WO2023036203A1 - Cs-4 fermented mycelium heteropolysaccharide, preparation method therefor and use thereof - Google Patents

Cs-4 fermented mycelium heteropolysaccharide, preparation method therefor and use thereof Download PDF

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WO2023036203A1
WO2023036203A1 PCT/CN2022/117637 CN2022117637W WO2023036203A1 WO 2023036203 A1 WO2023036203 A1 WO 2023036203A1 CN 2022117637 W CN2022117637 W CN 2022117637W WO 2023036203 A1 WO2023036203 A1 WO 2023036203A1
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heteropolysaccharide
fermented
treatment
ethanol
group
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PCT/CN2022/117637
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French (fr)
Chinese (zh)
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王红敏
高雯
王伟
金嘉丽
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江西济民可信集团有限公司
江西济民可信药业有限公司
江西金水宝制药有限公司
上海济煜医药科技有限公司
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Priority to CN202280043095.8A priority Critical patent/CN117500842A/en
Publication of WO2023036203A1 publication Critical patent/WO2023036203A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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

Definitions

  • the invention belongs to the field of traditional Chinese medicine pharmacy, and in particular relates to a Cs-4 fermented mycelia heteropolysaccharide prepared by using Paecilomyces mothii (Cs-4) mycelium as a raw material, a preparation method and application thereof.
  • Cs-4 fermented mycelia heteropolysaccharide prepared by using Paecilomyces mothii (Cs-4) mycelium as a raw material, a preparation method and application thereof.
  • Cordyceps sinensis is a complex of the subunits and larvae corpses of Cordyceps sinensis (Berk.) Sacc., a fungus of the family Ergotaceae, and is a complex of precious Chinese medicinal materials (hereinafter referred to as Cordyceps). Because the natural Cordyceps growth environment is special, and the price is higher, it cannot meet the demand of the market. Therefore, people use modern bio-fermentation technology to cultivate Cordyceps mycelium to replace natural Cordyceps.
  • Cs-4 Fermented Cordyceps fungus powder
  • the drug names are Jinshuibao Capsules and Jinshuibao Tablets.
  • Jinshuibao As a representative species of artificial Cordyceps, Jinshuibao has the functions of nourishing the lungs and kidneys, secreting essence and nourishing qi, and has been widely used clinically in the treatment of renal system diseases, respiratory system diseases, metabolic diseases, cardiovascular and cerebrovascular system diseases, endocrine system diseases and adjuvant therapy for tumors.
  • the material basis of its medicinal effect is not clear.
  • Cordyceps polysaccharide as one of the main active ingredients in Cordyceps sinensis, has attracted the attention of many researchers at home and abroad.
  • the current research focuses on the activity screening of crude polysaccharides from Cordyceps sinensis.
  • the research on polysaccharide structure is still in its infancy, and most of them only cover the composition of monosaccharides. and molecular weight information, or simply grading the polysaccharide without complete purification, so that the chemical structure of the polysaccharide cannot be accurately and deeply excavated, which severely limits the quality control and subsequent clinical transformation of the active polysaccharide.
  • the present invention proposes a Cs-4 fermented mycelia heteropolysaccharide.
  • the structural formula is as follows:
  • Manp mannose pyranose
  • Galp is galactopyranose
  • Glcp is glucopyranose
  • n is selected from 6-104.
  • the inventor extracted and identified the polysaccharide for the first time.
  • the polysaccharide has better effects of preventing or treating chronic renal failure, inhibiting immune rejection, preventing or treating hyperlipidemia, and preventing or treating acute kidney injury. Compared with fermented Cordyceps powder In other words, better results can be obtained with lower doses.
  • the above polysaccharide may further include one of the following additional technical features:
  • Manp is mannose pyranose
  • Galp is galactopyranose
  • Glcp is glucopyranose
  • n is selected from 6-68. According to an embodiment of the present invention, n is selected from 15-21.
  • n is selected from 15-104.
  • n is selected from 21-104.
  • n is selected from 6-15.
  • n is selected from 6-21.
