由 曾念开 吴璐伶 谢惠菁 张玉卓 徐畅 张絮 韩云霄 �田润 于 2021-08-05 设计创作，主要内容包括：本发明提供一种黄柄黄肉牛肝菌粗多糖的制备方法,包括以下步骤：将干燥的黄柄黄肉牛肝菌子实体粉碎,过筛,得干粉备用；称取干粉,按料液比1g∶25～40mL加入蒸馏水,在40～60℃下超声15～30min,然后于70～90℃热水下浸提3～5h,离心,得第一沉淀和第一上清液；在第一沉淀中加入第一次蒸馏水量的40～50％水,于70～90℃下浸提2.5～3.5h,将提取液离心；得第二沉淀和第二上清液；第一上清液和第二上清液减压浓缩,得浓缩液；在磁力搅拌器搅拌情况下缓缓将乙醇溶液加入浓缩液中,静置过夜；离心,将沉淀冷冻干燥24～28h,即得黄柄黄肉牛肝菌粗多糖。本发明提供的黄柄黄肉牛肝菌粗多糖的提取方法操作简单,易于产业化推广使用。采用上述超声波协同热水浸提法提取得到的粗多糖具有增重、加强脏器细胞分化等多方面的功效。(The invention provides a preparation method of boletus flavipes crude polysaccharide, which comprises the following steps: pulverizing dried Armillariella tabescens fruiting body, and sieving to obtain dry powder; weighing dry powder, adding distilled water according to the material-liquid ratio of 1 g: 25-40 mL, performing ultrasonic treatment at 40-60 ℃ for 15-30 min, then leaching with hot water at 70-90 ℃ for 3-5 h, and centrifuging to obtain a first precipitate and a first supernatant; adding water with the amount of 40-50% of the first distilled water into the first precipitate, leaching for 2.5-3.5 h at 70-90 ℃, and centrifuging the extracting solution; obtaining a second precipitate and a second supernatant; concentrating the first supernatant and the second supernatant under reduced pressure to obtain concentrated solution; slowly adding the ethanol solution into the concentrated solution under the stirring of a magnetic stirrer, and standing overnight; centrifuging, and freeze-drying the precipitate for 24-28 h to obtain the boletus flavipes crude polysaccharide. The extraction method of the boletus flavipes crude polysaccharide provided by the invention is simple to operate and easy to industrially popularize and use. The crude polysaccharide extracted by the ultrasonic wave and hot water extraction method has the effects of increasing weight, strengthening organ cell differentiation and the like.)

1. A preparation method of boletus flavipes crude polysaccharide is characterized by comprising the following steps:

(1) pulverizing dried Armillariella lutescens fruiting body, and sieving to obtain dry powder;

(2) weighing dry powder, adding distilled water according to the material-liquid ratio of 1 g: 25-40 mL, performing ultrasonic treatment at 40-60 ℃ for 15-30 min with the ultrasonic power of 500-600W, then leaching with hot water at 70-90 ℃ for 3-5 h, and centrifuging to obtain a first precipitate and a first supernatant;

(3) adding water accounting for 40-50% of the mass of the first distilled water into the first precipitate, leaching for 2.5-3.5 hours at 70-90 ℃, and centrifuging the extracting solution; obtaining a second precipitate and a second supernatant;

(4) concentrating the first supernatant and the second supernatant under reduced pressure to obtain concentrated solution; slowly adding the ethanol solution into the concentrated solution under the stirring of a magnetic stirrer, and standing overnight; centrifuging, and freeze drying the precipitate to obtain the boletus flavipes crude polysaccharide.

2. The method according to claim 1, wherein the temperature of the concentration under reduced pressure is 50 to 60 ℃.

3. The method according to claim 1, wherein the ethanol solution is added in an amount of 4 times by volume of the concentrate.

4. The method according to claim 1, wherein the ethanol solution has a volume concentration of 80 to 95%.

5. Use of the crude polysaccharide of suillus luteus obtained according to claim 1, characterized by the fact that it is used in the preparation of products for animal weight gain.

