CN117924533A - Cordyceps sinensis polysaccharide and application thereof - Google Patents
Cordyceps sinensis polysaccharide and application thereof Download PDFInfo
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- CN117924533A CN117924533A CN202410114607.6A CN202410114607A CN117924533A CN 117924533 A CN117924533 A CN 117924533A CN 202410114607 A CN202410114607 A CN 202410114607A CN 117924533 A CN117924533 A CN 117924533A
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- Prior art keywords
- cordyceps
- polysaccharide
- intestinal
- acid
- cordyceps sinensis
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Abstract
The scheme discloses Cordyceps militaris polysaccharide and application thereof in preparation of medicines or health products for regulating intestinal flora and improving intestinal barrier function in the technical field of biological medicine. The invention applies the Cordyceps sinensis polysaccharide, which is the main active ingredient of Cordyceps sinensis, to the regulation of intestinal flora and the improvement of intestinal barrier function. Experiments prove that the Cordyceps dyotidis Diffusae polysaccharide can regulate the structure and abundance of the intestinal flora of the diabetic mice induced by streptozotocin for the first time, including up-regulating the abundance of beneficial bacteria of the genus Roche (Roseburia), corynebacterium anaerobacter (Anaerosporipes), and Flavobacterium (Flavonifractor), reducing colon and serum inflammatory factor expression, increasing content of short chain fatty acid such as propionic acid and butyric acid in colon content, and up regulating intestinal canal compact connecting protein expression, thereby improving intestinal canal barrier function and reducing blood sugar.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to Cordyceps sinensis polysaccharide and application thereof.
Background
International diabetes alliance (International diabetes federation, IDF) statistics indicate that there are 4.25 million diabetics worldwide in 2017, as many as 3.52 million potential diabetics, and it is expected that there will be nearly 7 million diabetics in 2045 years. Wherein Type 1diabetes (Type 1diabetes mellitus,T1DM) accounts for about 5% -10% of the total amount of diabetes onset. At present, T1DM has no effective therapeutic drug, and clinical treatment mainly adopts insulin substitution therapy, but can control diabetes symptoms but can not improve islet inflammation; whereas the broad-spectrum immunosuppressant for immunotherapy of T1DM has very wide side effects; beta cell-specific immunotherapy against glutamate decarboxylase and heat shock protein 60 has entered phase 2 clinic, but has little efficacy. Therefore, it has become urgent to find an immunomodulatory drug that protects islet β cells from inflammatory injury with little side effects. The traditional Chinese herbal medicine in China is rich in resources, has a wide clinical application foundation, searches for a novel hypoglycemic drug with immunoregulatory activity, and is one of important ways for developing new drugs for diabetes.
The etiology and pathogenesis of T1DM are very complex, and are currently thought to be related to various factors such as genetics, immunity, environment, etc. In recent years, various studies show that the composition and structure of intestinal flora of T1DM patients and healthy people are different, and the change of the structural composition of the intestinal flora can influence the occurrence and development of diabetes. Recently, researchers have found that han nationality T1DM patients have significantly different intestinal flora characteristics compared to healthy subjects: the ratio of Bacteroides to firmicutes increased, the bacteria abundance of faixalbacterium was inversely related to glycosylated hemoglobin (HbA 1 c), and the bacteria abundance was positively related to autoantibodies. These studies suggest that the intestinal flora may play a role in the occurrence and development of T1DM, providing a new idea for studying pathogenesis and therapeutic strategies of T1DM, but the specific mechanism of the intestinal flora participating in T1DM is not completely understood.