  • n is selected from 15-68.
  • n is selected from 21-68.
  • n is selected from any integer between 6-104.
  • n is selected from any integer between 6-68.
  • n is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 100, 101, 102, 103, 104, 105, 106, 107.
  • the relative molecular weight of the polysaccharide is selected from 10-152kDa. It should be noted that the relative molecular weight used in the present invention refers to the weight average molecular weight, and the weight average molecular weight refers to the average weight of the weight, that is, the weighted average of molecular weight to molecular weight.
  • the relative molecular weight of the polysaccharide is selected from 10-100kDa.
  • the relative molecular weight of the polysaccharide is selected from 10-23.25kDa.
  • the relative molecular weight of the polysaccharide is selected from 10-30.12kDa.
  • the relative molecular weight of the polysaccharide is selected from 10-151.15kDa.
  • the relative molecular weight of the polysaccharide is selected from 23.25-30.12kDa.
  • the relative molecular weight of the polysaccharide is selected from 23.25-100kDa.
  • the relative molecular weight of the polysaccharide is selected from 30.12-100kDa.
  • the relative molecular weight of the polysaccharide is selected from 23.25-151.15kDa.
  • the relative molecular weight of the polysaccharide is selected from 30.12-151.15kDa.
  • the relative molecular weight of the polysaccharide is selected from 23.15kDa or 30.12kDa or 151.15kDa.
  • the polysaccharide composition includes: glucose, galactose and mannose, and the molar ratio of the glucose, galactose and mannose is 1:(1.0-2.0):(1.5-2.5).
  • the polysaccharide composition includes: glucose, galactose and mannose, and the molar ratio of the glucose, galactose and mannose is 1:(1.5-1.8):(2.0-2.5).
  • the polysaccharide composition includes: glucose, galactose and mannose, and the molar ratio of the glucose, galactose and mannose is 1:(1.71-1.74):(2.09-2.44).
  • the present invention proposes a method for extracting heteropolysaccharides from the aforementioned Cs-4 fermented mycelia.
  • the method includes: 1) degreasing the Cs-4 bacterial powder with ethanol, discarding the ethanol extract to obtain a degreasing residue, 2) extracting the residue in water to obtain a water Extraction, 3) subjecting the water extract to alcohol precipitation treatment, the solvent of the alcohol precipitation treatment is ethanol, 4) purifying the precipitate after the alcohol precipitation treatment in step 3), to obtain the fermented mycelia heteropolymer polysaccharides.
  • the method according to the embodiment of the present invention is simple in operation, high in extraction efficiency, and the purity of the extracted polysaccharide is high, which can reach more than 94%.
  • the method may further include at least one of the following additional technical features:
  • the degreasing treatment is performed at a temperature of 100°C.
  • the degreasing treatment is performed three times, and each time is 1 hour.
  • the amount of ethanol in the degreasing process is 10 times that of Cs-4.
  • the degreasing ethanol is 85%-100% ethanol.
  • the extraction treatment is carried out at a temperature of 75°C-78°C.
  • the extraction process is carried out three times, and each time is 1 hour.
  • the amount of water in the extraction process is 10 times that of the residue.
  • the present invention proposes a method for extracting heteropolysaccharides from the aforementioned Cs-4 fermented mycelia.
  • the method includes: 1) heating and extracting Cs-4 in 10 times the amount of 85% ethanol for 3 times, each time for 1 hour, discarding the ethanol extract to obtain a residue; 2) extracting the The residue was heated and extracted with 10 times the amount of water for 3 times, each time for 1 hour, and the water extracts were combined; 3) The water extract was subjected to 80% ethanol precipitation to obtain the precipitate Cs-4-P; 4) The precipitate was Purify Cs-4-P to obtain fermented mycelia heteropolysaccharide Cs-4-P1, and continue to purify to obtain 3 homogeneous polysaccharides, named Cs-4-P1-1, Cs-4-P1-2, Cs -4-P1-3.
