CN116903760A - Auricularia auricula polysaccharide, preparation method thereof and application thereof in inhibiting obesity - Google Patents
Auricularia auricula polysaccharide, preparation method thereof and application thereof in inhibiting obesity Download PDFInfo
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- CN116903760A CN116903760A CN202310598492.8A CN202310598492A CN116903760A CN 116903760 A CN116903760 A CN 116903760A CN 202310598492 A CN202310598492 A CN 202310598492A CN 116903760 A CN116903760 A CN 116903760A
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- polysaccharide
- auricularia
- auricularia auricula
- obesity
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Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Sustainable Development (AREA)
- Child & Adolescent Psychology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Diabetes (AREA)
- Materials Engineering (AREA)
- Obesity (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses auricularia auricula polysaccharide, a preparation method thereof and application of the auricularia auricula polysaccharide in obesity inhibition. The auricularia auricula polysaccharide is prepared by a water extraction and alcohol precipitation method, a repeated freeze thawing method and freeze drying. According to the evaluation method of the effect of inhibiting obesity of the auricularia auricula polysaccharide, the auricularia auricula polysaccharide can inhibit the generation of obesity, and the evaluation method comprises the steps of remarkably reducing the weight of an obese mouse, remarkably reducing the weight gain percentage, remarkably reducing the weight of liver, remarkably reducing the area of fat cells and remarkably reducing the content of serum triglyceride.
Description
Technical Field
The invention belongs to the field of biotechnology and polysaccharide extraction, and particularly relates to auricularia auricula polysaccharide, a preparation method thereof and application of the auricularia auricula polysaccharide in inhibiting obesity.
Background
Obesity is now a disease that affects human health and is highly developed. In the body, the balance of energy balance of the body is maintained by controlling the metabolism of energy. When the body ingests energy excessively, but does not take appropriate exercise for consumption, the body's energy is filled and thus is converted into a high-energy substance, i.e., fat, because it cannot be used and consumed rapidly, and obesity occurs with the prolongation of the state maintenance time. Obesity can cause a range of problems in the body including sleep disorders, dyspnea, joint and mobility problems, psychological distress, type ii diabetes, cerebral infarction, and gastrointestinal cancer. Obese patients suffer from a variety of complications, bear physical stress, and also suffer from psychological stress from discrimination by others in society, which adversely affects employment and quality of life. Thus, there is an urgent need for the prevention and treatment of obesity.
Current treatments for obesity mainly include lifestyle changes, medication, surgical treatments, and the like. Lifestyle changes are currently common and widely used weight loss methods, mainly by providing obese patients with healthy and reasonable dietary advice and regulations, e.g., meal fiber instead of greasy meat products, eating health products that have been reported and approved for use, etc. Some drugs acting on the central nervous system, metabolic pathways and regulating hormone levels have been developed and approved for use, which have provided some assistance in the treatment of obese patients and obesity, but their applicable population and lifespan do not have sufficient theoretical basis. Surgical treatment is considered for patients with BMI of 40 or more and severely obese patients, and although the surgical weight loss effect is better than conventional treatment, complications (such as pulmonary embolism), side effects such as heartburn, etc.) and even death may occur. In addition, obesity has poor compliance with weight loss and physical exercise intervention programs, so improvement of obesity by dietary therapy remains the currently accepted mainstay.
At present, ganoderan, cordyceps polysaccharide and hericium erinaceus polysaccharide have been reported to have good effects in relieving weight and adipose tissue weight of obese mice, reducing blood lipid parameters in serum and the like. The agaric is a large medicinal fungus, belongs to basidiomycetes, and has important roles in traditional medicine and modern medicine due to higher anti-tumor and immunoregulatory activity. WoodThe fruiting body of ear has biological activities such as anticoagulation, blood sugar reduction, cholesterol reduction, blood lipid reduction, anti-tumor and anti-inflammatory activity. The main reason for the effect of the Auricularia is that the Auricularia is rich in polysaccharide, phenolic compounds and vitamin D 2 Minerals, etc. In-vivo and in-vitro researches show that the polysaccharide extracted from the agaric has high nutritive value and various pharmacological activities, and has almost no toxic or side effect on human bodies. At present, researches on the role of auricularia auricula polysaccharide in relieving obesity, reducing blood fat and other related metabolic diseases and how auricularia auricula polysaccharide plays an anti-obesity mechanism through intestinal flora are shallow.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the auricularia auricula polysaccharide, the preparation method and the application thereof in inhibiting obesity, and the preparation method is simple, has high extraction efficiency and high purity, and provides a new raw material for researching and developing related products for improving obesity in the fields of biomedicine, health care products and the like.
