阅读说明：本技术 一种树舌多糖提取物的制备方法和应用 (Preparation method and application of Ganoderma applanatum polysaccharide extract ) 是由 王丽娜 姜海涛 杨蕾 张译文 刘磊 潘睿 郭海乐 杨景杰 曹钰茜 郑辉 陶静宇 于 2021-10-22 设计创作，主要内容包括：一种树舌多糖提取物的制备方法和应用,它涉及医药健康领域,本发明目的是针对现有降血糖降血脂药物副作用多且疗效不显著的问题,为了得到既能辅助降血糖降血脂,毒副作用又小的保健品。本发明是将树舌子实体烘干、粉碎；水提,醇沉；沉淀用水溶解,去除蛋白质,上清液进行脱色除杂,收集洗脱液；干燥,得精制树舌多糖提取物。本发明的树舌多糖制备用于降血糖降血脂的药物。本发明用于降血糖降血脂的药物领域。(The invention discloses a preparation method and application of a glossogyne polysaccharide extract, relates to the field of medical health, and aims to solve the problems of more side effects and unobvious curative effect of the existing hypoglycemic and hypolipidemic drugs so as to obtain a health-care product which can assist in reducing blood sugar and blood fat and has small toxic and side effects. The invention is that oven drying Ganoderma Applanatum fruiting body, pulverizing; extracting with water, and precipitating with ethanol; dissolving the precipitate with water, removing protein, decolorizing the supernatant, removing impurities, and collecting eluate; drying to obtain refined Ganoderma Applanatum polysaccharide extract. The Ganoderma Applanatum polysaccharide of the invention is used for preparing medicines for reducing blood sugar and blood fat. The invention is used in the field of medicines for reducing blood sugar and blood fat.)

1. The preparation method of the lingua polysaccharide extract is characterized by comprising the following steps of:

1) drying: drying the fruiting body of the Ganoderma Applanatum at 70-80 ℃ for 4 h;

2) crushing: pulverizing and sieving with 40 mesh sieve;

3) water extraction: adding pure water with the weight 5-8 times of that of the decoction pieces, heating and refluxing at 100 ℃, extracting for 3 times, each time for 1.5-2 hours, and combining water extract;

4) alcohol precipitation: concentrating the water extract obtained in the step 3), adding absolute ethyl alcohol to enable the alcohol content to reach 80%, standing for 24h, centrifuging to obtain a supernatant and a precipitate, wherein the precipitate is a primary alcohol precipitation Ganoderma Applanatum crude polysaccharide extract; dissolving the Ganoderma Applanatum polysaccharide extract subjected to the first alcohol precipitation in water until the concentration reaches 0.2-0.7 g/mL based on the crude drug amount, continuously adding anhydrous ethanol until the alcohol content reaches 80%, standing overnight, centrifuging to obtain supernatant and precipitate, and obtaining the precipitate as crude Ganoderma Applanatum polysaccharide subjected to the second alcohol precipitation;

5) deproteinizing by a Savage method: dissolving the crude polysaccharide of the Ganoderma Applanatum in step 4) with pure water, mixing with Sevage reagent, standing for layering, centrifuging, and collecting the uppermost polysaccharide water solution;

6) repeating the operation of the step 5) for 3-5 times to obtain a deproteinized polysaccharide solution;

7) and (3) decoloring and impurity removing by using AB-8 macroporous resin: A) macroporous resin pretreatment: soaking macroporous resin in ultrapure water, soaking and washing with absolute ethyl alcohol for multiple times, adding ultrapure water to avoid turbidity, and repeatedly washing with ultrapure water for later use; B) decoloring and removing impurities: adsorbing and decoloring the polysaccharide solution after deproteinization in the step 6) by using treated macroporous resin, wherein the flow rate of the polysaccharide solution on a column is 1.0mL/min, the eluent is ethanol with the volume percentage of 75%, and the elution volume is 3 times of the column volume;

8) and (3) drying: and (3) drying the polysaccharide solution subjected to decolorization and impurity removal in the step 7) under reduced pressure until the specific gravity is 1.08-1.12 to obtain the Ganoderma Applanatum polysaccharide extract.

2. The method for preparing the lingua polysaccharide extract according to claim 1, wherein the Sevag reagent in step 5) is chloroform: the n-butanol is 5: 1, and mixing the components in a ratio of 1.