  • the method may further include at least one of the following additional technical features:
  • the purification treatment includes protein removal treatment, decolorization treatment, and column purification treatment. It should be noted that protein removal treatment, decolorization treatment, and column purification treatment can be routine treatment methods, and an appropriate operation method can be selected according to specific needs.
  • the present invention also proposes a pharmaceutical composition.
  • the pharmaceutical composition comprises the aforementioned Cs-4 fermented mycelia heteropolysaccharide or the Cs-4 fermented mycelia heteropolysaccharide Cs-4- P1, and the three homogeneous polysaccharides (Cs-4-P1-1, Cs-4-P1-2, Cs-4-P1-3) obtained by further purification.
  • the present invention also proposes the aforementioned Cs-4 fermented mycelia heteropolysaccharide or the Cs-4 fermented mycelia heteropolysaccharide obtained according to the method described above or the aforementioned
  • the pharmaceutical composition of the invention is used in the preparation of medicines, and the medicines are used for preventing or treating chronic renal failure, suppressing immune rejection, preventing or treating hyperlipidemia, and preventing or treating acute kidney injury.
  • the inventors found that the polysaccharide has better effects of preventing or treating chronic renal failure, inhibiting immune rejection, preventing or treating hyperlipidemia, and preventing or treating acute kidney injury. Excellent effect.
  • the acute kidney injury is selected from cisplatin-induced acute kidney injury and/or LPS-induced acute kidney injury.
  • the pharmaceutical composition according to the present invention comprises the aforementioned Cs-4 fermented mycelia heteropolysaccharide or the Cs-4 fermented mycelia heteropolysaccharide obtained according to the aforementioned method. The inventors found that the polysaccharide has a better effect of preventing or treating acute kidney injury, and the effect is equivalent to that of the positive drugs amifostine and dexamethasone.
  • the present invention also proposes a pharmaceutical composition for preventing or treating hyperlipidemia.
  • the pharmaceutical composition comprises 1.0-10.0 ⁇ g/mL of the aforementioned Cs-4 fermented mycelia heteropolysaccharide or the Cs-4 fermented mycelia heteropolysaccharide obtained according to the aforementioned method.
  • Polysaccharides The inventors found that the polysaccharide has a better effect of preventing or treating hyperlipidemia, and compared with Jinshuibao, a lower dose can obtain a better effect.
  • the above pharmaceutical composition may further include at least one of the following additional technical features:
  • the concentration of the polysaccharide is 3.0 ⁇ g/mL.
  • the present invention at least proposes at least one of the following technical effects:
  • the present invention extracts and identifies polysaccharides with repeating structural units for the first time (Cs-4-P1-1, Cs-4-P1-2, Cs-4-P1-3 for short);
  • heteropolysaccharide of the present invention is preventing or treating chronic renal failure, it can be used in low doses (16mg/kg/day) than fermented Cordyceps fungus powder Cs-4 (1600mg/kg/day), Huangkui capsule (1000mg/kg/day) kg/day) is better, and compared with the clinical classic old drug dexamethasone, the effect is equivalent or better, and the present invention aims to provide a new alternative drug for the prevention or treatment of chronic renal failure;
  • the effect of the heteropolysaccharide of the present invention on inhibiting immune rejection is better than that of fermented Cordyceps powder Cs-4, and compared with the classic clinical drug cyclosporine, the effect is equivalent or better, and the safety is better.
  • the present invention aims to provide a new alternative drug for suppressing immune rejection;
  • the heteropolysaccharides of the present invention can lower triglycerides and total cholesterol at low doses (1.0-10.0 ⁇ g/mL) when preventing or treating hyperlipidemia. Compared with atorvastatin calcium, the effect is equivalent or better, and the present invention aims to provide a new alternative drug for preventing or treating hyperlipidemia.
  • Fig. 1 is a HPGPC spectrum according to an embodiment of the present invention.
  • Fig. 2 is an infrared spectrum of a sample after methylation according to an embodiment of the present invention.
  • Fig. 3 is a total ion current spectrum of a Cs-4-P1-2 derivative according to an embodiment of the present invention.
  • Fig. 4 is a 1 H NMR spectrum of Cs-4-P1-2 according to an embodiment of the present invention.