A preparation method of auricularia auricula polysaccharide comprises the following steps:
1) Extracting auricularia auricula polysaccharide;
2) Purifying auricularia auricula polysaccharide;
the extraction in the step 1) is as follows: soaking Auricularia in clear water to remove impurities, oven drying at 60deg.C, grinding, sealing, and storing for subsequent extraction of Auricularia polysaccharide; weighing 100 g Auricularia powder, adding 1L pure water, heating in 90 deg.C water bath for 2 h, and stirring with glass rod for 5-6 times; centrifuging at 2000 r/min for 5 min after heating, and collecting the obtained solution; adding 1L pure water into the centrifuged Auricularia auricula-judae residues, and repeatedly extracting once; mixing the solutions, concentrating with rotary evaporator, adding absolute ethanol to a final concentration of 75%, standing overnight at 4deg.C, centrifuging at 3500 r/min for 5 min, collecting precipitate, oven drying at 60deg.C, grinding, drying, sealing, and preserving;
purification of the auricularia auricula polysaccharide in the step 2): taking dried powder 2.0 g, adding pure water 500 mL, and sealing after auxiliary dissolution by a vortex meter; freezing at-20deg.C, and dissolving at 90deg.C for 20 times; 3000 Centrifuging at r/min for 10 min, collecting supernatant, concentrating by rotary evaporator, and lyophilizing to obtain Auricularia polysaccharide.
The method is characterized by the structural characterization of auricularia auricula polysaccharide; grinding 1mg dried Auricularia polysaccharide and 100-200 mg dried potassium bromide (KBr) powder, tabletting with tablet press, and performing Fourier transform infrared spectrum scanning analysis with scanning range of 4000 cm -1 - 400 cm -1 。
According to the method, purity is checked by measuring the total sugar content of the auricularia auricula polysaccharide and the content of auricularia auricula polysaccharide protein.
An agaric polysaccharide prepared according to the method.
An evaluation method of the obesity inhibiting effect of the auricularia auricula polysaccharide comprises the steps of selecting 18C 57BL/6 male mice with the age of 5 weeks, and randomly dividing the mice into three groups of 6 mice; the control group freely ingests the control feed D12450H, the 9% normal saline 10 mL/kg gastric lavage treatment, the high fat obesity model group freely ingests the high fat feed D12451, the 9% normal saline 10 mL/kg gastric lavage treatment, the auricularia auricula polysaccharide intervention group freely ingests the high fat feed D12451, and the auricularia auricula polysaccharide solution 200 mg/kg BW gastric lavage treatment; the tail tip of the mice is used for blood sampling after the mice are fasted by 12 h, the blood glucose content is detected by a rapid detection glucometer, and the blood glucose tolerance degree of the mice is analyzed; collecting mouse serum for blood biochemical detection; separating heart, liver, spleen and kidney, and weighing the viscera; adipose tissue was isolated and HE section stained; extracting DNA of intestinal bacteria in cecum contents by using EZNA cube genome DNA separation kit, and sequencing V1-V3 region of intestinal bacteria 16S rRNA; through the measurement and analysis of the indexes, the effect of the auricularia auricula polysaccharide on improving the obesity induced by high fat is evaluated.
The auricularia auricula polysaccharide is used for preparing obesity-inhibiting medicines or functional foods, and the indication is obesity.
The medicine or the functional food can prevent or treat obesity by reducing blood lipid and blood glucose.
The medicine or functional food changes the structure of intestinal flora, improves the relative abundance of Fusobacteria, muribaculum, negativibacillus, parabacteroides, desulfotomaculum guttoideum, eubacterium saphenum, muribaculum intestinale, catabacter hongkongensis, emergencia timonensis, parabacteroides goldsteinii, alistipes putredinis, papillibacter cinnamivorans and Massilimaliae massiliensis, and reduces the relative abundance of Microbacterium, faecalicatena contorta and Clostridium leptum.