In addition, studies have also found that damage to the intestinal barrier is also associated with the development of diabetes. The intestinal barrier is composed of intestinal mucosal epithelium, intestinal mucus, intestinal flora, intestinal associated lymphoid tissue, mucin, digestive enzymes, and immune factors, and can be divided into physical, chemical, immune, and biological barriers. The physical barrier is the most important, the structural basis is complete intestinal mucosa epithelial cells and tight connection among the epithelial cells, and four barriers are mutually crossed and jointly act to maintain the homeostasis of the intestinal tract. For example, the mucus proteins secreted by goblet cells may constitute a chemical barrier with other intestinal fluids, enzymes, etc.; goblet cells also participate in IgA (sIgA) synthesized by iga+b cells from the lamina propria, in the intestinal mucosal immune system; physiological bacteria in intestinal microorganisms can combine with sites of a mucin layer to generate colonization resistance to form a biological barrier of intestinal tracts, and can stimulate normal immune system response of a host, so that the biological barrier has important significance for constructing the immune system of the host. Recently, various natural active products have been demonstrated to have protective effects on intestinal barrier lesions, such as pinocembrin is capable of repairing lipopolysaccharide-induced intestinal epithelial Caco-2 cell monolayer barrier lesions, resveratrol is effective in improving the immunosuppressive mouse intestinal barrier damage caused by cyclophosphamide, and hesperetin can treat dextran sodium sulfate-induced enteritis in mice, etc. by improving intestinal barrier lesions. The mechanism of action involves a variety of pathways that regulate oxidative stress, inhibit intestinal inflammation, regulate intestinal flora composition and metabolites, and increase expression of claudins.
Therefore, for regulating intestinal flora and protecting intestinal barrier function, research and development of safe and effective personalized therapeutic drugs are indispensable.
Disclosure of Invention
The invention aims to provide Cordyceps wear polysaccharide, and researches show that the Cordyceps wear polysaccharide has the effects of better regulating intestinal flora and protecting intestinal barrier.
The Cordyceps sinensis polysaccharide in the scheme is derived from Cordyceps sinensis fermentation hypha, and is obtained by extracting the Cordyceps sinensis fermentation hypha in water bath, precipitating with absolute ethanol, purifying and decolorizing. Specific:
weighing 500g of dried Cordyceps dyotidis Diffusae fermented mycelium powder, adding 95% ethanol at a feed liquid ratio of 1:6, removing fat and impurities, and repeating for 5 times. Centrifuging at 5000rpm for 10min, removing supernatant, adding deionized water into the residue at a feed-liquid ratio of 1:5, extracting in water bath at 85deg.C for 2 hr, intermittently stirring, filtering, and repeating the operation for 5 times. Mixing the filtrates, concentrating to 1/5 of the original volume on a rotary evaporator, adding 4 times of absolute ethanol, precipitating with ethanol in a refrigerator at 4deg.C for 24h, centrifuging at 5000rpm for 10min, collecting precipitate, and washing with absolute ethanol and acetone for 2 times. Dissolving the precipitate with a certain volume of deionized water, removing protein by Sevag method until no obvious protein layer is formed at the junction of the water phase and chloroform phase, concentrating under reduced pressure, drying, decolorizing according to laboratory optimized conditions, and dialyzing (3500 Da) to obtain Cordyceps Dai water extract polysaccharide, namely Cordyceps Dai polysaccharide.
Through component analysis, the Cordyceps dorsalis polysaccharide monosaccharide composition provided by the application comprises mannose, glucose and galactose, and the molar ratio is 5.07:91.12:3.81.
The Cordyceps dyotidis polysaccharide of the invention can reduce the abundance of cyanobacteria (Cyanobacteria) and soft-wall fungus (Tenericutes) at the portal level, can obviously up-regulate the abundance of rochanteria (Roseburia), anaerobic corynebacteria (Anaerosporipes) and Flavobacterium (Flavonifractor) at the portal level (p < 0.05), down-regulate the abundance of Escherichia (Escherichia), klebsiella (Klebsiella) and Salmonella (p < 0.05), inhibit colon and serum inflammatory factor expression, increase the expression of short chain fatty acid propionic acid, butyric acid, isobutyric acid, isovaleric acid and valeric acid, further inhibit NF- κB/MLCK/p-MLC signal paths by up-regulating the expression of short chain fatty acid receptor GPR41 and GPR43, promote the expression of colon tightly-connected proteins ZO-1 and Occludin and improve the damage of intestinal barrier, regulate blood sugar level and weight loss of diabetic mice, and reduce the intestinal canal of the mice by antibiotics, and reproduce the same after the mouse intestinal canal group is cleared by the same, and the Cordyceps dydropsy has the effect of providing a new therapeutic model for the human diabetes, and a human has a new therapeutic effect of transplanting drug, and has a therapeutic effect of the present drug is provided.
