CN106511401A - Preparation method and application of cordyceps taii mycelia polysaccharides - Google Patents

Abstract

The invention discloses a preparation method and an application of cordyceps taii mycelia polysaccharides. According to the invention, a cordyceps taii GYYA0601 strain is screened, and it is discovered that the mycelia polysaccharides, which are prepared in accordance with the strain, have a good effect when used as a medicine for treating diabetics; the mycelia polysaccharides can inhibit the activity of [alpha]-glucosidase, significantly reduce the blood glucose level of a patient with the diabetics, improve the injury degree of pancreatic [beta] cells and increase the secretion of insulin; in addition, the mycelia polysaccharides also can boost body immunity; and the mycelia polysaccharides, on preventing and treating the diabetics, have the advantages of being good in curative effect, low in side effects and the like.

Description

Preparation method and application of polysaccharide from Cordyceps dai's mycelium

technical field

The invention relates to the technical field of biomedicine, in particular to a preparation method and application of the mycelia polysaccharide of Cordyceps daideniensis.

Background technique

Diabetes is a disorder of glucose and lipid metabolism characterized by hyperglycemia. It is the third most serious disease that endangers human health after cardiovascular and cerebrovascular diseases and cancer (AssociationAD.Diagnosis and classification of diabetes mellitus.Diabetes Care, 2012 , 35, Suppl(1):S42–7; http://www.idf.org/diabetesatlas). Drug therapy is the main treatment strategy for diabetes. However, the most commonly used clinical first-line hypoglycemic drugs, such as sulfonylureas, biguanides, glycosidase inhibitors, and insulin sensitizers, have many toxic and side effects and are prone to cause secondary complications. Glycemic control with diet, sulfonylurea, metformin, orinsulin in patients with type 2diabetes mellitus: progressive requirement for multiple therapies (UKPDS 49). Journal of the American Medical Association, 1999, 281(6):2005-12). Therefore, the development of new high-efficiency, low-toxic hypoglycemic drugs has become a research hotspot in the field of biomedicine.

Polysaccharides are the main effective components in natural medicines, with wide biological activities, high efficiency and low toxicity, and are ideal sources of medicines. In recent years, fungal polysaccharides have been found to have a good hypoglycemic effect. Cordyceps is a kind of fungal resources with important application prospects in the field of biomedicine. Studies have shown that the mycelium polysaccharides of Cordyceps sinensis and Cordyceps militaris have obvious hypoglycemic and antidiabetic effects (Kiho T, Ookubo K, Usui S, et al. Structural features and hypoglycemic activity of a polysaccharide (CS-F10) from the cultured mycelium of Cordyceps sinensis. Biological&Pharmaceutical Bulletin, 1999,22(9):966-70; Li SP, Zhang GH, Zeng Q, et al. Hypoglycemic activity of polysaccharide, with antioxidation, isolated from cultured Cordyceps mycelia. Phytomedicine 2006; 13(6): 428-33; Zhang G, HuangY, Bian Y, et al. Hypoglycemic activity of the fungi Cordyceps militaris, Cordyceps sinensis, Tricholoma mongolicum, and Omphalia lapidescensin streptozotocin-induced diabetic rats. Applied Microbiology and Biotechnology 2006): 25( 6), is a good resource for preparing anti-diabetic drugs and health products. Cordyceps taii (Metarhizium anisopliae) is a new Cordyceps fungus first reported by Chinese mycologist Professor Liang Zongqi in Guizhou Province. Guizhou folks have always mistakenly believed that Cordyceps sinensis is used for nourishing and strengthening the body.

Contents of the invention

The technical problem to be solved by the present invention is to provide a preparation method of the mycelia polysaccharide of Cordyceps daimerii.

The invention also provides an application of the polysaccharide of the Cordyceps daiburi mycelium in the preparation of antidiabetic drugs.

The present invention is achieved in that the preparation method of Cordyceps dai's mycelium polysaccharide comprises the steps:

1) using the Cordyceps daiurii GYYA0601 strain to prepare the Cordyceps daiwuli mycelium;

2) after drying the Cordyceps daiburi mycelium, crushing it to pass through a 60-100 mesh molecular sieve to obtain the Cordyceps daiburi mycelium powder;

3) Add petroleum ether to degrease the Cordyceps mycelium powder at a material-to-liquid ratio of 1:3-5. The unit of the material-to-liquid ratio is g/ml; Distilled water is added, and the unit of solid-liquid ratio is g/ml. The crude polysaccharide is extracted by water extraction and alcohol precipitation; the crude polysaccharide is deproteinized, decolorized and dialyzed, concentrated and alcohol-precipitated to obtain the polysaccharide of Cordyceps daiburi mycelium.