Compared with the prior art, the invention has the following advantages:
(1) The hot water extraction method for sampling has the advantages of environmental protection, energy conservation, high efficiency, no toxicity, easy realization and the like. The temperature is too low to damage the cell wall of the wood ear fruiting body, so that the polysaccharide is difficult to dissolve out, and the extraction rate is low. The extraction temperature is 90 ℃, so that the extraction rate is improved. The protein residue in the auricularia auricula polysaccharide prepared by the repeated freeze thawing method is lower. The agaric polysaccharide is prepared by a water extraction and alcohol precipitation method, a repeated freeze thawing method and freeze drying, and the total sugar content is 97.6 percent and the protein content is 2.4 percent.
(2) After the auricularia auricula polysaccharide is treated, the weight gain percentage, the liver weight and the heart weight of the mice are obviously reduced, the obesity degree of the mice is 19.24%, and the obesity level of the mice is not reached.
(3) After the auricularia auricula polysaccharide is treated, the abdominal fat content and the average area of fat cells of the mice are reduced, the blood sugar of the mice is reduced, the blood sugar tolerance degree is improved, and the blood fat concentration of the mice is reduced.
(4) The auricularia auricula polysaccharide can change the structure of intestinal flora, so that the relative abundance of Fusobacteria, muribaculum, negativibacillus, parabacteroides, desulfotomaculum guttoideum, eubacterium saphenum, muribaculum intestinale, catabacter hongkongensis, emergencia timonensis, parabacteroides goldsteinii, alistipes putredinis, papillibacter cinnamivorans and Massilimaliae massiliensis is obviously improved, and the relative abundance of Microbacterium, faecalicatena contorta and Clostridium leptum is obviously reduced.
Drawings
Fig. 1 is a schematic diagram of the preparation and evaluation of agaric polysaccharide.
Fig. 2 is an infrared spectrum of the auricularia auricula polysaccharide.
FIG. 3 (a) is the effect of auricularia auricula polysaccharide on the body weight of high fat diet mice;
FIG. 3 (b) is the effect of auricularia auricula polysaccharide on the percentage of weight gain;
FIG. 3 (c) is the effect of auricularia auricula polysaccharide on liver weight;
FIG. 3 (d) is the effect of auricularia auricula polysaccharide on heart weight;
FIG. 3 (e) is the effect of auricularia auricula polysaccharide on spleen weight;
FIG. 3 (f) is the effect of auricularia auricula polysaccharide on kidney weight;
wherein Chow: normal diet group; HFD: a high fat diet group; multiple hfd+ps: p <0.05 is significant, significant differences between Chow and HFD groups are marked as x, significant differences between HFD and hfd+ps groups are marked as #.
FIG. 4 (a) is the effect of auricularia auricula polysaccharide on the weight of mouse fat;
FIG. 4 (b) is the effect of auricularia auricula polysaccharide on adipocyte size;
fig. 4 (c) is the effect of agaric polysaccharide on fat status.
FIG. 5 (a) is the effect of auricularia auricula polysaccharide on blood glucose levels in high fat diet mice;
fig. 5 (b) is the effect of auricularia auricula polysaccharide on AUC.
FIG. 6 is the effect of auricularia auricula polysaccharide on serum biochemical indicators TG (a), T-CHO (b), LDL-C (C) and HDL-C (d) of high fat-induced obese mice.
FIG. 7 is the effect of auricularia auricula polysaccharide on microbial diversity in high fat-induced obese mice.
FIG. 8 shows the difference in changes in intestinal microorganisms at the genus level.
Fig. 9 is a differential analysis of intestinal microorganisms LEfSe.
Detailed Description
The present invention will be described in detail with reference to specific embodiments and drawings. The experimental methods used in the examples are conventional experimental methods unless otherwise specified. The materials, reagents and the like used in the examples are commercially available unless otherwise specified.
Example 1 preparation method of Auricularia auricula polysaccharide
1. Extraction of auricularia auricula polysaccharide
The whole preparation process is shown in figure 1. Soaking Auricularia in clear water to remove impurities, oven drying at 60deg.C, grinding, and sealing for storage. Weighing 100 g Auricularia fruit body powder, adding 1L pure water, heating in water bath at 90deg.C for 2 h, and stirring with glass rod for 5-6 times. After the heating was completed, the mixture was centrifuged (2000 r/min,5 min) and the resulting solution was collected. Adding 1L pure water into the centrifuged Auricularia auricula-judae residue, and repeatedly extracting once according to the above method. Mixing the solutions, concentrating with rotary evaporator, adding absolute ethanol to a final concentration of 75%, standing overnight at 4deg.C, centrifuging (3500 r/min,5 min), collecting precipitate, oven drying at 60deg.C, grinding, drying, sealing, and storing.