The Cordyceps dycepin polysaccharide has the following beneficial effects:
1. lowering blood glucose, increasing body weight, increasing islet beta cell numbers, and reducing apoptosis in diabetic mice.
2. Inhibit colon and serum inflammatory factor expression.
3. Promoting the content of short chain fatty acid.
4. Promoting expression of colonic tight junction proteins and improving damage to intestinal barrier.
5. Regulating intestinal flora structure.
6. Studies have found that 400mg/kg/d Cordyceps dyi polysaccharide can alleviate T1DM in type 1 diabetes (T1 DM), further exploring molecular mechanisms. Compared with the current clinical medicines for treating diabetes, the medicine and food homology of the experiment is low in price, and easy to popularize and accept.
The Cordyceps dycepin polysaccharide can be applied to preparing products for regulating intestinal flora or reducing blood sugar or promoting short chain fatty acid content or improving intestinal barrier injury.
Further, the product is one of a medicine, a health product and a functional food.
Further, the using dosage of the Cordyceps dorsalis polysaccharide is 50-800 mg/kg/d.
Further, the short chain fatty acid is at least one of propionic acid, butyric acid, isobutyric acid, isovaleric acid and valeric acid.
Drawings
Fig. 1 is a physicochemical property analysis of Cordyceps dycepin (CTP): (a) monosaccharide composition of CTP; (B) infrared spectroscopic analysis of CTP; (C) Component analysis of CTP (note: mannose Man, rhamnose Rha, glucuronic acid GlcA, galacturonic acid GalA, glucose Glc, galactose Gal, xylose Xyl, arabinose Ara, fucose Fuc);
FIG. 2 is a experimental procedure of CTP intervention in diabetic mice and effects on empty stomach blood glucose and body weight: (A) experimental design flow; (B) fasting blood glucose changes; (C) Weight change (note: n=8, mean±sd, **p<0.01vs CON,## p <0.01vs DM);
FIG. 3 is a graph showing the effect of CTP intervention on diabetic mouse islet beta cells observed by double staining of the mouse pancreatic tissue HE sections and insulin/TUNEL (note: scale bar = 100 μm);
FIG. 4 is the effect of CTP on intestinal flora diversity and at the portal level on intestinal flora: (A) comparison of intestinal flora alpha diversity index; (B) PCoA analysis based on an inter-sample distance matrix; (C) Venn plot of intestinal flora OTU; (D) Comparison of main phylum abundance of intestinal flora (note: n.gtoreq.6, mean+ -SD, *p<0.05vs CON,# p <0.05vs DM);
FIG. 5 is a graph showing the results of CTP intervention in the changes in intestinal flora levels in diabetic mice: (A) LDA distribution displays each group of characteristic bacteria; (B) Belongs to level main change bacteria (note: n is not less than 6, mean+ -SD, *p<0.05vs CON,# p <0.05vs DM);
FIG. 6 is a graph showing the results of CTP intervention in reducing colonic inflammation in diabetic mice (note: n.gtoreq.6, mean+ -SD, **p<0.01vs CON,## p <0.01vs DM);
FIG. 7 is a graph showing the results of CTP intervention in reducing serum inflammation in diabetic mice (note ：n≥6,Mean±SD,*p<0.05,**p<0.01vs CON,#p<0.05,##p<0.01vs DM);
FIG. 8 is a graph showing the results of CTP intervention in diabetic mice for increasing short chain fatty acids (note ：n≥6,Mean±SD,*p<0.05,**p<0.01vs CON,#p<0.05,##p<0.01vs DM);
Fig. 9 is a graph of colon tissue HE staining of mice observing CTP intervention to protect against colon barrier injury (note: scale bar = 200 μm);
FIG. 10 shows that CTP-mediated increase of colon part-associated protein and signaling pathway-associated protein expression in diabetic mice: (a) D a canonical protein band pattern; (B) Quantitative analysis of protein expression (note) ：n≥6,Mean±SD,*p<0.05,**p<0.01vs CON,#p<0.05,##p<0.01vs DM);