3. The method for preparing the lingua polysaccharide extract according to claim 1, wherein in the step 5), AB-8 macroporous resin is used for decoloring and impurity removal.

4. The method for preparing the Ganoderma applanatum polysaccharide extract as claimed in claim 1, wherein the Ganoderma applanatum polysaccharide extract obtained by the first ethanol precipitation in step 4) is dissolved in water to a concentration of 0.5g/mL based on crude drug.

5. The use of a lingua polysaccharide extract prepared according to claim 1 for preparing a medicament for lowering blood glucose and blood lipid levels.

Technical Field

The invention relates to the field of medical health, in particular to a preparation method and application of a Ganoderma applanatum polysaccharide extract.

Background

Hyperlipidemia: lipid metabolism or abnormal operation causes the blood lipid content in human blood to exceed the normal range, which is manifested as high cholesterol or triglyceride or low high density lipoprotein, and modern medicine is called as "dyslipidemia". Hyperlipidemia is a common disease, frequently encountered disease, and especially causes the violence of cardiovascular and cerebrovascular diseases, the damage of the disease to the body is secret, gradual, progressive and systemic, and the direct damage of the disease is the acceleration of systemic atherosclerosis. The hyperlipidemia has a long course, and needs to be treated by statins, fibrates, nicotinic acid, resins and other medicaments for a long time, and patients with serious disease and unsatisfactory medicament treatment effect can be treated by operation. However, chemical drugs generally have large side effects and are easy to cause symptoms such as nausea, vomiting, appetite reduction, physical strength reduction and the like.

Hyperglycemia: when fasting (no sugar and any sugar containing intake within 8 hours) blood glucose is above the normal range, called hyperglycemia, fasting blood glucose has a normal value of 4.0-6.1mmol/L, and two hours after a meal blood glucose is above the normal range of 7.8mmol/L, also called hyperglycemia. At present, diseases causing blood sugar elevation mainly include diabetes caused by islet dysfunction or insulin resistance, chronic diseases such as hyperthyroidism, hepatitis and liver cirrhosis, long-term glucocorticoid use, many genetic diseases and the like. The treatment method comprises drug treatment and surgery treatment. The drug therapy generally adopts oral hypoglycemic drugs, insulin injection and GLP-1 receptor agonist injection.

And (3) tongue of the tree: also named as the red old mother fungus and the flat glossy ganoderma. Is fruiting body of Ganoderma applanatum of Polyporaceae. Widely planted and distributed all over the country. Slightly bitter taste and mild nature. It enters spleen and stomach meridians. Has anti-inflammatory and anticancer effects. Can be used for treating pharyngolaryngitis, esophageal cancer, and nasopharyngeal carcinoma. Has the pharmacological effects of regulating immunity, resisting tumor, inhibiting bacteria, and regulating blood pressure.

Ganoderma Applanatum polysaccharide: ganoderma Applanatum polysaccharide is secondary metabolite of Ganoderma Applanatum, and is mainly present in fruiting body. Is a macromolecular compound with a molecular weight ranging from thousands to hundreds of thousands. The Ganoderma Applanatum polysaccharide is one of the most effective components of Ganoderma Applanatum. The Ganoderma Applanatum polysaccharide has various pharmacological activities: it has effects in improving immunity, accelerating blood microcirculation, improving oxygen supply capacity of blood, reducing ineffective oxygen consumption of body in static state, scavenging free radicals, improving cell membrane sealing degree, resisting radiation, and improving the ability of liver, bone marrow and blood to synthesize DNA, RNA, and protein.

At present, the Ganoderma Applanatum is mostly used as one of the medicinal components for treating hyperlipemia and hyperglycemia. No report of using the Ganoderma Applanatum polysaccharide alone is provided, and the existing Ganoderma Applanatum polysaccharide has the problem of poor extraction effect.

Disclosure of Invention

The invention aims to solve the problems of more side effects and unobvious curative effect of the existing hypoglycemic and hypolipidemic drugs, and aims to obtain a health-care product which can assist in reducing blood sugar and blood fat and has small toxic and side effects. The invention provides a preparation method of a glossogyne polysaccharide extract and application of the glossogyne polysaccharide extract in a health-care product for assisting in reducing blood sugar and blood fat.