6. Use of the crude polysaccharide of Flavobacterium flavum obtained in claim 1, characterized by the use in the preparation of products for enhancing the differentiation of animal organ cells, said crude polysaccharide of Flavobacterium flavum being used in combination with a Ganoderma spore powder.

7. Use of the crude polysaccharide of Flavobacterium lutescens obtained in claim 1, characterized by the use thereof for the preparation of a product for increasing the content of acidic substances in the intestinal tract of an animal.

8. Use of the crude polysaccharide of Flavobacterium lutescens obtained in claim 1, characterized by the use thereof for the preparation of a product for reducing the ammonia content in the intestinal tract of an animal.

9. Use of the crude polysaccharide of suillus luteus obtained according to claim 1, characterized by the fact that it is used for the preparation of products for reducing the serum diamine oxidase content of animals.

10. Use of the crude polysaccharide of Flavobacterium lutescens obtained in claim 1, characterized by the use thereof for the preparation of a product for reducing endotoxin in animal serum.

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to a preparation method and application of boletus flavipes crude polysaccharide.

Background

The fungus of the Boletaceae family is widely distributed in the world, and because the geographical position, the climatic conditions and other aspects of the distribution area have differences, the species of the fungus of the Boletaceae family in different areas have obvious differences. According to incomplete statistics, the edible species of the boletaceae fungi in China reach 199. The bolete is one of four famous boletes, has the advantages of low fat, high protein and carbohydrate, reasonable amino acid composition and the like, and is an edible fungus with commercial potential and research value.

Boletus also has important medicinal value. The main effects of the medicinal value of bolete are clearing heat and relieving restlessness, tonifying deficiency and refreshing, relaxing muscles and tendons and promoting blood circulation. Research shows that some kinds of powdered boletes raveenii (Pulveroboletus raveenii) complex can treat symptoms such as waist soreness and leg pain, tetany, numbness and the like; boletus huamei (B. specialiosus Frost) can be used for treating abdominal distention and dyspepsia; and boletes with toxicity such as bolete fuliginosus (Tylopilus fullleus) and bolete portulacea (Ruboboletus saratas) can be used for extracting toxin and applying in the field of medicine. Experiments with Suluol et al showed that extracts from the fruit body of Boletus edulis (Lanmaoa Asiatica G.Wu & Zhu L.Yang) could improve immunity. The shiyanhong and the like intervene a type 2 diabetes model rat by using a bolete polysaccharide extract, and the blood sugar of the rat is reduced.

The chemical components of Boletaceae fungus are most prominent in polysaccharide component. Researches of Sunliping et al find that the content of water-soluble crude polysaccharide in the extracted 6 Yunnan wild edible bolete is high. Aiming at the extraction of boletus polysaccharide, corresponding extraction methods are provided by research. For example, CN105273101A discloses a method for extracting bolete polysaccharide from bolete, which comprises the following steps: (1) extracting bolete with 0.25-0.35% hydrochloric acid solution at 30-40 deg.C for 1-3 hr to obtain extractive solution; (2) adding alkali into the extracting solution to adjust the pH value to 7.5-8.0, and then carrying out solid-liquid separation to remove insoluble substances to obtain a clear aqueous solution A; (3) adding acid into the aqueous solution A to adjust the pH value to 4.0-4.5, and then carrying out solid-liquid separation to remove insoluble substances to obtain a clear aqueous solution B; (4) adding the aqueous solution B into a macroporous adsorption resin column, and washing the macroporous adsorption resin column with water to remove unadsorbed impurities; washing the macroporous adsorption resin by using an ethanol solution with the volume fraction of 15-20% to remove impurities with higher polarity; washing macroporous adsorbent resin with 65-70% ethanol solution as desorption solution, collecting the washed desorption solution, concentrating under reduced pressure, and drying to obtain Boletus edulis extract. The method has high extraction efficiency, but needs to adjust pH for many times and adopts a macroporous adsorption resin column to elute under specific conditions, so the operation is relatively complex and the cost is relatively high.