FIG. 11 is the effect of CTP intervention on fasting blood glucose and body weight of diabetic mice after flora removal: (A) experimental design flow; (B) fasting blood glucose changes; (C) change in body weight (note: n=8, mean±sd);
Fig. 12 is an observation of HE staining of CTP-mediated pancreas and colon in diabetic mice after flora removal: (A) Pancreatic HE staining (B) colon HE staining (note: scale bar = 100 μm and 200 μm);
FIG. 13 is the effect of fecal transplantation on fasting blood glucose and body weight of diabetic mice for CTP-dry prognosis: (A) experimental design flow; (B) fasting blood glucose changes; (C) Weight change (note: n=8, mean±sd, #p <0.01vs DM-FMT);
fig. 14 is HE staining observations of pancreas and colon of diabetic mice for fecal fungus transplantation for CTP dry prognosis: (A) Pancreatic HE staining (B) colon HE staining (note: scale bar = 100 μm and 200 μm).
Detailed Description
The present invention will be further described in detail with reference to the drawings in conjunction with the detailed description of the invention, in order to make the objects, technical solutions and some of the present invention more apparent. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present invention.
Example 1: modeling and administration method
Male Kunming mice (20-30 g) were 24 in total, purchased from Changsha day Biotechnology Co., ltd (laboratory animal license number: SCXK (Hunan) 2019-0014), and after 7d acclimatization in a clean-standard laboratory (temperature: 25.+ -. 2 ℃, humidity: 60.+ -. 5%, light: 12h/d, well ventilated), the mice were randomized into 2 groups, with 8 control groups (CON group) and the remaining mice were molded, and the molded mice were intraperitoneally injected with STZ (50 mg/kg, dissolved in 0.1M citrate buffer, pH=4.6) for 5 consecutive days. After 1 week, the fasting blood glucose level of the mice after 12 hours of fasting was measured with a blood glucose meter, and if the fasting blood glucose exceeded 11.1mmol/L, it was judged as diabetic mice. Control mice were intraperitoneally injected with citrate buffer for 5 consecutive days, and then perfused with normal saline during the post-dosing treatment.
After successful modeling, the model mice were randomly divided into a diabetes model group (DM group) and a Cordyceps dycepstrae polysaccharide group (CTP group), 8 each. Group CON (normal saline, lavage); DM group (normal saline, lavage); CTP group (400 mg/kg/d polysaccharide solution, lavage) was followed for 4 weeks. Mice were checked for blood glucose at fixed time points weekly and weighed.
After 28 days, the materials are anesthetized, pathological sections of pancreatic tissues and colon tissues are manufactured, pathological changes of the pancreatic tissues and the colon tissues of the mice are observed through HE staining, and immunofluorescence staining is carried out to examine the damage condition of islet beta cells through insulin and TUNEL apoptosis; RT-PCR (reverse transcription-polymerase chain reaction) for examining the expression condition of colon tissue inflammatory factor mRNA; detecting the content of inflammatory factors in serum by using a Shanghai Jiang Lai biological ELISA kit; detecting the content of short chain fatty acid in colon contents of the mice by a GC-MS method; western blot experiments were performed to detect expression of the colon barrier zonulin ZO-1, occludin and signaling pathway-related proteins.