The preparation method of the lingual polysaccharide extract is carried out according to the following steps:

1) drying: drying the fruiting body of the Ganoderma Applanatum at 70-80 ℃ for 4 h;

2) crushing: pulverizing and sieving with 40 mesh sieve;

3) water extraction: adding pure water with the weight 5-8 times of that of the decoction pieces, heating and refluxing at 100 ℃, extracting for 3 times, each time for 1.5-2 hours, and combining water extract;

4) alcohol precipitation: concentrating the water extract obtained in the step 3), adding absolute ethyl alcohol to enable the alcohol content to reach 80%, standing for 24h, centrifuging to obtain a supernatant and a precipitate, wherein the precipitate is a primary alcohol precipitation Ganoderma Applanatum crude polysaccharide extract; dissolving the Ganoderma Applanatum polysaccharide extract subjected to the first alcohol precipitation in water until the concentration reaches 0.2-0.7 g/mL based on the crude drug amount, continuously adding anhydrous ethanol until the alcohol content reaches 80%, standing overnight, centrifuging to obtain supernatant and precipitate, and obtaining the precipitate as crude Ganoderma Applanatum polysaccharide subjected to the second alcohol precipitation;

5) deproteinizing by a Savage method: dissolving the crude polysaccharide of the Ganoderma Applanatum in step 4) with pure water, mixing with Sevage reagent, standing for layering, centrifuging, and collecting the uppermost polysaccharide water solution;

6) repeating the operation of the step 5) for 3-5 times to obtain a deproteinized polysaccharide solution;

7) and (3) decoloring and impurity removing by using AB-8 macroporous resin: A) macroporous resin pretreatment: soaking macroporous resin in ultrapure water, soaking and washing with absolute ethyl alcohol for multiple times, adding ultrapure water to avoid turbidity, and repeatedly washing with ultrapure water for later use; B) decoloring and removing impurities: adsorbing and decoloring the polysaccharide solution after deproteinization in the step 6) by using treated macroporous resin, wherein the flow rate of the polysaccharide solution on a column is 1.0mL/min, the eluent is ethanol with the volume percentage of 75%, and the elution volume is 3 times of the column volume;

8) and (3) drying: and (3) drying the polysaccharide solution subjected to decolorization and impurity removal in the step 7) under reduced pressure until the specific gravity is 1.08-1.12 to obtain the Ganoderma Applanatum polysaccharide extract.

Further, in step 5), the Sevag reagent is chloroform: the n-butanol is 5: 1, and mixing the components in a ratio of 1.

Further, in the step 5), AB-8 macroporous resin is used for decoloring and impurity removal.

Further, the Ganoderma Applanatum polysaccharide extract obtained by the first alcohol precipitation in the step 4) is dissolved in water to a concentration of 0.5g/mL based on crude drug.

The application of the prepared artist's conk polysaccharide extract in preparing the medicine for reducing blood sugar and blood fat is disclosed.

The invention has the following beneficial effects:

the Ganoderma Applanatum polysaccharide extract provided by the invention has the function of assisting in reducing blood fat; the extract can influence the blood pressure and blood lipid metabolism in vivo and influence the immunity of organism. The invention prepares the Ganoderma Applanatum polysaccharide extract into a health product with the functions of assisting in reducing blood sugar and blood fat.

Detailed Description

It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples for carrying out the invention, and that various changes in form and details may be made therein without departing from the spirit and scope of the invention in practice.

To make the objects, aspects and advantages of the embodiments of the present invention more apparent, the following detailed description clearly illustrates the spirit of the disclosure, and any person skilled in the art, after understanding the embodiments of the disclosure, may make changes and modifications to the technology taught by the disclosure without departing from the spirit and scope of the disclosure.

The exemplary embodiments of the present invention and the description thereof are provided to explain the present invention and not to limit the present invention.

