CN111253496A - Armillariella tabescens mycelium polysaccharide with blood sugar reducing effect - Google Patents
Armillariella tabescens mycelium polysaccharide with blood sugar reducing effect Download PDFInfo
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- CN111253496A CN111253496A CN202010068963.0A CN202010068963A CN111253496A CN 111253496 A CN111253496 A CN 111253496A CN 202010068963 A CN202010068963 A CN 202010068963A CN 111253496 A CN111253496 A CN 111253496A
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- polysaccharide
- blood sugar
- armillaria
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- mycelium
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- 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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- 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
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- 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
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
- A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
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- 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
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- 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
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Abstract
The invention discloses a Armillaria mellea mycelium polysaccharide with a blood sugar reducing effect, wherein the total sugar content of the Armillaria mellea mycelium polysaccharide is more than 86%, and the monosaccharide composition and the molar ratio are mannose, arabinose and fucose which are 1.58:1: 2.74. The armillariella mycelium polysaccharide has the activity of reducing the blood sugar and the blood fat of type 2 diabetic mice, particularly has obvious effect on improving insulin resistance, can be used as a novel natural blood sugar-reducing active substance, and is widely applied to the fields of health-care food and medicine.
Description
Technical Field
The invention belongs to the field of health-care food, and particularly relates to armillaria tabescens mycelium polysaccharide with a blood sugar reducing effect, and a preparation method and application thereof.
Background
In recent years, the number of people suffering from diabetes has rapidly increased due to aging of population, reduction in labor intensity, and change in dietary structure. It is estimated that about 2.46 hundred million diabetics exist in the world at present, about 9200 million diabetics exist in China, and diabetes becomes the 2 nd most chronic disease in the world after hypertension. At present, diabetes is mainly controlled by taking medicines for treatment and life style adjustment, such as insulin injection and taking therapeutic medicines. However, long-term administration of the medicine can cause adverse drug reactions and can also endanger life in severe cases. Therefore, the search for a natural product with high efficiency, low toxicity and capability of reducing blood sugar has become a hot research in the field of health and health food.
With the development of functional foods, edible fungi are widely concerned by people. Research reports that many edible mushrooms in nature, such as ganoderma lucidum, grifola frondosa, corious versicolor, phoenix mushroom and the like, have an anti-diabetic effect, wherein fungal polysaccharides play a key role. Armillariella tabescens (Scop. ex. Fr) Sing, also known as Armillariella pseudomela, is named because the mycelium is white when it is nascent on a culture medium and emits light blue fluorescence in the dark. The armillaria tabescens belongs to Agaricales, Tricholomataceae and clitocybe, is a precious edible and medicinal fungus, and has been widely used for treating acute and chronic hepatitis, appendicitis, biliary tract infection, otitis media, cholecystitis and the like as a traditional medicine. The armillarisin contains various active ingredients, such as armillarisin, armillarisin B, armillarisin polysaccharide and the like. The armillaria tabescens polysaccharide is used as an important active component and is found to have the functions of resisting tumors and regulating immunity. The polysaccharide of the mycelium of the armillaria tabescens, which is discovered in the invention, has the remarkable activities of reducing blood sugar and improving insulin resistance, and has not been reported in related documents.
Disclosure of Invention
The invention aims to provide a armillariella mycelium polysaccharide with the effect of reducing blood sugar, and a preparation method and application thereof. The research of the invention finds that the Armillariella tabescens mycelium polysaccharide has obvious activity of reducing blood sugar and blood fat, especially has obvious effect on improving insulin resistance, and has very important academic value and development prospect.
The polysaccharide of the mycelium of the armillaria tabescens has the effect of reducing blood sugar, the total sugar content is more than 86 percent, and the monosaccharide composition and the molar ratio of mannose to arabinose to fucose are 1.58 to 1 to 2.74.