At present, research on the yellow-handle yellow-fleshed beef liver fungi is few, the research on extraction and pharmacology of the yellow-handle yellow-fleshed beef liver fungi polysaccharide is less, and the knowledge on the yellow-handle yellow-fleshed beef fungi polysaccharide is not deep enough, so that the development of the yellow-handle yellow-fleshed beef fungi related industry is greatly restricted. Therefore, the invention has great significance in further exploring the extraction method and the pharmacological action of the boletus flavipes crude polysaccharide.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a preparation method and application of boletus flavipes crude polysaccharide.

The technical scheme of the invention mainly comprises the following contents:

a method for preparing boletus flavomarginatus crude polysaccharide comprises the following steps:

(1) pulverizing dried Armillariella tabescens fruiting body, and sieving to obtain dry powder;

(2) weighing dry powder, adding distilled water according to the material-liquid ratio of 1 g: 25-40 mL, performing ultrasonic treatment at 40-60 ℃ for 15-30 min, then leaching with hot water at 70-90 ℃ for 3-5 h, and centrifuging to obtain a first precipitate and a first supernatant;

(3) adding water with the amount of 40-50% of the first distilled water into the first precipitate, leaching for 2.5-3.5 h at 70-90 ℃, and centrifuging the extracting solution; obtaining a second precipitate and a second supernatant;

(4) concentrating the first supernatant and the second supernatant under reduced pressure to obtain concentrated solution; slowly adding the ethanol solution into the concentrated solution under the stirring of a magnetic stirrer, and standing overnight; centrifuging, and freeze drying the precipitate to obtain the boletus flavipes crude polysaccharide.

Preferably, the freeze drying time of the precipitate is 24-28 h.

Preferably, the ultrasonic power is 500-600W.

Preferably, the temperature of the reduced pressure concentration is 50-60 ℃; the amount of ethanol solution added was 4 times the volume of the concentrate.

Preferably, the volume concentration of the ethanol solution is 80-95%.

On the other hand, the invention also provides the application of the obtained boletus flavipes crude polysaccharide in various products. The related applications comprise the application in preparing products for increasing animal weight, the application in preparing products for strengthening the differentiation of animal organ cells, the application in preparing products for improving the content of acidic substances in animal intestinal tracts, the application in preparing products for reducing the content of ammonia in animal intestinal tracts, the application in preparing products for reducing the content of diamine oxidase in animal serum and the application in preparing products for reducing the content of endotoxin in animal serum.

The invention has the following effects:

the invention adopts ultrasonic wave and hot water extraction to extract the boletus flavipes crude polysaccharide with various pharmacological actions. Experiments prove that the crude polysaccharide has the effects of increasing weight, strengthening organ cell differentiation, improving the content of acidic substances in animal intestinal tracts, reducing the content of ammonia in animal intestinal tracts, reducing the content of diamine oxidase in animal serum and reducing the endotoxin in animal serum. The boletus flavipes crude polysaccharide has important medicinal value.

The method can lay a foundation for the development and utilization of the bolete flavipes resource and improve the additional value of the bolete flavipes.

The extraction method of the boletus flavipes crude polysaccharide provided by the invention is simple to operate and easy to industrially popularize and use.

Drawings

FIG. 1: photograph of fruiting body of Boletus flavus

FIG. 2: weight gain in mice

FIG. 3: standard curve of ammonia content

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

The boletus flavipes is Butyriboletus pseudopolypicicus. Butyl riboletus pseudofollicus Kuan Zhao & Zhu L. Yang, in Wu, Li, Zhu, Zhao, Han, Cui, Li, Xu & Yang, Fungal university 81:69 (2016). The medicinal material of the yellow-handle beef bolete used by the invention is purchased from the West double-banner farmer market in Yunnan province.