Example 1

The preparation method of the lingua polysaccharide extract of the embodiment is carried out according to the following steps:

1) drying: drying the fruiting body of the Ganoderma Applanatum at 70-80 ℃ for 4 h;

2) crushing: pulverizing and sieving with 40 mesh sieve;

3) water extraction: adding pure water 8 times the weight of the decoction pieces, heating and refluxing at 100 deg.C for 3 times, each for 1.5 hr, and mixing water extractive solutions;

4) alcohol precipitation: concentrating the water extract obtained in the step 3), adding absolute ethyl alcohol to enable the alcohol content to reach 80%, standing for 24h, centrifuging to obtain a supernatant and a precipitate, wherein the precipitate is a primary alcohol precipitation Ganoderma Applanatum crude polysaccharide extract; dissolving the Ganoderma Applanatum polysaccharide extract with water to make its concentration reach 0.5g/mL based on crude drug amount, adding anhydrous ethanol to make its alcohol content reach 80%, standing overnight, centrifuging to obtain supernatant and precipitate, and precipitating to obtain Ganoderma Applanatum crude polysaccharide;

5) deproteinizing by a Savage method: dissolving the crude polysaccharide of the Ganoderma Applanatum in step 4) with pure water, mixing with Sevage reagent, standing for layering, centrifuging, and collecting the uppermost polysaccharide water solution; repeating for 3-5 times;

6) and (3) decoloring and impurity removing by using AB-8 macroporous resin: A) macroporous resin pretreatment: soaking macroporous resin in ultrapure water, soaking and washing with absolute ethyl alcohol for multiple times, adding ultrapure water to avoid turbidity, and repeatedly washing with ultrapure water for later use; B) decoloring and removing impurities: adsorbing and decoloring the polysaccharide solution after deproteinization in the step 5) by using treated macroporous resin, wherein the flow rate of the polysaccharide solution on a column is 1.0mL/min, the eluent is ethanol with the volume percentage of 75%, and the elution volume is 3 times of the column volume;

7) and (3) drying: and (3) drying the polysaccharide solution subjected to decolorization and impurity removal in the step 6) under reduced pressure until the specific gravity is 1.08-1.12 to obtain the Ganoderma Applanatum polysaccharide extract.

Example 2

Verification of the Effect of the Ganoderma Applanatum polysaccharide extracted in example 1 on hyperglycemia

Experimental materials: 60 rats were taken, and 40 of them were used to establish a diabetic rat animal model. The first group was 20 normal controls, the second group was diabetic rats 20 using a successful early model, and the third group was diabetic rats 20 using a successful early model, which was the Ganoderma Applanatum polysaccharide group.

The experimental method comprises the following steps: the Ganoderma Applanatum polysaccharide group was administered by intragastric administration with Ganoderma Applanatum polysaccharide dissolved in water at a ratio of 0.2g/kg d, and the normal group and the diabetic control group were administered with the same volume of distilled water.

The test results are as follows:

TABLE 1 Effect of Ganoderma Applanatum polysaccharide on blood glucose concentration mmol/L in diabetic rats

Grouping	Day 1	Day 7	Day 14
				Normal group	4.22±0.21	4.34±0.19	4.57±0.23
Diabetes group	18.21±0.37	19.05±0.32	18.97±0.64
				Ganoderma Applanatum polysaccharide group	18.21±0.51	9.12±0.46	7.29±0.49

At the beginning of the experiment, there is no significant difference between the diabetes group and the Ganoderma Applanatum polysaccharide group, and the Ganoderma Applanatum polysaccharide group in the middle and later stages of the experiment can significantly reduce the hyperglycemia of the model-making rat.

TABLE 2 Effect of Ganoderma Applanatum polysaccharide on insulin concentration mU/L in diabetic rats

Grouping	Day 1	Day 7	Day 14
				Normal group	4.19±0.51	4.25±0.36	4.12±0.41
Diabetes group	0.91±0.24	0.93±0.45	0.89±0.72
				Ganoderma Applanatum polysaccharide group	0.87±0.24	1.54±0.26	2.01±0.42

At the beginning of the experiment, there is no significant difference between the diabetes group and the Ganoderma Applanatum polysaccharide group, and the Ganoderma Applanatum polysaccharide group in the middle and later stages of the experiment can significantly increase insulin of the model-making rat.

TABLE 3 Ganoderma Applanatum polysaccharide has an effect on glucokinase specific activity miIU/min. mgpro on hepatic glucokinase, TC, TG of diabetic rats.

Grouping	Day 14 glucokinase	Day 14 TC	TG on day 14
				Normal group	20.10±0.22	1.51±0.22	1.47±0.35
Diabetes group	2.15±0.46	1.68±0.41	1.59±0.28
				Ganoderma Applanatum polysaccharide group	10.28±0.47	1.14±0.56	0.98±0.39

The Ganoderma Applanatum polysaccharide group can significantly increase hepatic glucokinase of model-making rat, and significantly reduce TC and TG in late stage of test.