Example 2: antibiotic bacterial cleaning experiment
Male Kunming mice (20-30 g) were obtained from Changsha day Biotechnology Co., ltd (laboratory animal license number: SCXK (Hunan) 2019-0014), and after 7d of conditioning in a clean standard laboratory (temperature: 25.+ -. 2 ℃, humidity: 60.+ -. 5%, light: 12h/d, well ventilated), the mice were molded by intraperitoneal injection of STZ (50 mg/kg, dissolved in 0.1M citrate buffer, pH=4.6) for 5 consecutive days, and after 1 week, fasting blood glucose levels of the mice after 12 hours of fasting were measured with a glucometer, and if fasting blood glucose exceeded 11.1mmol/L, diabetic mice were judged.
After successful modeling, the model mice were randomly divided into Diabetes model groups (DM), antibiotic groups (Antibiotic mixture, abx), antibiotic and polysaccharide combination groups (Antibiotic mixture +CTP, abx+CTP), 8 mice in each group, wherein the Abx and Abx+CTP groups were treated daily with 200. Mu.L of antibiotic mixture (10 g/L ampicillin, 10g/L neomycin, 8g/L metronidazole and 5g/L vancomycin) after modeling, the Abx+CTP groups mice were additionally given a 400mg/kg/d dose of CTP for lavage, and the DM groups mice were control groups, lavage saline. Fasting blood levels and weight changes were measured at each of experiments 2,3,4, and 5 weeks.
After 28 days, the materials are anesthetized, pathological sections of pancreatic tissues and colon tissues are prepared, and pathological changes of the pancreatic tissues and the colon tissues of the mice are observed by HE staining.
Example 3: fecal fungus transplanting experiment
Male Kunming mice (20-30 g) were obtained from Kyowa day Biotechnology Co., ltd (laboratory animal license number: SCXK (Hunan) 2019-0014), and after 7d of conditioning in a clean standard laboratory (temperature: 25.+ -. 2 ℃, humidity: 60.+ -. 5%, light: 12h/d, well ventilated), the mice were molded by intraperitoneal injection of STZ (50 mg/kg, dissolved in 0.1M citrate buffer, pH=4.6) for 5 consecutive days, and after 1 week, fasting blood glucose levels of the mice after 12 hours of fasting were measured with a glucometer, and if fasting blood glucose exceeded 11.1mmol/L, diabetic mice were judged. After successful construction of DM model, 200. Mu.L of a mixture of gastric lavage antibiotics (10 g/L ampicillin, 10g/L neomycin, 8g/L metronidazole and 5g/L vancomycin) was treated every day for 2 weeks to construct a sham-sterile diabetic mouse model.
After successful modeling, model mice were randomly divided into Diabetes group fecal transplant recipients (Diabetes model-fecal microbiota transplantation, DM-FMT) and CTP intervention group fecal transplant recipients (CTP-fecal microbiota transplantation, CTP-FMT), 8 per group. After the end of the mouse experiment in example 1, colon faeces of the mice of the DM group and CTP group were collected as donors, 200mg of the donor faeces particles were suspended in 4mL of physiological saline, and then centrifuged at 100rpm for 1 minute to remove insoluble substances, and the supernatant was collected for use. DM-FMT group and CTP-FMT group were perfused with DM group fecal supernatant and CTP group fecal supernatant, respectively, 200. Mu.L, for 28 days. Fasting blood glucose and body weight changes were measured at weeks 4, 5, 6, 7, 8, respectively.
After 28 days, the materials are anesthetized, pathological sections of pancreatic tissues and colon tissues are prepared, and pathological changes of the pancreatic tissues and the colon tissues of the mice are observed by HE staining.
Example 4: HE staining
Firstly, paraffin sections are dewaxed to water in dimethylbenzene, absolute ethyl alcohol and alcohol in sequence, hematoxylin is dyed, dehydration is carried out firstly after hematoxylin is dyed, eosin is dyed for 5min, dehydration is carried out in absolute ethyl alcohol, n-butyl alcohol, dimethylbenzene and the like in sequence finally, and image acquisition and analysis are carried out under a microscope after airing and sealing.