The extraction method of the boletus flavipes crude polysaccharide comprises the following steps:

(1) crushing the dried boletus flavipes fruiting body by using a crusher, and sieving by using a 40-mesh sieve to obtain dry powder;

(2) weighing 20g of dry powder, adding distilled water according to the material-liquid ratio of 1g to 25-40 mL, and performing ultrasonic treatment at 40-60 ℃ for 15-30 min, wherein the ultrasonic power is 500-600W; then, leaching the mixture for 3-5 hours in hot water at the temperature of 70-90 ℃, and centrifuging the mixture for 15min at 4000rpm to obtain a first precipitate and a first supernatant;

(3) adding water with the amount of 40-50% of the first distilled water into the first precipitate, leaching for 2.5-3.5 h at 70-90 ℃, and centrifuging the extracting solution for 15min at 4000 rpm; obtaining a second precipitate and a second supernatant;

(4) concentrating the first supernatant and the second supernatant at 50-60 ℃ under reduced pressure to one third of the original volume to obtain a concentrated solution; slowly adding 80-95 vt% ethanol solution into the concentrated solution under the stirring condition of a magnetic stirrer, wherein the adding amount of the ethanol solution is 4 times of the volume of the concentrated solution, and standing overnight; centrifuging at 4000rpm for 15min, and freeze-drying the precipitate for 24-28 h to obtain the boletus flavipes crude polysaccharide.

Experiments prove that the pharmacological action effect of the boletus flavipes crude polysaccharide prepared within the parameter range is not obviously different from that of the boletus flavipes crude polysaccharide prepared in the embodiment 1.

Example 1 extraction of crude polysaccharide from Boletus flavus

(1) Crushing the dried boletus flavipes fruiting body by using a crusher, and sieving by using a 40-mesh sieve to obtain dry powder;

(2) weighing 20g of dry powder, adding distilled water according to the feed-liquid ratio of 1g to 25mL, and performing ultrasonic treatment at 50 ℃ for 15min with the ultrasonic power of 500W; then leaching under hot water at 85 ℃ for 3h, and centrifuging at 4000rpm for 15min to obtain a first precipitate and a first supernatant;

(3) adding 50% of water of the first distilled water amount into the first precipitate, leaching at 85 deg.C for 2.5h, and centrifuging the extractive solution at 4000rpm for 15 min; obtaining a second precipitate and a second supernatant;

(4) concentrating the first supernatant and the second supernatant at 50 deg.C under reduced pressure to one third of the original volume to obtain concentrated solution; slowly adding 95 vt% ethanol solution into the concentrated solution under the stirring of a magnetic stirrer, wherein the adding amount of the ethanol solution is 4 times of the volume of the concentrated solution, and standing overnight; centrifuging at 4000rpm for 15min, and freeze drying the precipitate for 24h to obtain boletus flavipes crude polysaccharide.

Example 2 research on the pharmacological action of boletus flavomarginatus crude polysaccharide-animal experiment

Animals: healthy male Kunming mice (provided by Henan Scout Biotech company) weigh 20-30 g, 6-8 weeks old, laboratory animal production license: SCXK 2020-0005.

2.1 animal grouping and feeding

The male Kunming mice are randomly divided into 6 groups of 7 mice per group, namely a blank control group (K), a low-dose group (PL) of Flavobacterium lutescens polysaccharide, a high-dose group (PH) of Flavobacterium lutescens polysaccharide, a ganoderma spore powder group (G), a low-dose group (PLG) of Flavobacterium lutescens polysaccharide combined with ganoderma spore powder group (PLG) and a high-dose group (PHG) of Flavobacterium lutescens polysaccharide combined with ganoderma spore powder group. The experimental animal groups are shown in Table 1.

Mouse feeding conditions and treatment: feeding at 20-25 deg.C for 7 days. The same amount of feed was dosed daily and water was fed freely during the experiment. The medicine is powder and is dissolved and diluted by deionized water. Each group was gavaged once every morning at the same time, the continuous gavage time was 14 days, and the gavage volume was 0.3mL each time. Mice were sacrificed at 7d by gavage, 3 mice per group randomly, and the remainder at 14d were all sacrificed. After last gastric lavage before each group of mice is sacrificed, fasting is carried out for 8-12h without water prohibition, and the weight of each male Kunming mouse is weighed; CO is carried out the next day₂Sacrifice, dissect, and draw blood from eyeball. Serum and colonic feces were collected.