  • Fig. 5 is a 13 C NMR spectrum of Cs-4-P1-2 according to an embodiment of the present invention.
  • Fig. 6 is a 1 H- 1 H COZY spectrum of Cs-4-P1-2 according to an embodiment of the present invention.
  • Fig. 7 is a 1 H- 1 H TOCSY spectrum of Cs-4-P1-2 according to an embodiment of the present invention.
  • Fig. 8 is a 1 H- 13 C HSQC chart according to an embodiment of the present invention.
  • Fig. 9 is a 1 H- 13 C HMBC spectrum according to an embodiment of the present invention.
  • Fig. 10 is a 1 H- 1 H NOESY spectrum according to an embodiment of the present invention.
  • Fig. 11 is a diagram of a pathological slice according to an embodiment of the present invention.
  • Dexamethasone acetate tablets (batch number: 015200406, Shanghai Pharmaceutical Xinyi Pharmaceutical Co., Ltd.), ambrette (batch number: 20110210, Jiangsu Suzhong Pharmaceutical Group Co., Ltd.), fermented Cordyceps powder (batch number: 190800810-1, Jiangxi Sinopharm Co., Ltd.), Xinsaisiping cyclosporine soft capsules (batch number: 200825, Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd.).
  • Glcp stands for glucopyranose
  • Galp stands for galactopyranose
  • Manp stands for mannose
  • h stands for hour
  • min stands for minute
  • W stands for week
  • rpm stands for the number of revolutions per minute of the device
  • DEAE diethylaminoethyl Cellulose
  • Seph stands for dextran gel
  • HPGPC high performance gel chromatography
  • Mw stands for weight average molecular weight
  • Mp stands for peak molecular weight
  • t R stands for retention time
  • BUN stands for blood urea nitrogen
  • CREA stands for blood creatinine
  • UA stands for Uric acid
  • CsA stands for cyclosporine
  • MTC stands for minimum toxic concentration
  • LPS stands for lipopolysaccharide.
  • Deproteinization and decolorization 2% crude polysaccharide aqueous solution extracted with water, equal amount of Sevage reagent, shake vigorously for 10 minutes each time, 4000rpm, 10min, repeat decolorization 3-4 times until no obvious white layer appears, and combine the upper solution.
  • Decolorization Add 2% activated carbon powder to the white polysaccharide solution, keep warm at 50°C for 30 minutes, filter twice with double-layer filter paper, pass through a 0.45 micron membrane, concentrate until there is no organic reagent smell, and prepare for the next step of purification.
  • Standard curve drawing Weigh 5.417mg of glucose, weigh it accurately, place it in a 50mL volumetric flask, add water to dissolve and make it to volume, and obtain a glucose standard solution of 0.1mg/mL. Pipette standard solutions 0, 0.2, 0.4, 0.6, 0.8, 1.0mL into 20mL stoppered test tubes, make up to 1.0mL with distilled water, add 1.0mL of 5% phenol solution and 5.0mL of concentrated sulfuric acid, shake well, and place at room temperature After 10 minutes, use a vortex shaker to fully mix the reaction solution, then place the test tube in a water bath at 30°C for 20 minutes, and measure the absorbance at 490 nm. With the glucose concentration as the abscissa and the absorbance as the ordinate, perform linear regression to obtain the standard curve equation.
  • Test of the test product Weigh 50mg of Cs-4-P1, weigh it accurately, put it in a 10mL measuring bottle, dissolve it by ultrasonic, add water to make it up to the mark, shake well, and you get it. Take 0.2 mL each of the above prepared solutions in a 20 mL stoppered test tube, and add water to make up to 1.0 mL. Make up to 1.0mL with distilled water, add 1.0mL of 5% phenol solution and 5.0mL of concentrated sulfuric acid, shake well, leave at room temperature for 10min, mix the reaction solution thoroughly with a vortex oscillator, and then place the test tube in a 30°C water bath After reacting for 20min, measure the absorbance at 490nm.