2. Purification of auricularia auricula polysaccharide
Taking dried powder 2.0 g, adding pure water 500 mL, and sealing after auxiliary dissolution by a vortex machine. Freezing at-20deg.C, and dissolving at 90deg.C for 20 times. Centrifuging (3000 r/min,10 min) to obtain supernatant, concentrating with rotary evaporator, and lyophilizing to obtain Auricularia polysaccharide.
3. Purity test of Auricularia auricula polysaccharide
Determination of the total sugar content of the auricularia auricula polysaccharide: accurately weighing 20 mg glucose standard sample, and adding water to fix the volume to 500 ml. Configuration of
Glucose with the concentration of 0, 0.004, 0.008, 0.012, 0.016 and 0.020 mg/mL respectively, 6% phenol 1.0 mL and 5.0 mL concentrated sulfuric acid are sequentially added, shaking and cooling are carried out, optical density is measured at 490 nm after boiling water bath for 15 min, and a standard curve is drawn. The agaric polysaccharide was diluted, the same procedure was followed, and the total sugar content was calculated as absorbance values. Determination of the auricularia auricula polysaccharide protein content: blank tubes, standard tubes, and assay tubes were each configured according to the coomassie brilliant blue kit method, with 5 replicates per tube. 595 Absorbance values were measured at nm. Protein content was calculated from absorbance values.
The absorbance values of glucose are shown in table 1. The standard curve equation is calculated as y=8.251x+0.06711, r 2 = 0.9917, indicating that the standard curve fits well within the experimental range, and that the total sugar content of the sample can be calculated accurately from the absorbance values. In the experiment, 2.6. 2.6 mg auricularia auricula polysaccharide is added with water to a constant volume of 5 mL, and the concentration of auricularia auricula polysaccharide solution is 0.52. 0.52 mg/mL. In the measurement, the sample to be measured is diluted by 100 times and then is subjected to the following methodThe absorbance value is 0.109, and the sugar content of the auricularia auricula polysaccharide is 97.6% through calculation.
TABLE 1 glucose standard solution concentration and absorbance at 490 and 490 nm
| Glucose concentration (mg/mL) | 0 | 0.004 | 0.008 | 0.012 | 0.016 | 0.020 |
| Absorbance (OD 490 value) | 0.064 | 0.103 | 0.136 | 0.164 | 0.196 | 0.234 |
The effect of repeated freeze thawing to remove protein was examined using coomassie brilliant blue. Detection of residues in polysaccharides by a two-point method
Protein content. The absorbance values at standard protein concentration (281.5. Mu.g/mL) and corresponding 595-nm for the blank are shown in Table 2 for each concentration set 5 replicates. The result shows that the 5 times of repetition has smaller difference, the system is stable, and the sample concentration in the concentration range of the two can be accurately detected. In the experiment, 5 mg/mL auricularia auricula polysaccharide is prepared, absorbance is detected at 595, 595 nm after sample addition, and the absorbance of the sample to be detected is 0.513. The protein concentration of the sample to be detected is calculated to be 120.7 mug/mL according to the method for calculating the concentration of the sample to be detected provided by the kit, and the protein content in the auricularia auricula polysaccharide is 2.4%. The result shows that the protein residue in the auricularia auricula polysaccharide prepared by the repeated freeze thawing method is lower.
TABLE 2 protein standard solution concentration and absorbance at 595 and 595 nm
| Protein concentration (μg/mL) | 0 | 281.5 |
| Absorbance (OD 595 value) | 0.272±0.002 | 0.834±0.005 |
4. Structural characterization of auricularia auricula polysaccharide
The infrared absorption spectrum of Auricularia polysaccharide is shown in figure 2. By comparing with the infrared absorption spectrum of the auricularia auricula polysaccharide reported before, most of absorption peaks are the same, and the infrared spectrum reading and main functional groups in the auricularia auricula polysaccharide are shown in table 3. The result shows that based on the infrared spectrum of the auricularia auricula polysaccharide, the detected substance is a polysaccharide compound, and the auricularia auricula polysaccharide obtained by the water extraction method is an acidic polysaccharide containing a pyran ring structure, beta-D-glucopyranose and glycoprotein.
TABLE 3 Infrared spectroscopic analysis of Auricularia auricula polysaccharides
EXAMPLE 2 inhibition study of Auricularia auricula polysaccharides on mouse obesity
1. Experimental materials
Specific pathogen free (Specified pathogens free, SPF) grade C57BL/6 male mice: purchased from the university of western security traffic animal center (shanxi, china); high fat feed (D12451), control feed (D12450H) (detailed feed formulation and caloric content are shown in Table 4, purchased from Shanghai Saipoi Biotechnology Co., ltd.) cell tissue fixative (30%), total cholesterol (Total cholesterol, T-CHO), triglyceride (TG), low Density lipoprotein (Low density lipoprotein chesterol, LDL-C), high Density lipoprotein (High density lipoprotein chesterol, HDL-C) content detection kit (purchased from Nanjing's institute of biological engineering, nanjing, china).
Table 4 mouse feed formulation and caloric status
| Component (g) | Control feed (#D12450H) | High-fat feed (# D12451) |
| Casein (Casein) | 200 | 200 |
| L-methionine (L-Cystine) | 3 | 3 |
| Corn starch (Corn) Starch） | 452.2 | 72.8 |
| Maltodextrin (Malto xtrin 10) | 75 | 100 |
| Sucrose (Sucrose) | 172.8 | 172.8 |
| Cellulose (Cellulose) | 50 | 50 |
| Soybean Oil (Soybean Oil) | 25 | 25 |
| Lard (Lard) | 20 | 177.5 |
| Mineral (Mineral Mix S10026) | 10 | 10 |
| Dicalcium phosphate (dicalcium phosphate) | 13 | 13 |
| Calcium carbonate (Calcium Carbonate) | 5.5 | 5.5 |
| Potassium citrate (Potassium Citrate) | 16.5 | 16.5 |
| Vitamin (Vitamin Mix V10001) | 10 | 10 |
| Choline tartrate (Choline Bitartrate) | 2 | 2 |
| Pigment (FD)&C Yellow Dye #5） | 0.04 | 0 |
| Pigment (FD)&C Red Dye #40） | 0.01 | 0.05 |
| Aggregate (g) | 1055.05 | 858.15 |
| Protein (Protein) | 20 | 20 |
| Carbohydrates (carbohydrates) | 70 | 35 |
| Fat (Fat) | 10 | 45 |
| Feed caloric value (Kcal/g) | 3.85 | 4.73 |
2. Experimental method
(1) Test animal feeding and grouping
18C 57BL/6 male mice at 5 weeks of age were kept in SPF at 22.+ -. 2 ℃ in 3 cages. After one week of pre-feeding, groups of 6 were randomized. Control diet group (Chow-fat diet group, chow): free drinking water, free feeding control feed (D12450H), 9% normal saline (10 mL/kg) gastric lavage treatment (1 time/day), feeding for 8 weeks. High-fat diet group (HFD): free drinking water, free ingestion of high fat feed (D12451), 9% normal saline (10 mL/kg) gastric lavage treatment (1 time/day), and feeding for 8 weeks. Auricularia polysaccharide intervention group (High-fat set+ Auricular auricula polysaccharide group, HFD+PS): free drinking water, free feeding of high fat feed (D12451), gastric lavage treatment of Auricularia polysaccharide solution (200 mg/kg BW) (1 time/day), and feeding for 8 weeks.
(2) Serum collection and preservation
Serum collection: the mouse orbit was bled, and the blood was allowed to stand at 4℃for 30 min and then centrifuged (3000 rmp) for 15 min. Preservation of serum: sealing the upper serum layer in a 1.5 mL sterile centrifuge tube, quickly quick freezing in liquid nitrogen, and storing in a-80 ℃ ultralow temperature refrigerator for subsequent blood biochemical detection.
(3) Organ collection and weighing
Immediately after completion of orbital bleeding, the heart, liver, spleen and kidney were isolated. PBS washes the blood on the surface of the viscera and peels off the surface-attached fat. The filter paper was blotted to dry the surface water and the weights of the organs were weighed.
(4) Isolation preservation of adipose tissue and HE section staining
The visceral fat of the mice was removed, and after weighing and recording with an analytical balance, the mice were stored with a tissue fixative. And (3) cutting the paraffin embedded tissue into sections, placing the fat sections in an inverted fluorescent microscope bright field for observation and photographing, and introducing 200 x tissue section pictures into a computer to measure the cell area.
(5) Detection of serum biochemical index
The content of the serum biochemical index is measured by adopting a corresponding kit.
(6) Detection of blood glucose tolerance level of mice
The experimental mice fasted by 12 h and then blood is collected from the tail tip, and the blood glucose content detected by the rapid detection glucometer is the fasting blood glucose. The mice were then perfused with a glucose solution (100 mg/kg BW) and the blood glucose levels were collected and recorded at 0 min,15 min,30 min,60 min, 90 min,120 min post-lavage. The blood glucose content of the mice at 6 time points is plotted, the area under the curve (Area under the curve, AUC) is calculated, and the blood glucose tolerance degree of the mice is analyzed.
(7) Cecal microorganism detection and analysis
The DNA of the intestinal bacteria in the cecum content was extracted using EZNA-genomic DNA isolation kit (omega BioTek, duraville, USA) and the V1-V3 region of the intestinal bacteria 16S rRNA was sequenced. PCR was performed using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 533R (5'-TTACCGCGGCTGCTGGCAC-3') with a higher degree of conservation. Based on an IonS5TMXL sequencing platform, a Single-ended sequencing (Single-End) method is utilized to construct a small fragment library for Single-ended sequencing. The reading process and quality control are performed using QIIME pipes. The mass filtered reads were clustered into operational taxa (Operational taxonomic units, OTU) using usearchh. Sequences were taxonomically characterized from phylum to genus using the ribosomal database project (Ribosomal Database Projec, RDP). Determining the degree of intestinal microflora differentiation using Principal co-ordinates analysis, PCoA; the difference in intestinal microbiota and genus levels between groups of mice was detected using a two-tailed T-test; and identifying the classification units with rich differences by a linear discriminant analysis effect size method.
3. Experimental results
(1) Establishment of high-fat diet induced high-fat obesity mouse model and influence of auricularia auricula polysaccharide on high-fat diet mice
After 8 weeks of high fat diet treatment, the body weight, percentage of body weight gain and organ index of the mice are shown in fig. 3 (a) to 3 (f). Compared with Chow group, the high-fat diet significantly improves the weight and weight gain percentage of HFD group miceP<0.05 The liver weight and the heart weight of the mice are obviously increasedP<0.05 But the effect on spleen and kidney weight was insignificant. As shown in table 5, at the end of the experiment, mice in the HFD group had an obesity degree of 37.21% and reached an obese state. The result shows that the high-fat diet for 8 weeks can lead the mice to reach an obese state, and the high-fat obese mouse model is successfully established. While the high fat obesity model is established, the other group of mice treated by the same treatment are subjected to the gastric lavage treatment of auricularia auricula polysaccharide (200 mg/kg BW), compared with the HFD group, the weight of the mice, the weight increase percentage, the liver weight and the heart weight are obviously reducedP<0.05 No significant differences in liver and spleen weights. After 8 weeks of agaric polysaccharide treatment, mice had a degree of obesity of 19.24% and did not reach the level of obesity. The results show that the auricularia auricula polysaccharide can significantly inhibit obesity of mice caused by high-fat diet.
TABLE 5 degree of obesity in mice in HFD group and HFD+PS group
| Degree of obesity | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| HFD group | 11.59% | 11.58% | 12.21% | 17.02% | 20.31% | 21.70% | 26.38% | 37.21% |
| HFD+PS group | 5.27% | 2.47% | 3.32% | 7.25% | 8.87% | 9.04% | 11.59% | 19.24% |
(2) Effect of Auricularia polysaccharide on fat of high-fat diet mice
The effect of auricularia auricula polysaccharide on fat of high-fat diet mice is shown in fig. 4 (a) to 4 (c). After 8 weeks of high fat diet, mice in the HFD group had significantly fatter body type, significantly increased fat content and significantly increased adipocytes, average area [ ] compared to Chow groupP<0.05). Mice in the HFD+PS group had a reduced body size and fat mass compared to mice in the HFD group, but did not reach a significantly different level, but the average area of adipocytes was observed to be significantDecrease down to%P<0.05). The results show that the auricularia auricula polysaccharide can influence the accumulation of abdominal fat of the high-fat diet mice, and has the capability of reducing the abdominal fat content and the average area of fat cells.
(3) Influence of auricularia auricula polysaccharide on fasting blood glucose and blood glucose tolerance degree of high-fat diet mice
The effect of auricularia auricula polysaccharide on fasting blood glucose and blood glucose tolerance of high-fat diet mice is shown in fig. 5 (a) and 5 (b). Compared with Chow group, the HFD group has obviously increased blood sugar content of mice at 6 time points of blood sugar level collectionP<0.05 After glucose supplementation, the blood glucose level of the mice rises rapidly and drops slowly, and in addition, the AUC value rises markedly [ ]P<0.05). The blood glucose levels were lower in the HFD + PS group than in the HFD group at 6 test time points, and the AUC values were also lower than in the HFD group, but none reached significant differential levels. The result shows that the long-term high-fat diet can obviously raise the blood sugar level of the organism and reduce the blood sugar tolerance level, and the auricularia auricula polysaccharide has the capability of reducing the blood sugar of mice and improving the blood sugar tolerance level.
(4) Influence of auricularia auricula polysaccharide on serum biochemical index of high-fat induced obese mice
The effect of auricularia auricula polysaccharide on serum biochemical index of high-fat induced obese mice is shown in figure 6. After high-fat diet treatment, the content of T-CHO and LDL-C in serum of HFD group mice is obviously increased compared with that of Chow group miceP<0.05 TG content in serum was increased and HDL-C content was decreased, but none reached significant differential levels. Compared with HFD group, the TG content in serum of HFD+PS group mice is obviously reducedP<0.05 Although there were no significant differences in the remaining indices, they all brought them closer to the Chow group normal level. The result shows that the hyperlipidemia appears in the high-fat diet mice, and the auricularia auricula polysaccharide can reduce the blood fat concentration of the mice.
(5) Influence of Auricularia auricula polysaccharides on intestinal microbial diversity of obese mice
As shown in fig. 7, the diversity results indicate that although three groups of samples did not significantly differ by the significance analysis, the HFD group richness index (ACE, chao 1) had an upward trend compared to the other two groups; the diversity index (Shannon) has a decreasing trend compared to the other two groups. This suggests that long-term high-fat diets have a trend to increase host gut microflora species abundance and decrease diversity; the ingestion of auricularia auricula polysaccharides has a trend of improving the richness and diversity of the host intestinal microflora. As can be seen from the PCoA analysis result based on the Bray-Curtis distance algorithm, the three groups of samples are distinguished obviously, and the anoim statistical analysis shows that the differences between the three groups are obvious (P < 0.001). The results show that the microbial communities among the three groups differ significantly.
(6) Analysis of the structure of a genus-level microbial community
Statistical analysis of the genus level microorganisms among groups using T-test is shown in FIG. 8. The difference of the genus level microorganism genus among the three groups is obvious. Chow group and HFD group share 19 differential bacteria; the HFD group and HFD+PS group had 7 different bacteria. After ingestion of the auricularia polysaccharide, the abundance of a total of 4 bacteria is reversed. The HFD group significantly reduced the relative abundance of Negativibacillus, parabacteroides and Muribaculum compared to Chow or hfd+ps groups; the relative abundance of Microbacterium is significantly improved.
(7) Biomarker analysis
To examine the effect of auricularia polysaccharide intake on specific microorganisms, total 71 and 83 OTUs were screened from Chow and HFD groups and HFD and hfd+ps groups, respectively, using the LEfSe assay at OTU level. The HFD group was 19 total OTUs with significant differences from the other two groups, and were retrieved and annotated in the NCBI database, as shown in fig. 9. From the graph, the HFD group has 5 OTU abundance increase and 14 OTU abundance decrease compared with the Chow group; there were 5 OTU abundances rising and 14 OTU abundances falling compared to the hfd+ps group.
In summary, agaric polysaccharide is capable of inhibiting the development of obesity, including significantly reducing body weight in obese mice; reducing the percentage of weight gain and liver weight; reducing adipocyte area and significantly reducing serum triglyceride content. Furthermore, using microbiology techniques, it was found that the intake of agaric polysaccharides significantly altered intestinal flora diversity, while having the ability to boost Shannon index, compared to high fat obese mice. Studies have shown that ingestion of auricularia auricula polysaccharides significantly increases the relative abundance of Desulfotomaculum guttoideum, eubacterium saphenum, muribaculum intestinale, catabacter hongkongensis, emergencia timonensis, parabacteroides goldsteinii, alistipes putredinis, papillibacter cinnamivorans, and Massilimaliae massiliensis, and significantly decreases the relative abundance of Faecalicatena contorta and Clostridium leptum. These intestinal microorganisms may play an important role in the mechanism of agaric polysaccharide against high lipid-induced obesity.
The above list is merely a few examples of the present invention and is not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, improvement, etc. that comes within the spirit and principle of the claims of the present invention should be included in the protection scope of the claims of the present invention.
Claims (8)
1. A preparation method of auricularia auricula polysaccharide is characterized by comprising the following steps:
1) Extracting auricularia auricula polysaccharide;
2) Purifying auricularia auricula polysaccharide;
the extraction in the step 1) is as follows: soaking Auricularia in clear water to remove impurities, oven drying at 60deg.C, grinding, sealing, and storing for subsequent extraction of Auricularia polysaccharide; weighing 100 g Auricularia powder, adding 1L pure water, heating in 90 deg.C water bath for 2 h, and stirring with glass rod for 5-6 times; centrifuging at 2000 r/min for 5 min after heating, and collecting the obtained solution; adding 1L pure water into the centrifuged Auricularia auricula-judae residues, and repeatedly extracting once; mixing the solutions, concentrating with rotary evaporator, adding absolute ethanol to a final concentration of 75%, standing overnight at 4deg.C, centrifuging at 3500 r/min for 5 min, collecting precipitate, oven drying at 60deg.C, grinding, drying, sealing, and preserving;
purification of the auricularia auricula polysaccharide in the step 2): taking dried powder 2.0 g, adding pure water 500 mL, and sealing after auxiliary dissolution by a vortex meter; freezing at-20deg.C, and dissolving at 90deg.C for 20 times; 3000 Centrifuging at r/min for 10 min, collecting supernatant, concentrating by rotary evaporator, and lyophilizing to obtain Auricularia polysaccharide.
2. The method of claim 1, wherein the structural characterization of the agaric polysaccharide; grinding 1mg dried Auricularia polysaccharide and 100-200 mg dried potassium bromide (KBr) powder, tabletting with tablet press, and performing Fourier transform infrared spectrum scanning analysis with scanning range of 4000 cm -1 - 400 cm -1 。
3. The method according to claim 1, wherein the purity test is performed by measuring the total saccharide content of the agaric polysaccharide and measuring the protein content of the agaric polysaccharide.
4. An agaric polysaccharide prepared according to the method of claim 1.
5. An evaluation method of obesity inhibitory effect of the agaric polysaccharide according to claim 4, wherein 18C 57BL/6 male mice 5 weeks old are selected, and randomly divided into three groups of 6; the control group freely ingests the control feed D12450H, the 9% normal saline 10 mL/kg gastric lavage treatment, the high fat obesity model group freely ingests the high fat feed D12451, the 9% normal saline 10 mL/kg gastric lavage treatment, the auricularia auricula polysaccharide intervention group freely ingests the high fat feed D12451, and the auricularia auricula polysaccharide solution 200 mg/kg BW gastric lavage treatment; the tail tip of the mice is used for blood sampling after the mice are fasted by 12 h, the blood glucose content is detected by a rapid detection glucometer, and the blood glucose tolerance degree of the mice is analyzed; collecting mouse serum for blood biochemical detection; separating heart, liver, spleen and kidney, and weighing the viscera; adipose tissue was isolated and HE section stained; extracting DNA of intestinal bacteria in cecum contents by using EZNA cube genome DNA separation kit, and sequencing V1-V3 region of intestinal bacteria 16S rRNA; through the measurement and analysis of the indexes, the effect of the auricularia auricula polysaccharide on improving the obesity induced by high fat is evaluated.
6. Use of the agaric polysaccharide according to claim 4 for the preparation of a medicament or a functional food for inhibiting obesity, characterized in that the indication is obesity.
7. The use according to claim 6, wherein the medicament or functional food is for preventing or treating obesity by reducing blood lipid and blood glucose.
8. The use of claim 6, wherein the medicament or functional food alters the structure of the intestinal flora, increases the relative abundance of Fusobacteria, muribaculum, negativibacillus, parabacteroides, desulfotomaculum guttoideum, eubacterium saphenum, muribaculum intestinale, catabacter hongkongensis, emergencia timonensis, parabacteroides goldsteinii, alistipes putredinis, papillibacter cinnamivorans and Massilimaliae massiliensis, and decreases the relative abundance of Microbacterium, faecalicatena contorta and Clostridium leptum.
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