Example 5: insulin/TUNEL double staining
Firstly, paraffin sections are dewaxed to water in xylene, absolute ethyl alcohol and alcohol in sequence, antigen retrieval is carried out, 20 mug/mL protease K without DNase is dripped on a slide, 50 mug TUNEL detection liquid is dripped, and the slide is incubated for 60 minutes at 37 ℃ in a dark place; washing with PBS for 3 times and 3min each time; incubating for 60min with 5% BSA, and spin-drying without washing; incubation for primary antibody, wherein the temperature is 4 ℃ overnight and is protected from light; incubating the secondary antibody, carrying out water bath at 37 ℃ and keeping away from light for 1h; adding DAPI-containing anti-quencher to act for 90s; observed under a fluorescence microscope and photographed.
Example 6: colonic microorganism 16S rRNA sequencing analysis
Samples of colon contents were delegated to the Sichuan panomide Biotechnology Co.Ltd for microbiology analysis. Extracting total DNA of bacteria in intestinal contents, amplifying sequences of V4 regions of the total DNA, and performing high-throughput sequencing and library construction. And (3) performing quality control and analysis on the data obtained by sequencing, performing OTU clustering by using UPARSE software according to the similarity of 97% of the sequence, comparing the OTU with a database, and performing species annotation on the OTU. Evaluating the sequencing quantity by using a sparse curve (Rarefaction curve); calculating alpha and beta diversity, and evaluating the diversity of the microorganism composition by adopting an alpha diversity index Shannon-Wiener index (Shannon index), and evaluating the richness of the microorganism composition by adopting a super 1 index (Chao 1 index); meanwhile, comparing and analyzing the microbial community structure difference between different samples by using a principal coordinate analysis (PRINCIPAL COORDINATE ANALYSIS, PCoA); the differentiation analysis was performed using LEFSe (LDAEffect Size) analysis method.
Example 7: real-time fluorescent quantitative PCR (RT-PCR) analysis of colon inflammatory factor mRNA expression
Total RNA from the colon of the mice was extracted using Trizol lysate, and the concentration of total RNA was determined by spectrophotometry and was diluted to a uniform concentration of 250 ng/. Mu.L and reverse transcribed. The reaction conditions for RT-PCR were as follows: 95 ℃, 5s,60 ℃, 30s,72 ℃ and 30s. All gene primers were designed according to Prime3.0 software and synthesized by the Productivity company (Table 1), and the relative mRNA expression of all genes of interest was calculated according to the method of 2 -ΔΔCt.
Table 1 primer sequences of genes
Example 8: ELISA analysis of serum inflammatory factor content
Serum was removed from the-80 ℃ freezer and run according to the instructions of the ELISA detection kit (Shanghai Jiang Lai): setting a standard substance hole, a blank hole and a sample hole to be detected, adding 50 mu L of standard substances with different concentrations into the standard substance hole, adding 50 mu L of sample diluent into the blank hole, and adding 10 mu L of mouse serum sample and 40 mu L of sample diluent into the sample to be detected. Then adding 100 mu L of HRP-labeled detection antibody into each hole, sealing the reaction holes by a sealing plate film, and incubating for 60min in a water bath at 37 ℃; diluting 20mL of concentrated washing liquid with distilled water to prepare 400mL of washing liquid with working concentration; removing liquid in the reaction hole, beating dry on the water-absorbing paper, filling 300 mu L of washing liquid, standing for 1min, beating dry on the water-absorbing paper, and repeating the plate washing for 5 times; adding 50 mu L of substrate into each hole, and incubating for 15min at 37 ℃ in dark place; adding 90 mu L of stop solution into each hole, and measuring the OD value of each hole at the wavelength of 450 nm; and (5) preparing a standard curve, and calculating the concentration of the sample hole.
Example 9: GC-MS analysis of colon content short chain fatty acid content
An appropriate amount of colon contents (feces) was weighed, and 20. Mu.L of 0.5mol/L NaOH solution, 20. Mu.L of internal standard solution and 460. Mu.L of methanol were added to each sample; homogenizing in a cell homogenizer for 2min; preparing an 80% aqueous methanol solution with a methanol-water ratio of 4:1, and adding 400 mu L to the sample; vortex mixing for 30s, and freezing at-20deg.C for 30min; ultrasonic treatment of the sample in ice bath at 4deg.C for 10min; centrifuging at 12,000rpm and 4deg.C for 10min; transferring the supernatant into a new centrifuge tube, and placing the centrifuge tube in a vacuum drying oven at 37 ℃ to dry; adding 40 mu L of derivatization liquid into the sample, and placing the sample in a water bath kettle at 60 ℃ for 90min to achieve the purpose of derivatization; 60 μl of N-tert-butyldimethylsilyl-N-Methyltrifluoroacetamide (MTBSTFA) was added and incubated in a 60℃water bath for 30min; vortex mixing for 30s and centrifuging at 22,000rpm, 4deg.C for 10min; transferring 70 μl of the supernatant into new 1.5mL EP tube, diluting with 140 μl of pyridine, sealing with sealing film, and storing in-20deg.C refrigerator; samples were subjected to GC-MS analysis by Sichuan panomik Biotechnology Co.
Example 10: western blot analysis of intestinal tract tight junction proteins and related signal pathway protein expression
Extracting total protein of the colon of the mouse by using a lysis solution containing PMSF, detecting the protein concentration by a BCA method, adjusting the protein concentration to be consistent, performing polyacrylamide gel electrophoresis, transferring to filter paper to remove redundant liquid after membrane transfer, BSA blocking and primary and secondary antibody incubation, absorbing a proper amount of ECL luminescent solution to cover the PVDF membrane, placing the PVDF membrane into a Bio-RadChemiDocTM MP IMAGE SYSTEM darkroom for development, and preserving the strip. And detecting the gray value of the protein band by adopting Image J software, and calculating the relative expression quantity of the target protein.
The experimental innovation finds: the Cordyceps dyceps polysaccharide CTP 400mg/kg/d intervenes on T1DM mice for 28 days, can effectively reduce fasting blood glucose, increase weight of the mice, reduce islet beta cell apoptosis and reduce organism inflammation, and we explore the molecular mechanism thereof to determine the specific action mechanism thereof. CTP increases the content of short chain fatty acids such as propionic acid and butyric acid by intervening the composition and abundance of intestinal flora, activates the expression of its receptor GPR41 and 43, and further up regulates the expression of claudin ZO-1 and Occludin by inhibiting NF- κb/MLCK/p-MLC signaling pathway, improving damage of intestinal barrier, exerting an effect of improving T1 DM; after the intestinal flora of the mice is cleared by antibiotics, the diabetic mice model is replicated and the drug intervention is carried out, so that the drug effect is weakened; the mice treated by transplanting the fecal bacteria after the intervention of CTP have the same treatment effect, and no report is provided at present, so that a new treatment drug selection and mechanism are provided for treating T1 DM.
Example 11: cordyceps dycepin polysaccharide preparation
Weighing 500g of dried Cordyceps dyotidis Diffusae fermented mycelium powder, adding 95% ethanol at a feed liquid ratio of 1:6, removing fat and impurities, and repeating for 5 times. Centrifuging at 5000rpm for 10min, removing supernatant, adding deionized water into the residue at a feed-liquid ratio of 1:5, extracting in water bath at 85deg.C for 2 hr, intermittently stirring, filtering, and repeating the operation for 5 times. Mixing the filtrates, concentrating to 1/5 of the original volume on a rotary evaporator, adding 4 times of absolute ethanol, precipitating with ethanol in a refrigerator at 4deg.C for 24h, centrifuging at 5000rpm for 10min, collecting precipitate, and washing with absolute ethanol and acetone for 2 times. Dissolving the precipitate with a certain volume of deionized water, removing protein by Sevag method until no obvious protein layer is present at the junction of the water phase and chloroform phase, concentrating under reduced pressure, drying, decolorizing according to laboratory optimized conditions, and dialyzing (3500 Da) to obtain Cordyceps dyceps dorsalis water-extracted polysaccharide.