Application of the polysaccharide of Cordyceps dai's mycelium in the preparation of antidiabetic drugs.

The results of modern pharmacological studies suggest that the crude polysaccharides of Cordyceps dai's mycelium have various activities such as immune regulation, anti-tumor, and anti-mutation (Liang Zongqi. Chinese Fungi. Vol. 32, Cordyceps. Beijing: Science Press, 2007) . The applicant collected and isolated Cordyceps taii GYYA0601 strain (Metarhizium taiiGYYA0601 strain) from a tea farm in Guiyang, and the preservation number is CGMCC No.2880. Through further research, it was found that its mycelium polysaccharides can delay aging through anti-oxidative stress and immune regulation, and the polysaccharides are a kind of galactoglucomannan (Xiao JH, Xiao DM, Chen DX, et al. Medicinal mushroom Cordyceps taii show antioxidant and immunoenhancing activities in a D-galactose-induced mouse model. Evidence-Based Complementary and Alternative Medicine Volume, 2012 (2012), Article ID 273435, 15 pages). It is worth mentioning that oxidative stress is one of the pathological mechanisms of diabetes. It is difficult for the body's antioxidant defense system to suppress the oxidative stress response caused by a large number of free radicals caused by high-glucose conditions, resulting in damage to islet cells, and at the same time reducing the sensitivity of peripheral tissues to insulin, resulting in insulin resistance. These factors promote diabetes and complications. Oxidative stress as a major culprit in kidney disease indiabetes. Diabetes 57:1446–54; Stadler K. Oxidative stress in diabetes. Advances in Experimental Medicine and Biology.2012 ; 771:272-87). Moreover, oxidative stress and insulin resistance are complementary, and insulin resistance promotes oxidative stress (Dong F, Fang CX, Yang X, et al. Cardiac overexpression of catalaserescues cardiac contractile dysfunction induced by insulin resistance: Role of oxidative stress, protein carbonyl formation and insulinsensitivity. Diabetologia. 2006;49(6):1421-33). Therefore, reducing the oxidative stress in the body may be an effective method to prevent and treat diabetes and its complications, and it is a new direction to develop new anti-diabetes and its complications drugs using antioxidant natural products. Therefore, the applicant took the mycelia polysaccharide of Cordyceps daideli with obvious anti-oxidative stress ability as the research object, and systematically studied its preparation method and its effect on α-glucosidase and animal models of diabetes for the first time. The preparation method of the Cordyceps daishi mycelium polysaccharide provided by the present invention and its application in antidiabetic drugs or auxiliary hypoglycemic health care products have not been reported so far.

The present invention prepares the Cordyceps daiburi mycelium polysaccharide through the Cordyceps daizi GYYA0601 strain, and the applicant finds that the mycelium polysaccharide has a good effect as a medicine for treating diabetes, and it can inhibit the activity of α-glucosidase, and can significantly It can reduce the blood sugar level of diabetic patients, improve the degree of damage of pancreatic islet β cells, increase insulin secretion, and enhance the body's immunity. It has the advantages of good curative effect and small side effects for the prevention and treatment of diabetes.

Description of drawings

Accompanying drawing 1 is the influence (200×) of mycelia polysaccharide of Cordyceps daibuli on pancreatic islet tissue cells of diabetic mouse model;

A-normal group NG; B-model group MCG; C-positive control group PG; D-high-dose group HG; E-middle-dose group MG; F-low-dose group LG.

detailed description

Embodiment 1 of the present invention: production of Cordyceps dai's fermented product

1. The polysaccharide is isolated from the fermentation mycelium of the Cordyceps fungus Cordyceps daimani (Metarrhizia daimani) GYYA0601 strain. The preservation number of the Cordyceps daimli strain is CGMCC NO.2880, and the authorized patent (ZL 200910301440) previously applied by the applicant has been published.