  • High-performance gel chromatography can be used to determine the relative molecular weight and distribution of macromolecular substances, and is the preferred method for the relative molecular weight of polysaccharides.
  • the principle is that according to the characteristics that polysaccharides with different relative molecular masses have a certain relationship with the elution retention time (t R ) on the gel column, a standard curve is first prepared with a standard sample of known relative molecular mass, and then the t of the sample is R obtains the relative molecular mass from the curve.
  • the peak molecular weight Mp represents the molecular weight corresponding to the highest peak retention time
  • the peak start and peak end molecular weights represent the molecular weight corresponding to the peak start time and peak end time, respectively.
  • the weight-average molecular weight Mw is obtained based on the statistical average of the molecular weight, which means that molecules of the same weight are distributed on both sides.
  • Preparation of 0.71% sodium sulfate Weigh 14.20g of sodium sulfate into a 2L beaker, add 2000ml of purified water to dissolve and dilute, pass through a 0.22um microporous filter membrane, and ultrasonically degas for about 10min.
  • test solution Take 50mg to 10ml measuring bottles of each sample, weigh them accurately, add an appropriate amount of mobile phase obtained from the above filtration, and after ultrasonication for 10min, set the volume to the mark and shake well to obtain the test solution.
  • the test solution was stored at 4°C.
  • Preparation of reference solution Weigh 10 mg each of dextran D0, D1, D2, D3, D4, D5, D6, D7, D8, D2000, and dextran 410,000 into each 1.5ml disposable centrifuge tube, accurately weigh and pipette 1ml Put 1ml of 0.71% sodium sulfate solution in each centrifuge tube and shake well.
  • the column temperature is 35°C
  • the flow rate is 0.8mL/min
  • the injection volume is 20ul.
  • sugar residue A is ⁇ 4,6)- ⁇ -D-Manp-(1 ⁇ .
  • HMBC spectrum (Fig. 9) was further used to clarify the position and sequence of connections between sugar residues. From the HMBC signal, it can be seen that H-1 of residue A and C-4 of residue B and C-6 of residue E have obvious correlation signals, and H-1 of residue B has correlation with C-6 of residue A Signals, H-1 of residue C and C-4 of residue B have related signals, H-1 of residue D and C-4 of residue A have related signals, H-1 of residue E and residues There is a correlation signal at C-4 of A, and the long-range correlation data of each sugar residue is shown in Table 5.
  • the connection mode between sugar residues can be judged, and it can be inferred that the 1st position of residue A is connected with the 4th position of residue B and the 6th position of E, and the 1st position of residue B is connected with The 6th position of residue A is connected, the 1st position of residue C is connected with the 4th position of residue B, and the 1st position of residue D and residue E are respectively connected with the 4th position of residue A.
  • the polysaccharide is ⁇ -(1 ⁇ 4)-D-mannose, ⁇ -(1 ⁇ 6)-D-mannose and ⁇ -(1 ⁇ 4)-D-galactose are alternately linked as the main chain, and in mannose
  • the 6th and 4th positions of the sugar are respectively linked to ⁇ -D-galactose and ⁇ -D-glucose branched heteropolysaccharides.
  • This repeat segment is 9 sugars, according to which according to the weight average molecular weight (Mw) of Cs-4-P1-1, Cs-4-P1-2, Cs-4-P1-3 in Table 1, its n value is calculated to be about : 104, 21, 16.
  • Test method 70 SD rats, half male and half male, were randomly divided into groups according to body weight, 10 rats in each group: normal control group, model group, Cs-4-P1-2 group, fermented Cordyceps powder group, Huangkui capsule group, Dexamethasone group. Before the test, the rats were put into metabolic cages, fasted for 24 hours (water intake as usual), recorded the urine output for 24 hours, and detected urine protein and urine creatinine. After the rats were weighed, 1 mL of blood was taken from the orbit, placed in a 1.5 mL centrifuge tube, centrifuged at 3000 rpm for 10 min, and the blood was taken to detect urea nitrogen (BUN), creatinine (Scr) and uric acid, as the basic value.