The preparation method of the armillaria mycelium polysaccharide with the blood sugar reducing effect comprises the following steps:
step 1: inoculating 3-5g Armillariella tabescens (shop.EX. Fr) Sing strain (the strain is purchased from Hefeicheng Biopharmaceutical Co., Ltd.) into 300-500mL glucose-potato liquid culture medium (200 g peeled potato is cut up and boiled, then filtered by four layers of gauze, 20g glucose is added, water is added to 1000mL, sterilization is carried out at 121 ℃ for 20min to obtain glucose-potato culture medium), and standing and culturing at 26 ℃ for 20 days to obtain a armillaria mycelium;
step 2: adding distilled water into the armillaria mycelia (5000 g) obtained in the step 1 according to a liquid-material ratio (g/mL) of 1:3-5, crushing, leaching at 80 ℃ for 2-3 h, repeatedly leaching for 2-3 times, sieving by a 60-mesh sieve, and combining extracting solutions;
and step 3: concentrating the extracting solution obtained in the step 2 to 1/200-250 of the original volume, adding 95 vt% ethanol with 4 times of volume until the final concentration of the ethanol is 75 vt%, standing for 12h at 4 ℃, centrifuging at 4500rpm/min, and collecting the precipitate;
and 4, step 4: dissolving the precipitate obtained in the step 3 with distilled water, adding an equal volume of Sevag reagent (n-butyl alcohol: chloroform v/v ═ 1:4), magnetically stirring for 2h, and centrifuging to obtain a supernatant; repeating until protein is completely removed, concentrating the polysaccharide solution, dialyzing (molecular weight cut-off of a dialysis bag is 3500) to remove small molecules, performing rotary evaporation and concentration to obtain a thick liquid, and performing vacuum freeze drying to obtain the polysaccharide of the mycelia of the armillaria tabescens.
The invention relates to an application of armillaria tabescens mycelium polysaccharide in preparing foods, health-care products or medicines with the effects of reducing blood sugar and blood fat.
The invention relates to an application of armillariella mycelium polysaccharide in preparing a medicament for treating type 2 diabetes, which has the obvious activities of reducing blood sugar and blood fat and improving insulin resistance.
The invention takes a type 2 diabetes mouse induced by high-sugar high-fat diet combined with streptozotocin as a research object, and animal experiments prove that the armillaria tabescens mycelium polysaccharide has the functions of reducing blood sugar and blood fat.
The index of the armillariella mycelium polysaccharide to insulin resistance of type 2 diabetic mice is as follows: the model group is (12.33 + -2.15), the metformin (positive control) group is (6.26 + -1.00), and the polysaccharide group of the mycelia of the armillaria tabescens is (6.77 + -1.05).
Drawings
FIG. 1 is a standard monosaccharide composition HPLC chromatogram (. about. -solvent peak, 1-mannose, 2-rhamnose, 3-glucose, 4-galactose, 5-arabinose, 6-fucose).
FIG. 2 is a high performance liquid chromatogram of polysaccharide monosaccharide composition of mycelia of Armillariella tabescens (x-solvent peak, 1-mannose, 5-arabinose, 6-fucose).
Figure 3 is the effect of armillaria mycelium polysaccharide on glucose tolerance in type 2 diabetic mice (n ═ 8, x ± s).
Detailed Description
The preparation, blood sugar and lipid reducing and insulin resistance improving activities of the present invention are described in the following examples, which are only illustrative and not intended to limit the scope of the present invention.
Example 1: preparation of Armillariella tabescens mycelium polysaccharide
1. Inoculating 3-5g of armillariella mellea strain into 300-500mL of glucose-potato liquid culture medium, and standing and culturing at 26 ℃ for 20 days to obtain the armillariella mellea mycelium.
2. Adding the mycelia of the armillaria tabescens into distilled water according to the liquid-material ratio of 1:5, crushing for 10min by using a flash extractor, then leaching for 3h at 80 ℃, repeatedly leaching for 3 times, sieving by using a 60-mesh sieve, and combining the extracting solutions;
3. concentrating the obtained extractive solution to 1/250 of original volume, adding 4 times volume of 95% ethanol until the final concentration of ethanol is 75%, standing at 4 deg.C for 12h, centrifuging at 4500rpm/min, and collecting precipitate;
4. dissolving the precipitate obtained in the step 3 with distilled water, adding an isovolumetric Sevag reagent (n-butyl alcohol: chloroform: 1:4), magnetically stirring for 2h, and centrifuging to obtain a supernatant; repeating until protein is completely removed, concentrating the polysaccharide solution, dialyzing to remove small molecules, performing rotary evaporation and concentration to obtain a thick liquid, and performing vacuum freeze drying to obtain the armillariella mycelium polysaccharide.
Example 2: determination of total sugar and monosaccharide composition of Armillariella tabescens mycelium polysaccharide
1. The total sugar content of the polysaccharide of the mycelia of the armillaria tabescens is determined by a phenol-sulfuric acid method. Accurately sucking 2mL of armillaria mycelia polysaccharide solution (50 mu g/mL) and glucose standard solution, adding 1mL of 6% phenol and 5mL of concentrated sulfuric acid, setting three groups of parallels, measuring the light absorption value at 490nm, and calculating the total sugar content of the armillaria mycelia polysaccharide according to a formula of a standard curve.