2. The formula of Cordyceps slant seed culture medium includes 25g of sucrose, 3g of soybean, 5g of yeast extract, 2g of peptone, 3g of wheat bran, 1g of KH ₂ PO ₄ , 0.5g of MgSO ₄ 7H ₂ O, 18g of agar, 1L of distilled water, initial pH 7.0; Cordyceps Daikleri liquid seed or/fermentation medium formula includes 30g sucrose, 5g peptone, 3g yeast extract, 1g KH ₂ PO ₄ , 0.5g MgSO ₄ ·7H ₂ O, 1L distilled water, initial pH 7.0.

3. The slanted seeds of Cordyceps daishii were cultured at 23-28°C for 7-15 days, and stored at 4°C for later use. Transplant the slant strain into a 500mL shaker flask filled with 120mL liquid seed medium, and then place it on a rotary shaker at 120-180r/min at 25-30°C for 4 days to prepare fermented liquid seeds. A 50L fermenter tank is filled with 33L fermentation medium, inoculated with fermented liquid seeds at a ratio of 4% (v/v) by volume coefficient, fermented at 150-250r/min, and fermented at 28°C for 60-80h. After the fermentation is terminated, the fermentation product is filtered under reduced pressure, washed with distilled water for 3 times, placed in an oven, dried at 45-60°C until constant weight, and the dry weight of the fermentation product is weighed. The dried fermented product is crushed into powder, passed through a 60-100 mesh molecular sieve, sealed and refrigerated at -20°C for later use.

Embodiment 2 of the present invention: Preparation of Cordyceps dai's mycelium polysaccharide

Add petroleum ether to degrease the Cordyceps mycelium powder at a material-to-liquid ratio of 1:3-5, and the unit of the material-to-liquid ratio is g/ml; add the degreased mycelium powder at a material-to-liquid ratio of 1:5-15 Distilled water, the unit of solid-liquid ratio is g/ml, and leaching in a boiling water bath for 1-3 hours, repeated 2-4 times, suction filtration, combined filtrate, and then concentrated under reduced pressure to 1/3-1/4 volume, Put the concentrated filtrate in a beaker, add ethanol with a mass percentage of 80-90%, let it stand overnight in a refrigerator at 4°C, and centrifuge to obtain the precipitate; then add distilled water at a solid-liquid ratio of 1:1-3 (unit: g/ml) Dissolve the precipitate, then add ethanol with a mass percentage of 80-90%, let it stand overnight in a refrigerator at 4°C, repeat this operation until the ethanol liquid has no obvious color; dry the total precipitate obtained from separation, and obtain crude polysaccharide; use water to extract Extract crude polysaccharides by alcohol precipitation; use Sevag to remove free protein, then decolorize with activated carbon or hydrogen peroxide, and then dialyze with 3500kD molecular weight convective water for 24 hours.

Example 3 of the present invention: Inhibitory activity of polysaccharides from Cordyceps daideli mycelia to α-glucosidase

1. The activity of the positive drug acarbose on α-glucosidase: construct an enzyme reaction system, and micro-screen the standard reaction system with a total of 140 μL, add 20 μL of phosphate buffer to each well, and then take 0.001, 0.01, 0.1, Add 20 μL of 1 mg/mL acarbose solution to the standard reaction system, and finally add 60 μL of 20 U/ml α-glucosidase solution and 10 μL of 10 mmol/L PNPG solution in sequence, mix well, and keep warm at 37°C for 30 minutes, and then Add 30 μL of 1mol/L Na ₂ CO ₃ to the well to terminate the reaction. At this time, it turns yellow. Measure the OD value at 405 nm with a microplate reader. At the same time, set a blank control and a background control. Make 3 replicate wells for each sample, and take the average value. Calculate the inhibitory activity of acarbose to α-glucosidase according to the following formula (see Table 1):

2. The activity of polysaccharides from Cordyceps daiburi mycelium on α-glucosidase: use the α-glucosidase inhibitor screening model constructed above to screen and study the optimal dose of polysaccharides from Cordyceps daiburi mycelium, and obtain out _IC50 . Add 20 μL of phosphate buffer to each well, then take 20 μL of polysaccharide solutions with concentrations of 5, 10, 20, 40, 80, 200, and 300 mg/ml and add them to the enzyme standard reaction system, and mix with 60 μL of 20 U/mL α-glucosidase After the solution and 10 μL 10 mmol/L PNPG solution were mixed evenly, they were incubated together at 37°C for 30 minutes, the stop solution was used to terminate the reaction, the OD value at 405 nm was measured by a microplate reader, and a blank control and a background control were set at the same time, and each sample was replicated 3 times. hole, take the average value. Similarly, the inhibitory activity of the polysaccharides of the Cordyceps daizii mycelium to α-glucosidase was calculated according to the above formula. The results are shown in Table 1, and the calculated IC ₅₀ value is 105.95 mg/mL.