  • BUN urea nitrogen
  • Scr creatinine
  • uric acid as the basic value.
  • the rats in the other groups were given 200 mg/kg of adenine (dissolved in normal saline) by intragastric administration, once a day, for 4 consecutive weeks, and in the last week, only administration was performed without modeling.
  • adenine dissolved in normal saline
  • the rats were dissected to take out the kidneys, the renal sebum was separated on ice, weighed, and the organ index was calculated. Then fixed in 4% neutral formaldehyde solution, embedded in paraffin, stained with hematoxylin-eosin (HE), examined with light microscope, and read the slides.
  • HE hematoxylin-eosin
  • the number of rats 0d is 10/group, half male and half male, compared with the model group * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001
  • Table 7 shows that: compared with the model group on the 7th, 11d, and 14d of the test, the body weight of the rats in the normal control group increased significantly; The body weight of the rats increased significantly (P ⁇ 0.001); the body weight of the dexamethasone group was lower than that of the model group from the 11th day, and the body weight was significantly reduced on the 18th and 28th days, suggesting that it was related to the side effects of hormones; Cs-4-P1-2 group, Cs-4-P group, and fermented Cordyceps powder group had no significant effect on body weight.
  • the body weight of the model group is lower than that of the normal control group, and the kidney weight and kidney organ coefficient are higher than that of the normal control group, all of which have extremely significant differences (P ⁇ 0.001); compared with the model group, the rats in the dexamethasone group The kidney weight decreased significantly (P ⁇ 0.05), and the kidney weight of rats in the Cs-4-P1-2, Cs-4-P and fermented Cordyceps powder groups showed a downward trend; compared with the model group, the normal control group, Cs-4- The kidney coefficient of P1-2 and fermented Cordyceps powder group decreased significantly (P ⁇ 0.05), and Cs-4-P group showed a downward trend.
  • BUN Blood urea nitrogen
  • CREA Serum creatinine
  • Adenine-induced chronic renal failure can cause severe renal tissue lesions, simulating the end-stage state of renal failure.
  • the renal tissue structure was normal, the glomerular vascular loops were thin and clear, the numbers of endothelial cells and mesangial cells were normal, and the surrounding renal tubules were normal.
  • glomerular fibrosis, renal tubule atrophy or dilation, foreign body filling in the renal tubule and glomerulus a large amount of fibrous tissue proliferation and inflammatory cell infiltration in the interstitium were found in the renal tissue of rats in the model group.
  • Cs-4-P1-2 Compared with the model group, the residual glomerular units in the Cs-4-P1-2 administration group increased significantly, and the pathological score decreased significantly (P ⁇ 0.01), and the pathological score in the ambrette and fermented Cordyceps powder group decreased significantly (P ⁇ 0.05) . It shows that Cs-4-P1-2 and fermented Cordyceps powder can improve and restore the renal pathological changes caused by long-term adenine gavage, and Cs-4-P1-2 is the best.
  • Cs-4-P1 The effect of Cs-4-P1 on the kidney was judged from the remaining glomeruli and nephrons. It can be seen from Table 12 that, compared with the model group, the pathological score of the normal control group was 0, the pathological score of the rats in the ambrette group and the fermented Cordyceps powder group decreased, and the Cs-4-P group had a downward trend, and the Cs-4-P1 The pathological score of the rats in the -2 group was significantly reduced.
  • Cs-4-P1-2 of the present invention on adenine-induced chronic renal failure is significantly better than that of dexamethasone, yellow sunflower, fermented Cordyceps fungus powder and its crude polysaccharide (Cs-4-P).
  • Transplant rejection is expressed as the percentage of necrotic skin in total skin, and skin damage caused by sutures, suture materials, skin displacement, and other trauma is not included in the statistics.
  • grade 5 When the grade of skin graft rejection reaches grade 5, it can be defined as the death of the grafted skin.
  • the clinical dose of cyclosporine used as an immunosuppressant for humans is 15mg/kg/day, the dose for mice is 150mg/kg, the dosage of fermented Cordyceps powder for humans is 3g ⁇ 6g/day for humans, and the dose of fermented Cordyceps powder is set to 3000mg/kg/day for mice, and set the dose of Cs-4-P1-2 as 30mg/kg/day for mice according to the yield.
  • the skin was scored from the fourth day, from
  • Example 6 Effect research on hyperlipidemia zebrafish
  • the 7dpf melanin allele mutant translucent Albino strain zebrafish was randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). The samples were dissolved in water respectively (see Table 17 for concentration), and a normal control group was set at the same time, and the volume of each well was 3 mL. After treatment at 28°C for 48 hours, the MTC of the samples against normal zebrafish was measured.
  • the zebrafish of the 5dpf melanin allele mutant translucent Albino strain were randomly selected, and given egg yolk powder in water to establish a hyperlipidemia model of the zebrafish. After 16 hours of feeding, the zebrafish were randomly assigned to 6-well plates, and 30 zebrafish were treated in each well. The samples were dissolved in water (see Table 17-18 for concentration), and the positive control was atorvastatin calcium at a concentration of 0.240 ⁇ g/mL. A normal control group and a model control group were set at the same time, and the volume of each well was 3 mL.
  • the MTC (minimum toxic concentration) of fermented Cordyceps powder and Cs-4-P1-2 to normal zebrafish were 300 and 10.0 ⁇ g/mL, respectively. See Table 17 for details.
  • mice were fasted for 12 h before operation and had free access to water. Except for the control group, the other groups received a single intraperitoneal injection of cisplatin solution at a dose of 10 mg/kg, and the control group was injected with an equal volume of normal saline. After 15 minutes, amifostine, Cs-4-P1 and Cs-4-P1-2 were administered in groups. Blood was collected on the 1st, 3rd and 5th days after the administration, and blood urea nitrogen (BUN) and creatinine (Scr) in serum were measured.
  • BUN blood urea nitrogen
  • Scr creatinine
  • Test results The results are shown in Table 22.
  • the serum urea nitrogen and creatinine in the control group tended to be stable on the 1st, 3rd and 5th day, and the serum urea nitrogen and creatinine in the model group began to increase on the 3rd day and remained at a high level on the 5th day , Compared with the control group, the serum urea nitrogen and creatinine of the model group on the 3rd and 5th days showed statistical differences (p ⁇ 0.05, p ⁇ 0.01).
  • Cs-4-P1-2 can treat and improve acute kidney injury in mice.
  • mice were fasted for 12 h before operation and had free access to water. Except for the control group, the other groups received a single intraperitoneal injection of 20 mg/kg of LPS solution, and the control group was injected with an equal volume of normal saline. After 15 minutes, dexamethasone and Cs-4-P1-2 were administered in groups respectively. After 36 hours, the animals were sacrificed, and blood was collected to measure serum creatinine and urea nitrogen.
  • mice in the model group were 17.69 ⁇ 1.13mmol/L and 45.35 ⁇ 5.85 ⁇ mol/L, respectively, showing a statistical difference (p ⁇ 0.01).
  • the positive drug dexamethasone was administered at a dose of 10mg/kg, and the serum urea nitrogen and creatinine levels of the mice in the positive drug group were 12.29 ⁇ 4.42mmol/L and 24.03 ⁇ 13.81 ⁇ mol/L, respectively, which showed significant differences compared with the model group difference (p ⁇ 0.05); the serum urea nitrogen levels of the mice in the Cs-4-P1-2 low, medium and high dose groups decreased to 14.41 ⁇ 1.73, 13.20 ⁇ 1.72 and 12.72 ⁇ 0.55mmol/L respectively; the serum creatinine levels were respectively decreased to 35.15 ⁇ 26.22, 26.34 ⁇ 2.75, 24.33 ⁇ 9.65 ⁇ mol/L, and compared with the model group, the serum urea nitrogen and creatinine of the mice in the Cs-4-P1-2 middle and high dose groups showed statistical differences (p ⁇ 0.05, p ⁇ 0.01, p ⁇ 0.001).
  • Cs-4-P1-2 can treat and improve acute kidney injury in mice.
  • