CN111635417A - Preparation method and application of tremellodon gelatinosum polyphenol for regulating intestinal flora of obese people - Google Patents
Preparation method and application of tremellodon gelatinosum polyphenol for regulating intestinal flora of obese people Download PDFInfo
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- CN111635417A CN111635417A CN202010537814.4A CN202010537814A CN111635417A CN 111635417 A CN111635417 A CN 111635417A CN 202010537814 A CN202010537814 A CN 202010537814A CN 111635417 A CN111635417 A CN 111635417A
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Abstract
The application relates to a preparation method and application of tremellodon gelatinosum polyphenol for regulating intestinal flora of obese people. The method comprises the following steps: drying fresh Argyrophora gelatinosum fruiting body, drying, and pulverizing to obtain medicinal powder; adding anhydrous ethanol into the medicinal powder to obtain mixed solution; carrying out ultrasonic treatment on the mixed solution for 20-40 minutes, standing for 8-16 hours, and filtering to obtain a filtrate; concentrating and drying the filtrate to obtain an ethanol extract; adding an ethanol water solution with the ethanol volume fraction of 70% into the ethanol extract to obtain a redissolution; passing the redissolved solution through a chromatographic column filled with D-101 macroporous resin to carry out column hanging; then, sequentially eluting by using distilled water, an ethanol water solution with the ethanol volume fraction of 20 percent and an ethanol water solution with the ethanol volume fraction of 80 percent; and drying the eluent obtained by eluting with an ethanol water solution with the ethanol volume fraction of 80% to obtain the tremellodon gelatinosum polyphenol.
Description
Technical Field
The application relates to the technical field of traditional Chinese medicines, in particular to a preparation method and application of tremellodon gelatinosum polyphenol for regulating intestinal flora of obese people.
Background
The metabolic syndrome comprises a series of interrelated chronic diseases including central obesity, insulin resistance, hypertension, type 2 diabetes, atherosclerosis, Non Alcoholic Fatty Liver Disease (NAFLD), and chronic low grade inflammation. The ideal anti-metabolic syndrome drug should have the effects of reducing blood sugar, blood fat, weight and improving cardiovascular health, and there is increasing evidence that changing the structure and composition of intestinal microorganisms is closely related to obesity and diseases related to metabolic disorders. Intestinal microorganisms have become potential drug targets for the treatment of metabolic diseases. The regulation of intestinal microorganisms by drugs, probiotics and prebiotics etc. has numerous clinical benefits for patients with metabolic syndrome.
Disclosure of Invention
The application provides a preparation method and application of tremellodon gelatinosum polyphenol for regulating intestinal flora of obese people.
The application provides a preparation method of ardisia albuginea polyphenol for regulating intestinal flora of obese people, wherein the ardisia albuginea polyphenol comprises episacoviolin beta, episacoconin alpha, sarcovidin beta and sarcodonin alpha; the method comprises the following steps:
(1) drying fresh tremellodon gelatinosum sporocarp, and drying; pulverizing dried fruiting body of Hypsizygus marmoreus to obtain medicinal powder;
(2) adding anhydrous ethanol into the medicinal powder according to the proportion of adding 10g of anhydrous ethanol into every 1g of medicinal powder to obtain a first mixed solution;
(3) carrying out ultrasonic treatment on the first mixed solution for 20-40 minutes, standing for 8-16 hours, and filtering to obtain a filtrate;
(4) concentrating and drying the filtrate to obtain an ethanol extract;
(5) adding an ethanol water solution with the ethanol volume fraction of 70% into the ethanol extract to obtain a redissolution;
(6) passing the redissolved solution through a chromatographic column filled with D-101 macroporous resin to carry out column hanging; then, sequentially eluting by using distilled water, an ethanol water solution with the ethanol volume fraction of 20 percent and an ethanol water solution with the ethanol volume fraction of 80 percent;
(7) and concentrating and drying an eluent obtained by eluting with an ethanol water solution with the ethanol volume fraction of 80% to obtain the tremellodon gelatinosum polyphenol.
In some embodiments, the chromatography column has a volume of 1500 ml, a diameter to height ratio of 1: 10;
in the step (6), the volume of each eluent was 2 liters.
In some embodiments, the ultrasound has a power of 500W, a temperature of 30 ℃, and a time of 30 minutes;
in the step (5), 35-38 ml of an ethanol aqueous solution with an ethanol volume fraction of 70% is added to every 1g of the ethanol extract.
In a second aspect of the application, there is provided a tremellodon gelatinosum polyphenol obtained by the preparation method of the first aspect.
In a third aspect of the application, there is provided the use of the Polyphenol of Carum albolanum of the second aspect in the preparation of a food or a pharmaceutical product for modulating the intestinal flora of obese people.
In some embodiments, the use comprises use of said sarcodictyon polyphenol in the manufacture of a food or pharmaceutical product for reducing the abundance of harmful bacteria in the gut of a human, including intestinomonas, anoetruncus, buthriccicoccus, Papillibacter, Harryflintia, and angelakisela in the family ruminococcus.
In a fourth aspect of the present application, there is provided a use of the sarcodon gelatinosum polyphenol of the second aspect in the preparation of a medicament for preventing or treating metabolic syndrome.
In some embodiments, the pharmaceutical product achieves treatment or prevention of metabolic syndrome by modulating gut flora architecture in a recipient.
In a fifth aspect of the present application, there is provided the use of a combination of epilacoviolin β, epilaccodonin α, sarcovidin β and sarcodonin α in the manufacture of a food or pharmaceutical product for modulating the intestinal flora of an obese human.
In a sixth aspect of the present application, there is provided the use of a combination of epilacoviolin β, epilacoconin α, sarcovidin β and sarcodonin α in the manufacture of a medicament for the prophylaxis or treatment of metabolic syndrome.
The tremellodon gelatinosum polyphenol provided by the application can be prepared into clinically acceptable oral dosage forms with pharmaceutically acceptable auxiliary materials according to the traditional or modern production process. Such as powder, tea, granule, capsule, tablet, concentrated pill, dripping pill, pellicle, soft extract, oral liquid or oral milk, etc. The medicinal adjuvants can be starch, dextrin, lactose, microcrystalline cellulose, gelatin, sucrose, magnesium stearate, erythritol, xylitol, sorbitol, mannitol, maltitol, isomaltitol, hydrogenated starch hydrolysate, stevioside, steviolbioside, dihydrochalcone, glycyrrhizin, xylitol, aspartame, alitame, saccharin, sodium cyclamate, acesulfame potassium, sucralose, etc. The medicine needs to be taken for a long time clinically, is prepared into a solid preparation which is convenient to store and take by patients, and is more suitable for market demands. The preferable dosage forms are capsules, granules and tablets.
The prepared tremellodon gelatinosum polyphenol has the following advantages:
the Polyphenol of Sarcodon albus prepared in the embodiment of the application can remarkably reduce the abundance of Intestiminonas, Anaerotruncus, Butyricicoccus, Papilibacter, Harryflintia and Angelakissella in Ruminoccaceae. The Polyphenol of Hypnea insignis can regulate intestinal flora, inhibit growth of harmful bacteria, and improve intestinal environment. The Polyphenol of Hypnea insignis can also improve sugar metabolism and lipid metabolism disorder of mice, improve liver steatosis, and improve inflammation state of organism, thereby achieving the effect of improving obesity-related metabolic syndrome. Therefore, the polyporus albus polyphenol can improve the symptom of the metabolic syndrome by regulating intestinal flora, and can be used as a new medicine, a new health-care product food additive or a new feed additive.
Drawings
Fig. 1A shows the results of an oral glucose tolerance test;
FIG. 1B shows the area under the curve of the oral glucose tolerance test time-blood glucose curve;
fig. 2A shows the results of an insulin tolerance test;
fig. 2B shows the area under the curve of the insulin tolerance test time-insulin curve.
Detailed Description
In the following, in specific examples, the solutions provided in the present specification are described by way of example.
Polyphenols are important antioxidants, mainly present in fruits, tea and mushrooms. Has effects in regulating immunity, relieving inflammation, protecting liver, lowering blood pressure, and lowering blood sugar. In recent years, the beneficial effects of ingestion of polyphenols on disturbances of lipid and carbohydrate metabolism have been attributed to their effect on the intestinal flora.
Because the natural products have various structural characteristics and wide biological activity, the natural products are good lead compounds for developing anti-metabolic syndrome medicines targeting intestinal microorganisms. The Sarcodon aspratus (Sarcodonleucopus) belongs to Basidiomycota (Basidiomycota), Agaricaceae (Agaricamycetes), Hymenomycetes (Thelephorales), Hydnaceae (Bank ceraceae) and Sarcodactyla (Sarcodon) and is a very rare dual-purpose fungus for food and medicine.
The inventor of the application separates the tremellodon gelatinosum Polyphenol (PAE) from the acephate extract of the tremellodon gelatinosum in earlier experiments, and finds out the potential health promotion effect of the tremellodon gelatinosum polyphenol. Further, the inventors of the present application prepared a polyphenol extract from the sporocarp of tremella mesenterica and further studied its function and effect of improving metabolic syndrome by regulating intestinal flora.
Example 1 extraction of Polyphenol of Hypnea insignis
Collecting fresh Hypsizygus marmoreus fruiting body, drying, pulverizing, weighing 200g, ultrasonically extracting with anhydrous ethanol at a material-liquid ratio of 1:10 for 30min, soaking overnight, filtering, collecting filtrate, repeatedly extracting for 3 times, mixing filtrates, concentrating under reduced pressure at 36-40 deg.C, and drying to obtain ethanol extract 13.8 g.
Redissolving the ethanol extract with 500mL of ethanol/water solution (70:30), separating the obtained solution with D-101 macroporous resin (1500 mL in total volume and 1:10 in diameter-height ratio), repeatedly adsorbing the filtrate for 4 times, and eluting with distilled water, 20% ethanol water and 80% ethanol water, each elution volume being 2L. Mixing eluates eluted with 80% ethanol water, concentrating under reduced pressure at 36-40 deg.C, vacuum drying to obtain crude polyphenol extractive solution PAE component 3.79g rich in alkaloid, and storing at-20 deg.C for animal experiment.
For convenience, the Polyphenol extracted from Pleurotus Nebrodensis in example 1 may also be referred to as PAE.
Example 2 extraction of Polyphenol of Hypnea insignis
In this embodiment, the method for extracting sapodilla officinalis polyphenol comprises the following steps:
(1) drying fresh tremellodon gelatinosum sporocarp, and drying; pulverizing dried fruiting body of Hypsizygus marmoreus to obtain medicinal powder;
(2) adding anhydrous ethanol into the medicinal powder according to the proportion of adding 10g of anhydrous ethanol into every 1g of medicinal powder to obtain a first mixed solution;
(3) carrying out ultrasonic treatment on the first mixed solution, standing for 12 hours, and filtering to obtain a filtrate; wherein the power of the ultrasonic wave is 500W, the temperature is 30 ℃, and the time is 30 minutes.
(4) Concentrating and drying the filtrate to obtain an ethanol extract;
(5) adding ethanol water solution with the ethanol volume fraction of 70% into the ethanol extract according to the proportion that 35-38 ml of ethanol water solution with the ethanol volume fraction of 70% is added into every 1g of the ethanol extract to obtain a re-dissolved solution;
(6) passing the redissolved solution through a chromatographic column (volume 1500 ml, diameter-height ratio 1:10) filled with D-101 macroporous resin for column hanging; then, 2 liters of distilled water, 2 liters of ethanol water solution with the ethanol volume fraction of 20 percent and 2 liters of ethanol water solution with the ethanol volume fraction of 80 percent are used for elution in sequence;
(7) and concentrating and drying an eluent obtained by eluting with an ethanol water solution with the ethanol volume fraction of 80% to obtain the tremellodon gelatinosum polyphenol.
For convenience, the Polyphenol extracted from example 2 can also be referred to as PAE.
Example 3 analysis of Polyphenol component of Hypnea muscovi
GC-MS analysis is carried out on the polyporus albus polyphenol extracted in the embodiment 1 and the embodiment 2, and the result shows that the polyporus albus polyphenol comprises episacoviolin beta, episacoconin alpha, sarcovidin beta, sarcodonin alpha and the like.
Example 4 construction of obese mouse model
4.1 Experimental animals
The C57BL/6J male mice are purchased from the center of laboratory animals of Chinese academy of medical sciences, are fed freely and fed with water in an alternating environment of 25 ℃, 40-60% of humidity and 12/12h day and night, and are adaptively fed for one week.
4.2 model building experiment of obese mice
Blank Control (Control group): 10 male C57BL/6J mice were given normal standard mouse diet.
Model set (HFD set): 30C 57BL/6J male mice were given a high fat diet.
The mice were fed with normal diet and high fat diet (HFD diet) for 8 weeks, and when the body weight gain of the HFD diet mice was more than 20% of the body weight of the mice fed with normal diet, molding was considered successful.
Example 5 administration experiment
The mice in the model group were randomly divided into 3 groups of 10 mice each. Two groups were experimental groups, and the other group was model control (DIO). Wherein the experimental components are PAE-H group and PAE-L group. Each group was administered according to the administration regime of table 1. After 7 weeks of dosing treatment, mouse feces were frozen at-80 ℃. At the end of the experiment, 10% chloral hydrate was anesthetized and the mice were sacrificed after 12h of fasting. The dosing experimental groups are shown in table 1.
TABLE 1 grouping and handling of dosing experiments
Example 6 Polyphenol of Hyphance tiger palm regulates intestinal flora
Stool samples were taken from each mouse prior to dosing, and from each mouse at the end of the dosing experiment. Total DNA in each stool sample was extracted and metagenomic (meta) sequencing was performed. The sequencing results were clustered by OUT (operational Taxomic units) and then aligned in the RDP database. Thus, the intestinal flora structure of each mouse before and at the end of the administration was obtained.
By comparing the intestinal flora structure before and at the end of the administration of the mice in the experimental group, it was found that the levels of intestinomonas, anametruncus, Butyricicoccus, Papillibacter, Harryflintia and angelakisela genera in the intestinal tract of the mice at the end of the administration were significantly reduced relative to the levels before and at the end of the administration. The intestinal flora structures of mice of the blank control group and the model control group have no obvious change before and after administration.
Studies have shown that levels of the family Ruminococcaceae are significantly elevated in obese patients and irritable bowel syndrome patients. The prepared polyporus albus Polyphenol (PAE) can obviously reduce the levels of Intestiminonas, Anaerotruncus, Butyricicoccus, Papilibacter, Harryflintia and Angelakissella in Ruminoccaceae, and shows that the PAE can regulate intestinal flora. Further studies showed that the abundances of Anaerotruncus and Intestiminonas were positively correlated with the concentration of low density lipoprotein cholesterol (LDL-C), that of Anaerotruncus, Butyricoccus and Papilibacter were positively correlated with the concentration of Lipopolysaccharide (LPS), that of Anaerotruncus, Butyricoccus and Intestiminonas were positively correlated with the concentration of IL-6, and that of Anaerotruncus, Angelakisel, Butyricoccus and Intestiminonas was negatively correlated with the insulin sensitivity index. The abundance of Papillibacter shows a significant positive correlation with the concentrations of aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT). Thus, the beneficial effects of PAE on HFD diet-induced obesity and its associated complications are closely related to the intestinal flora.
Example 7 Polyphenol of Hyphance tiger palm improves sugar metabolism by modulating gut flora
Fasting blood glucose was measured at 0, 7, 14, 21, 28, 35, 42, and 48 days of administration for each group of mice, and the results are shown in table 2.
TABLE 2 fasting blood glucose values for the mice of each group
Note: represents p < 0.5; represents p < 0.01. The same applies hereinafter.
As can be seen from table 2, PAE can significantly reduce fasting blood glucose levels in mice.
At the end of dosing, each mouse was subjected to an oral glucose tolerance test (OGTTtest) and an insulin tolerance test (itttest) before the mice were sacrificed. Wherein the results of the oral glucose tolerance test are shown in FIG. 1A, the area under the curve of the time-blood glucose curve of the oral glucose tolerance test ((OGTT-AUC) is shown in FIG. 1B, the results of the insulin tolerance test are shown in FIG. 2A, and the area under the curve of the time-insulin curve of the insulin tolerance test ((OGTT-AUC) is shown in FIG. 2B.
At the end of dosing, before sacrifice of the mice, the insulin level, insulin sensitivity index, glycated hemoglobin/hemoglobin (HbA1c/Hb) were measured separately for each mouse, and the results are shown in Table 3.
TABLE 3 insulin, insulin sensitivity index and HbA1c/Hb for each group of mice
As shown in fig. 1A, 1B, 2A, 2B, table 3, PAE can significantly reduce HbA1c, insulin, and blood glucose levels; PAE may also improve glucose tolerance and insulin tolerance. PAE was shown to ameliorate the sugar metabolism disorder in mice induced by HFD diet by modulating gut microbiology.
Example 8 Polyphenol of Hyphance tiger palm improves lipid metabolism by modulating gut flora
At the end of the administration, blood samples were taken before the mice were sacrificed, and serum levels of Total Cholesterol (TC), Triglycerides (TG), high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) were measured in each mouse, and the results are shown in Table 4.
TABLE 4 serum TC, TG, HDL-C and LDL-C
As shown in Table 4 above, the plasma TG, TC and LDL-C levels in the mice on the HFD diet were increased by 1.33, 5.19 and 2.76 fold, respectively, compared to the normal diet, indicating that PAE significantly inhibited HFD-induced increases in plasma TG, TC and LDL-C levels.
Example 9 Polyphenol of Hyphance sanctum improves hepatic steatosis by modulating gut flora
The liver levels of TC, TG and HDL-C, LDL-C were measured in each mouse, and the results are shown in Table 5.
TABLE 5 TC, TG, HDL-C and LDL-C in the liver
As shown in table 4 above, long-term intake of high fat diet usually results in accumulation of fat in the liver, resulting in diseases such as non-alcoholic fatty liver. HFD group mice showed some degree of liver damage and PAE could significantly reduce TC, TG and LDL-C levels in the liver. It was shown that PAE can improve hepatic steatosis in HFD mice.
Example 10 Polyphenol of Hyphance tiger palm improves inflammation by modulating intestinal flora
At the end of the administration, before the mice were sacrificed, blood samples were taken and serum levels of TNF-. alpha.IL-6, IL-1. beta. and LPS were measured in each mouse, and the results are shown in Table 6.
TABLE 6 serum TNF-alpha, IL-6, IL-1 beta and LPS in groups of mice
As shown in Table 6, chronic inflammation has been shown to be a key factor in the development and progression of obesity and metabolic-related diseases. The PAE can obviously reduce the levels of LPS (lipopolysaccharide) and inflammatory factors IL-6 and TNF-alpha in the serum of the mice, and shows that the PAE improves the inflammatory symptoms of the obese mice by regulating intestinal flora.
The above examples show that the Polyphenol of Sarcodon albus prepared in the examples of the present application can significantly reduce the abundance of Intestiminonas, Anaerotruncus, Butyricicoccus, Papilibacter, Harryflintia and Angelakissella in Ruminococcaceae. The Polyphenol of Hypnea insignis can regulate intestinal flora, inhibit growth of harmful bacteria, and improve intestinal environment. The Polyphenol of Hypnea insignis can also improve sugar metabolism and lipid metabolism disorder of mice, improve liver steatosis, and improve inflammation state of organism, thereby achieving the effect of improving obesity-related metabolic syndrome. Therefore, the polyporus albus polyphenol can improve the symptom of the metabolic syndrome by regulating intestinal flora, and can be used as a new medicine, a new health-care product food additive or a new feed additive.
The above-mentioned embodiments, objects, technical solutions and advantages of the present application are further described in detail, it should be understood that the above-mentioned embodiments are only examples of the present application, and are not intended to limit the scope of the present application, and any modifications, equivalent substitutions, improvements and the like made on the basis of the technical solutions of the present application should be included in the scope of the present application.
Claims (10)
1. A preparation method of ardisia albuginea polyphenol for regulating intestinal flora of obese people is characterized in that the ardisia albuginea polyphenol comprises episacoviolin beta, episacoconin alpha, sarcovidin beta and sarcodonin alpha; the method comprises the following steps:
(1) drying fresh tremellodon gelatinosum sporocarp, and drying; pulverizing dried fruiting body of Hypsizygus marmoreus to obtain medicinal powder;
(2) adding anhydrous ethanol into the medicinal powder according to the proportion of adding 10g of anhydrous ethanol into every 1g of medicinal powder to obtain a first mixed solution;
(3) carrying out ultrasonic treatment on the first mixed solution for 20-40 minutes, standing for 8-16 hours, and filtering to obtain a filtrate;
(4) concentrating and drying the filtrate to obtain an ethanol extract;
(5) adding an ethanol water solution with the ethanol volume fraction of 70% into the ethanol extract to obtain a redissolution;
(6) passing the redissolved solution through a chromatographic column filled with D-101 macroporous resin to carry out column hanging; then, sequentially eluting by using distilled water, an ethanol water solution with the ethanol volume fraction of 20 percent and an ethanol water solution with the ethanol volume fraction of 80 percent;
(7) and concentrating and drying an eluent obtained by eluting with an ethanol water solution with the ethanol volume fraction of 80% to obtain the tremellodon gelatinosum polyphenol.
2. The method of claim 1, wherein the chromatography column has a volume of 1500 ml, a diameter to height ratio of 1: 10;
in the step (6), the volume of each eluent was 2 liters.
3. The method of claim 1, wherein the ultrasound has a power of 500W, a temperature of 30 ℃, and a time of 30 minutes;
in the step (5), 35-38 ml of an ethanol aqueous solution with an ethanol volume fraction of 70% is added to every 1g of the ethanol extract.
4. A Hypnea muscovi polyphenol obtained by the method according to any one of claims 1 to 3.
5. Use of the sapodilla alba polyphenol of claim 4 in the manufacture of a food or medicament for modulating the gut flora of obese people.
6. Use according to claim 5, characterized in that said use comprises the use of said Polyphenol of Sarcodon albus in the manufacture of a food or pharmaceutical product for reducing the abundance of harmful bacteria in the gut of a human population, including Intestiminomonas in the family Ruminoccaceae, Anaerotruncus, Butyricicoccus, Papilibacter, Harryflintia and Angelakissella.
7. Use of the sarcodictyon formosanus polyphenol of claim 4 in the preparation of a medicament for the prevention or treatment of metabolic syndrome.
8. The use of claim 7, wherein the medicament achieves the treatment or prevention of metabolic syndrome by modulating gut flora architecture in a recipient.
Use of epidecoviolin β, epidecodonin α, sarcovidin β and sarcodonin α in combination in the manufacture of a food or pharmaceutical product for modulating the intestinal flora in obese humans.
Use of epidecoviolin β, epidecodonin α, sarcovidin β and sarcodonin α in combination for the preparation of a medicament for the prophylaxis or treatment of metabolic syndrome.
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| CN112843068A (en) * | 2021-01-07 | 2021-05-28 | 西藏自治区高原生物研究所 | Application of polyporus albus polyphenol in preparation of product for prolonging service life of nematodes |
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| CN112843068A (en) * | 2021-01-07 | 2021-05-28 | 西藏自治区高原生物研究所 | Application of polyporus albus polyphenol in preparation of product for prolonging service life of nematodes |
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