TABLE 1 groups of experimental animals

Note: PB is yellow-handle bolete crude polysaccharide, and GLCP is Ganoderma spore powder.

2.2 index determination

(1) Vital signs of mice

The weight of each male Kunming mouse is measured before the 4 th day and the 7 th day of the mice are fed adaptively, the weight and the number of the Kunming mice in each group are recorded before the 7 th day of the feeding and before the gavage in the experimental period, and the weight and the number are recorded once per week for 3 times. The mean body weight per stage (one stage at 7 d) was calculated for each group of male Kunming mice during the experiment. Changes in body weight of each group of mice in the experimental period 14d were observed and compared for differences.

② mouse organ index

CO₂Killing each male Kunming xiaoAfter the mice, organs (liver, spleen, thymus, colon, and cecum) of the mice were separated during dissection, and the mass thereof was precisely measured in g.

The organ index was calculated as follows: caecum index ═ caecum weight/mouse weight × 100%

(2) Mouse feces

(ii) pH value

An average of 4 feces with a total weight of 0.1g were collected per mouse at 14d of the experiment. Dissolving with 1mL deionized water, mixing in a grinding instrument, centrifuging at 4 deg.C for 8min (4000r/min), and measuring the pH value of the supernatant with a pH meter.

Ammonia content

An average of 4 feces of approximately 0.1g weight were collected from each mouse at 14d of the experiment and kept in a refrigerator at-80 ℃ until use.

The ammonia content in the feces is detected by adopting a nano-grade reagent spectrophotometry. Measuring the ammonia content in the feces of Kunming mice according to the method for measuring ammonia in the air in public places (GB T18204.25-2000) and making a standard curve of the ammonia content.

(3) Mouse serum index

At experiment 14d, 5 replicates per group, mice were bled approximately 1mL per eyeball and centrifuged for 8min (4000r/min) before supernatant was obtained. The contents of diamine oxidase and endotoxin in the serum of mice were measured according to the instructions for diamine oxidase (DAO) and Endotoxin (ET) in the ELISA kit.

2.3 results and analysis

(1) Mouse vital sign index

Weight: the weight gain of the mice is shown in fig. 2, and analysis shows that the high-dose flavokernella lutescens polysaccharide group and the flavokernella lutescens polysaccharide combined ganoderma lucidum spore powder group have obvious weight increase compared with the control group in the second stage (7 th to 14 th days), which indicates that the mice have better absorption when being administered with the high-dose flavokernella lutescens polysaccharide or the combined administration.

Organ index: the mouse organ index profiles are shown in table 2. In the aspect of liver index, compared with a blank control group, the liver index of other administration groups is increased, but only the high-dose flavokeratius flavomarginatus and the bolete polysaccharide combined with the ganoderma lucidum spore powder have significant difference. In the aspect of spleen index, compared with a blank control group, spleen indexes of other administration groups are obviously increased, wherein the ganoderma lucidum spore powder group, the high-dose flavokereus flavomarginatus polysaccharide group and the high-dose flavokereus flavokeratitus polysaccharide combined ganoderma lucidum spore powder group have significant difference. In the aspect of thymus index, the thymus index of other administration groups is obviously increased compared with that of a blank control group, wherein the mythic fungus spore powder group, the low-dose flavokeratium flavomarginatum polysaccharide group and the high-dose flavokeratium polysaccharide combined mythic fungus spore powder group have obvious difference, and the result is probably related to that the weight of the mice of the low-dose flavokeratium polysaccharide group is slightly increased in the second stage (7 th-14 th days). The result of organ index is summarized, the use of the high-dose flavokeratia flavokeri polysaccharide group and the high-dose flavokeratia polysaccharide combined with the ganoderma lucidum spore powder can obviously improve the organ indexes, and play a role in enhancing the physique of the mouse and strengthening the organ cell differentiation of the mouse.