Table 1 Inhibition of α-glucosidase by the polysaccharides of Cordyceps daiburi mycelium

Embodiment 4 of the present invention: anti-diabetic effect of polysaccharides from Cordyceps dai's mycelium

1. Construction and administration of diabetes models: Male Kunming mice (20-30g) were raised in a clean standard laboratory (temperature: 25±2°C, humidity: 60±5%, light: 12h/d, well ventilated) After 7 days to adapt to the environment, the mice were randomly divided into two groups, including 8 normal control groups (NC), and the rest of the mice were used to create models. The mice in the model group were fasted for 12 hours overnight (free drinking water) Afterwards, 100mg/kg one-time intraperitoneal injection of STZ (citrate buffer solution, pH=4.44), after STZ injection 4h, feed the mouse 5% glucose solution, avoid the death of mouse because of hypoglycemia, after 5d, tail vein After the blood was taken, the fasting blood glucose was tested with a blood glucose meter for 3 consecutive times. If the blood glucose value was >11.1mmol/L, the modeling was successful. The normal group of mice was injected with citrate buffer once, and the drug control group received 0.5ml of 0.9% normal saline. The successful modeling mice were randomly divided into 5 groups, which were normal group (NG, 0.9% normal saline), model control group (MCG, 0.9% normal saline), positive control group (PG, 300mg/kg/d metformin ), low-dose polysaccharide group (LG, 100mg/kg/d), medium-dose polysaccharide group (MG, 200mg/kg/d), high-dose polysaccharide group (HG, 400mg/kg/d), 12 rats in each group.

2. The influence of Cordyceps dai's mycelium polysaccharide on the blood sugar of diabetic mice: as shown in Table 2, after a single intraperitoneal injection of 100 mg/kg STZ in the mice of the modeling group for 5 days, compared with the normal group, the fasting blood sugar of the mice Significantly increased (P<0.01), indicating that the diabetes model was established successfully; compared with the model group, the blood glucose value of the CTP intragastric administration group decreased significantly during the administration period, and it was statistically significant. Therefore, the mycelia polysaccharide of Cordyceps daizi can significantly reduce the blood sugar level of diabetic mice.

The effect of table 2 Cordyceps dai's mycelium polysaccharides on the blood sugar of STZ-induced diabetic mice ( n=12)

Note: Compared with MCG, ^a P<0.05, ^b P<0.01; compared with NG: ^c P<0.01

3. The effect of polysaccharides from Cordyceps daizii on serum insulin in diabetic mice: as shown in Table 3, compared with the normal group, the serum insulin content of the model group decreased significantly ( ^a P<0.05); compared with the model group, all Serum insulin levels in the administration group were significantly increased, and there was statistical significance at ^b P<0.01 level. Therefore, the mycelia polysaccharide of Cordyceps daizi can promote the secretion of insulin in diabetic mice. In addition, the results of pathological analysis (see Figure 1) indicated that the pancreatic islet cells in the NG group were distributed in clusters in the pancreatic exocrine glands, the shape of the edge of the islets was regular, and the boundary between the islets and the exocrine glands was clear, and the number of cells in the islets was large. The nuclei are stained in round blue, and the cytoplasm is rich in light pink staining; in the MCG group, the shape of the islets is irregular, the islets are shrunk, the vacuoles in the center are degenerated, the number of islet β cells is reduced, the distribution of islet cells is disordered, and some hyperplasia is arranged Glandular tube-like structure, and compensatory hypertrophy of some cells; the pancreas of the mice in the four administration groups including PG is relatively complete, and the number of islet β cells is more than that of the model group and the distribution is even, indicating that the polysaccharides of Cordyceps mycelia can protect The function of islet cells, and the effect of the high-dose group was better than that of the middle-dose and low-dose groups.