2. The monosaccharide composition analysis of the armillaria tabescens mycelium polysaccharide adopts an acid hydrolysis-pre-column PMP derivatization method to process a sample, and the analysis and the determination are carried out by a high performance liquid chromatography. The Armillaria tabescens mycelium polysaccharide (10mg) is accurately weighed, dissolved in 5mL of 2mol/L trifluoroacetic acid, sealed by filling nitrogen and hydrolyzed in an oven at 110 ℃ for 6 h. Removing water by rotary evaporator, adding appropriate amount of deionized water, rotary evaporating to dryness, and repeating for several times until the pH value of the solution is neutral. 1mL of distilled water was added for use.
Adding 50 μ L, 0.5mol/L PMP methanol solution and 50 μ L, 0.3mol/L NaOH solution into the standard monosaccharide and hydrolyzed sample solution, reacting in a water bath at 70 deg.C for 30min for pre-column derivatization of PMP, and neutralizing with 50 μ L, 0.3mol/L HCl to neutrality. The obtained product is detected by high performance liquid chromatography, and a DAD detector is selected. HPLC column temperature is 30 ℃, and chromatographic column is Zorbox Eclipse XDB-C18The column (4.6 mm. times.250 mm, 5 μm) was examined at a wavelength of 245 nm. Two detection mobile phases are selected, wherein the mobile phase A is acetonitrile, and the mobile phase B is 0.05mol/L phosphate buffer solution. Time gradient elution 0-50min, initial set to mobile phase a: mobile phase B is 17%: 83%, finally eluted to the ratio mobile phase a: mobile phase B is 20%: 80% and the sample amount is 10 μ L.
Example 3: blood sugar reducing activity determination of Armillariella tabescens mycelium polysaccharide
The prepared armillariella mycelium polysaccharide is used as a raw material, and a type 2 diabetes mouse model is utilized to perform experimental evaluation on the blood sugar reducing effect of the armillariella mycelium polysaccharide.
1. Preparation of diabetic mouse model
Selecting 32 SPF male C57BL/6J mice with the weight of 22 +/-2 g, randomly dividing the mice into 4 groups of 8 mice each, and dividing the mice into a blank group, a model group, a positive group and a armillaria polysaccharide group. The blank group was given normal diet, and the remaining three groups were given high-sugar high-fat diet (HFD). After feeding for 4 weeks, the HFD mice are fasted for 12 hours, and are subjected to intraperitoneal injection of 50mg/kg of Streptozotocin (STZ) in 4 times of small dose, so that a type 2 diabetes model is established; and (3) after 72h of molding, tail vein blood sampling is carried out to determine fasting blood glucose, the fasting blood glucose is more than or equal to 11.1mmol/L for 3 consecutive days, and a person who has polydipsia, polyphagia and polyuria is successfully copied as a model, and the mouse is a type 2 diabetic mouse after continuing high-sugar high-fat diet for 3 weeks. From 9 weeks, the blank group and the model group were gavaged with physiological saline of equal volume every day, the positive group was gavaged with 200mg/kg metformin every day, and the armillaria polysaccharide group was gavaged with 400mg/kg armillaria mycelium polysaccharide every day, 1 time every day, for 4 weeks. Measuring the weight and fasting blood glucose of the mice 1 time per week, fasting for more than 12h after last gastric lavage, measuring the oral glucose tolerance, and collecting blood to prepare serum samples. All experimental data were analyzed for variance using SPSS17.0 software.
2. Results of the experiment
(1) As can be seen from Table 1, HFD feeding at 4 weeks significantly increased the body weight of the mice compared to the normal diet group. After STZ injection, the body weight of the mice of the model group, the positive group and the armillaria polysaccharide group are all obviously reduced, and the obvious characteristics of diabetes are shown. The gavage treatment with physiological saline, metformin and armillaria mycelia polysaccharide was performed from week 9, and showed that the body weight of the model group was still continuously decreased; the positive group and the Armillaria mellea polysaccharide group have weight reduction but are relieved, which shows that the Armillaria mellea mycelium polysaccharide has the function of obviously relieving the emaciation symptom of the diabetic mice.
Table 1 change in body weight of mice (g) (n-8,
)
| group of | 0 week | 4 weeks | 8 weeks | For 12 weeks |
| Blank group | 21.89±0.71 | 25.10±0.85 | 27.69±0.64 | 29.18±0.67 |
| Model set | 22.00±0.89 | 29.14±0.92** | 28.18±0.85 | 25.88±0.81** |
| Positive group | 22.34±0.83 | 29.63±0.91 | 27.36±0.89 | 27.78±0.94## |
| Armillariella tabescens polysaccharide group | 22.68±0.88 | 30.26±0.95 | 28.54±1.04 | 28.24±0.88## |
Note: in comparison to the blank set, the data is,**P<0.01, very significant; in comparison to the model set,##P<0.01, very significant
(2) As can be seen from Table 2, the fasting blood glucose value of the mice in the blank group was always stabilized at 4-6 mmol/L during the experiment, and the fasting blood glucose of the mice was significantly increased after STZ injection. After 4 weeks of intervention treatment, compared with the model group, the blood sugar values of the positive group and the armillaria polysaccharide group are obviously reduced (P is less than 0.01).
Table 22 change in fasting plasma glucose value (mmol/L) in diabetic mice (n-8,
)
| group of | 9 weeks | For 10 weeks | 11 weeks | For 12 weeks |
| Blank group | 4.68±1.06 | 4.95±0.98 | 4.61±1.11 | 4.84±0.94 |
| Model set | 18.88±1.52** | 19.71±1.77** | 20.36±1.67** | 20.86±1.85** |
| Positive group | 19.63±1.57 | 19.09±1.73 | 17.94±1.46## | 16.46±1.47## |
| Armillariella tabescens polysaccharide group | 18.74±1.58 | 19.21±1.76 | 18.40±1.76# | 17.66±1.53## |
Note: in comparison to the blank set, the data is,**P<0.01, very significant; in comparison to the model set,##P<0.01, very significant;#P<0.05, remarkable
(3) Glucose tolerance is the body's ability to tolerate glucose. Compared with the blank group, the oral glucose tolerance of the model group mice is abnormal, the blood sugar is increased at 30min so as to reach the upper limit of detection, and the blood sugar is slowly reduced within 120 min. Compared with the model group, the positive group and the armillaria polysaccharide group reach a peak value at 30min, then slowly decline, and the overall decline trend is obvious (fig. 3). The results show that: the armillariella mycelium polysaccharide has certain improvement effect on the oral glucose tolerance injury of type 2 diabetic mice.
(4) As can be seen from Table 3, compared with the blank group, the serum Insulin (INS) and the insulin resistance index (HOMA-IR) of the model group mice are significantly increased (P <0.01), which indicates that the insulin resistance phenomenon of the diabetic mice is obvious. Both the positive and leucrose polysaccharide groups reduced insulin resistance (P <0.01) in diabetic mice to a different extent than the model group.
Table 3 effect of armillaria mycelium polysaccharide on insulin resistance in type 2 diabetic mice (n-8,
)
| group of | INS(mIU/L) | HOMA-IR |
| Blank group | 5.84±1.32 | 1.24±0.49 |
| Model set | 12.57±1.70** | 12.33±2.15** |
| Positive group | 9.02±1.43## | 6.26±1.00## |
| Armillariella tabescens polysaccharide group | 8.85±1.27## | 6.77±1.05## |
Note: in comparison to the blank set, the data is,**P<0.01, very significant; in comparison to the model set,##P<0.01, very significant
(5) The contents of total Triglyceride (TG), Total Cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) in serum are important detection indexes of hyperlipidemia. The results in Table 4 show that compared with the blank group, indexes such as TG, TC, LDL-C and the like of the mice in the model group are all obviously increased (P <0.01), and HDL-C is obviously reduced (P < 0.01); compared with the model group, TG, TC and LDL-C of mice in the positive group and the armillaria polysaccharide group are obviously reduced (P <0.05 or P <0.01), and HDL-C is obviously increased (P < 0.01). The results show that: the HFD and the STZ can cause the blood fat of mice to be obviously abnormal, and the armillariella mycelium polysaccharide has obvious improvement effect on the blood fat level of the mice with type 2 diabetes.
Table 4 effect of armillaria mycelium polysaccharide on blood lipids in type 2 diabetic mice (n-8,
)
note: in comparison to the blank set, the data is,**P<0.01, very significant; in comparison to the model set,##P<0.01, very significant;#P<0.05, it should be noted that the above description is only a preferred embodiment of the present invention, and should not be construed as limiting the present invention, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (2)
1. The polysaccharide of the mycelium of the armillaria tabescens with the effect of reducing blood sugar is characterized in that:
the polysaccharide of the mycelia of the armillaria tabescens has the total sugar content of over 86 percent, and the monosaccharide composition and the molar ratio of mannose to arabinose to fucose are 1.58 to 1 to 2.74.
2. Use of the polysaccharide of the mycelium of armillaria tabescens according to claim 1, wherein: is applied to preparing the medicine for treating type 2 diabetes, and has the obvious activities of reducing blood sugar and blood fat and improving insulin resistance.
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