TABLE 2 mouse organ index

Note: compared with the blank control group, the data represents significant difference (P < 0.05), and the data represents very significant difference (P < 0.01).

(2) Results of pH value and ammonia content of feces

pH value: as can be analyzed by comparing the data in Table 3, the pH of the feces of the mice in the administration group was decreased after the mice were gavaged for 14 d. Compared with a blank control group, the high-dose flavokereus flavokeri polysaccharide, the low-dose flavokereus flavokeri polysaccharide combined with the ganoderma lucidum spore powder group and the high-dose flavokereus flavokeri polysaccharide combined with the ganoderma lucidum spore powder group have the advantages that the pH value in the mouse excrement is remarkably reduced, and the low-dose flavokereus flavokeri polysaccharide group and the ganoderma lucidum spore powder group have the remarkable pH value reduction. The combination of the high-dose flavopulp bolete polysaccharide and the two can reduce the pH value of the excrement, and the effect is better than that of the ganoderma lucidum spore powder. Meanwhile, the combination of the Flavobacterium flavum polysaccharide and the ganoderma lucidum spore powder has better effect than the Flavobacterium flavum polysaccharide or the ganoderma lucidum spore powder, and the pH value of the excrement is related to the dosage of the Flavobacterium flavum polysaccharide. The results show that the use of the boletus flavipes polysaccharide or the boletus flavipes polysaccharide combined with the spore powder can increase the acidic substances such as short-chain fatty acid and the like in the intestinal tract of the mouse.

Ammonia content: the standard curve of ammonia content is shown in fig. 3, and comparing the data of ammonia content in table 3, it can be seen that the ammonia content of the feces of male Kunming mice in the administration group is reduced after the gavage 14 d. Compared with a blank control group, the ganoderma lucidum spore powder group is remarkably reduced, and the yellow-handle yellow-fleshed beef liver fungus polysaccharide alone group and the high-dose yellow-handle yellow-fleshed beef liver fungus polysaccharide combined ganoderma lucidum spore powder group are remarkably reduced. The results show that the combination of the flavokeratia lutescens polysaccharide alone or the high-dose flavokeratia lutescens polysaccharide and the ganoderma lucidum spore powder can reduce the content of the ammonia converted from the protein or other substances in the intestinal tract, and the combination effect is better.

TABLE 3 pH of feces from mice, Ammonia content

Note: compared to the blank control group: marked differences (P < 0.05) and marked differences (P < 0.01).

(3) Analysis of mouse serum measurements

The measurement results of diamine oxidase and endotoxin in mouse serum are shown in table 4. In terms of DAO, the table shows that the concentration of DAO in the administration group is extremely reduced compared with the control group, wherein the DAO concentration in the high-dose boletus polysaccharide and ganoderma lucidum spore powder group is the lowest, and the effect of the combination is better than that of the single boletus polysaccharide. Under normal physiological conditions, blood DAO levels are very low in vivo. When the barrier function of the intestinal mucosa is abnormal, DAO enters the intestinal tract after entering blood in a large amount, so that the DAO level in the intestinal tract and peripheral blood is abnormally increased and expressed. This indicator of DAO activity and circulating levels is commonly used to assess clinical damage to the intestinal mucosa. In terms of ET, the results analyzed in table 4 show that the low dose of boletus polysaccharide significantly reduced ET content compared to the control, whereas the high dose of boletus polysaccharide showed no significance, indicating that it is likely that the low dose has reached an effective concentration to reduce ET. The combined use of boletus and ganoderma lucidum spore powder can obviously improve the content of ET, which is related to the possible inhibition effect of the combined use, but the inhibition mechanism of the boletus and the ganoderma lucidum spore powder is to be further determined. The normal human intestinal tract contains a large amount of endotoxin, the endotoxin has no dangerous influence on the human body under the normal condition, and only when the barrier function of the intestinal tract is abnormal, the large amount of endotoxin can enter the blood circulation, so that endotoxemia occurs.

TABLE 4 content of DAO and ET in mouse sera

Note: compared to the blank control group: marked differences (P < 0.05) and marked differences (P < 0.01).

Comprehensive analysis of the weight growth condition, organ index, pH value and ammonia content of excrement, DAO and ET content in serum and intestinal flora sequencing of a mouse shows that each index indicates that the optimal administration mode is to use the high-dose boletus flavipes crude polysaccharide alone and the low-dose boletus flavipes crude polysaccharide in combination with ganoderma lucidum spore powder.

Comparative example 1 extraction of crude polysaccharide from Boletus flavus

The main differences between this comparative example and example 1 are: the conditions of hot water extraction are different.

(1) Crushing the dried boletus flavipes fruiting body by using a crusher, and sieving by using a 40-mesh sieve to obtain dry powder;

(2) weighing 20g of dry powder, adding distilled water according to the feed-liquid ratio of 1g to 25mL, and performing ultrasonic treatment at 50 ℃ for 15min with the ultrasonic power of 500W; then leaching under hot water at 50 ℃ for 3h, and centrifuging at 4000rpm for 15min to obtain a first precipitate and a first supernatant;

(3) adding 50% of water of the first distilled water amount into the first precipitate, leaching at 50 deg.C for 2.5h, and centrifuging the extractive solution at 4000rpm for 15 min; obtaining a second precipitate and a second supernatant;

(4) concentrating the first supernatant and the second supernatant at 50 deg.C under reduced pressure to one third of the original volume to obtain concentrated solution; slowly adding 95 vt% ethanol solution into the concentrated solution under the stirring of a magnetic stirrer, wherein the adding amount of the ethanol solution is 4 times of the volume of the concentrated solution, and standing overnight; centrifuging at 4000rpm for 15min, and freeze drying the precipitate to obtain boletus flavipes crude polysaccharide.

Comparative example 2

The main differences between this example and example 1 are: the ultrasound conditions were different.

(1) Crushing the dried boletus flavipes fruiting body by using a crusher, and sieving by using a 40-mesh sieve to obtain dry powder;

(2) weighing 20g of dry powder, adding distilled water according to the feed-liquid ratio of 1g to 25mL, and performing ultrasonic treatment at 80 ℃ for 15min with the ultrasonic power of 300W; then leaching under hot water at 85 ℃ for 3h, and centrifuging at 4000rpm for 15min to obtain a first precipitate and a first supernatant;

(3) adding 50% of water of the first distilled water amount into the first precipitate, leaching at 85 deg.C for 2.5h, and centrifuging the extractive solution at 4000rpm for 15 min; obtaining a second precipitate and a second supernatant;

(4) concentrating the first supernatant and the second supernatant at 50 deg.C under reduced pressure to one third of the original volume to obtain concentrated solution; slowly adding 95 vt% ethanol solution into the concentrated solution under the stirring of a magnetic stirrer, wherein the adding amount of the ethanol solution is 4 times of the volume of the concentrated solution, and standing overnight; centrifuging at 4000rpm for 15min, and freeze drying the precipitate to obtain boletus flavipes crude polysaccharide.

The crude polysaccharides extracted in comparative examples 1 and 2 were subjected to the animal experiment of example 2 described above, and the results thereof were compared with example 1. The main difference in the results is shown in: the organ indexes of the low-dose groups are all obviously lower than that of example 1(P < 0.05), the ammonia content of the low-dose groups is all obviously higher than that of example 1(P < 0.05), and the endotoxin content of the low-dose group of comparative example 1 is higher than that of example 1(P < 0.05). The main reason for the above differences may be related to the content and structure of polysaccharides and the content of other components such as proteins and flavonoids in the crude polysaccharides obtained under different extraction conditions.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that are within the spirit and principle of the present invention are intended to be included in the scope of the present invention.