Table 3 The effect of Cordyceps dai's mycelium polysaccharides on serum insulin in diabetic mice ( n=12)

Note: Compared with NG: ^a P<0.01; compared with MCG ^b P<0.01;

Model group MCG; C-positive control group PG; D-high-dose group HG; E-middle-dose group MG; F-low-dose group LG.4, the effect of mycelium polysaccharides of Cordyceps daideni on peripheral blood leukocytes of diabetic mice : As can be seen from Table 4, the number of white blood cells in the diabetic model group mice is lower than that of the normal group, but there is no significant difference; compared with the model group, the number of white blood cells in the Cordyceps mycelia polysaccharide administration group was significantly increased (P< 0.01); the increase of white blood cell count and the mycelia polysaccharide of Cordyceps daiburi showed a certain dose-dependent relationship; when the intragastric dose was 400mg/kg/d, the effect was better than that of the positive drug metformin, indicating that the polysaccharides of Cordyceps daiburi mycelia Can enhance the immune protection of the body.

Table 4 The effect of Cordyceps dai's mycelium polysaccharides on the number of peripheral blood leukocytes in diabetic mice ( n=12)

Note: ^a P<0.01 compared with MCG;

5. The effect of polysaccharides from Cordyceps daizii on the immune organs and viscera index of mice: Compared with the normal group, the thymus index of the mice in the model group was significantly reduced (P<0.05); compared with the model group, high-dose administration group can significantly improve the thymus index of mice (P<0.05), therefore, the polysaccharide of Cordyceps daiziensis mycelium can improve the cellular immune function of the body.

Table 8 Effects of polysaccharides from Cordyceps daideli mycelium on immune organ viscera index in diabetic mice

( n=8-12)

Note: Compared with NG, ^a P<0.05; compared with MCG, ^b P<0.05

Embodiment 5 of the present invention: the polysaccharide of Cordyceps dai's mycelia alone or in combination with medicines (including pharmaceutical carriers or excipients or antidiabetic drugs) is added with appropriate amount of auxiliary materials according to the prescription amount, mixed and pressed into disc shape or heterogeneous sheet shape Tablets (including plain tablets, sugar-coated tablets, film-coated tablets, etc.); or capsule preparations (including hard capsules, soft capsules, enteric-coated capsules, etc.) ); or add an appropriate amount of auxiliary materials according to the prescription amount to formulate a granule preparation of a certain size; or add an appropriate amount of binder or other auxiliary materials according to the prescription amount to make a pill-shaped or pill-like pill; or add an appropriate amount of concentrated sucrose aqueous solution according to the prescription amount. or a spherical or quasi-spherical drop pill made by shrinking and condensing after heating, melting and mixing with a suitable substrate according to the prescription amount, then dropping into the immiscible condensate; or adding tea according to the prescription amount Or other oral liquid preparations made by mixing other auxiliary materials.

The dosage of the present invention depends on various factors, including the nature and severity of the disease to be prevented or treated, sex, age, body weight, individual response, administration route, administration frequency and the like of the animal or patient. Accordingly, the dosage administered can vary widely. According to the actual drug quantity contained in the preparation produced from the compound of the present invention, it can be adjusted appropriately to achieve its therapeutic purpose.

Claims (2)

1. a preparation method of Cordyceps dai's mycelium polysaccharide, is characterized in that: comprise the steps: 1) Using the Cordyceps dai's GYYA0601 strain to prepare Cordyceps dai's mycelium; 2) After drying the mycelium of Cordyceps daiburi, crushing it to pass through a 60-100 mesh molecular sieve to obtain a powder of the mycelium of Cordyceps daiburi; 3) Add petroleum ether to degrease the Cordyceps mycelium powder at a material-to-liquid ratio of 1:3-5. Distilled water is added, and the unit of solid-liquid ratio is g/ml. The crude polysaccharide is extracted by water extraction and alcohol precipitation; the crude polysaccharide is deproteinized, decolorized and dialyzed, concentrated and alcohol-precipitated to obtain the polysaccharide of Cordyceps daiburi mycelium. 2. an application of the polysaccharide of Cordyceps daimli mycelium as claimed in claim 2 in the preparation of antidiabetic drugs.