Example 12: physicochemical property analysis of Cordyceps Dai polysaccharide
The CTP total sugar content is measured by adopting a phenol-sulfuric acid method; the uronic acid content is determined by m-hydroxybiphenyl method; the content of the sulfuric acid group is measured by adopting a barium sulfate turbidimetry method; protein content was determined using BCA method.
The molecular weight of CTP was measured by high performance size exclusion chromatography (High perfomance size exclusion chromatography, HPSEC), agilent1260 high performance liquid chromatography (Agilent, SANTA CLARA, CA, USA) and RID detector using a BioCore SEC-500 column (5 μm,7.8 mm. Times.300 mm). The mobile phase was pure water at a flow rate of 0.8mL/min with a column oven at 30deg.C.
CTP monosaccharide composition analysis was determined using 1-phenyl-3-methyl-5-pyrazolone (1-phenyl-3-methyl-5-pyrazolone, PMP) pre-column derivatization. CTP (5.0 mg) was hydrolyzed in 100. Mu.L 4mol/L trifluoroacetic acid at 110℃for 120min. After 200. Mu.L of methanol was added to the hydrolysate and trifluoroacetic acid was removed by vacuum evaporation at 50℃100. Mu.L of distilled water was added to the hydrolysate for dissolution to prepare a derivative. 100. Mu.L of a 0.6mol/L NaOH solution was added thereto, and 100. Mu.L of the above mixture was mixed and 100. Mu.L of a 0.5mol/L PMP solution was added thereto. Derivatizing in a water bath at 70 ℃ for 100min, and cooling to room temperature. The pH was adjusted to neutral (pH 6-7) with 50. Mu.L of 0.3mol/L HCl solution. After rotary evaporation to dryness at 50℃1mL of distilled water was added to redissolve the residue, 1mL of chloroform was added for extraction, after 1min of vortex shaking extraction, the aqueous layer was extracted after centrifugation at 3000rpm for 5min, and the lower chloroform layer was discarded to remove residual PMP.
HPLC detection conditions: the detection wavelength of the high performance liquid chromatograph was 254nm using a DAD ultraviolet detector. The column was ChromCore C column (4.6X105 mm,5 μm) with mobile phase 0.02mol/L ammonium acetate solution: acetonitrile=83:17 (v/v), flow rate 0.6mL/min, sample volume 20 μl, column temperature 30 ℃.
CTP fourier transform infrared spectroscopy (FTIR) analysis: CTP (1.0 mg) was mixed with KBr powder (100 mg) and pressed into granules. The FTIR spectrum of CTP was then analyzed using a Fourier infrared spectrometer in the range of 400-4000 cm -1.
TABLE 2 physicochemical Properties of CTP
Claims (6)
1. Cordyceps sinensis polysaccharide, which is characterized in that: the Cordyceps sinensis polysaccharide is derived from Cordyceps sinensis fermentation hypha, and is obtained by extracting the Cordyceps sinensis fermentation hypha in water bath, precipitating with absolute ethanol, purifying and decolorizing.
2. The Cordyceps dyi polysaccharide according to claim 1, wherein: the monosaccharide composition of the Cordyceps dycepin polysaccharide comprises mannose, glucose and galactose, and the molar ratio is 5.07:91.12:3.81.
3. Use of Cordyceps dycepin polysaccharide according to claim 1 or 2 for preparing a product for regulating intestinal flora or lowering blood glucose or promoting short chain fatty acid content or improving intestinal barrier injury.
4. A use according to claim 3, characterized in that: the product is one of medicines, health products and functional foods.
5. The use according to claim 4, characterized in that: the dosage of the Cordyceps dyotidis Diffusae polysaccharide is 50-800 mg/kg/d.
6. The use according to claim 5, characterized in that: the short chain fatty acid is at least one of propionic acid, butyric acid, isobutyric acid, isovaleric acid and valeric acid.
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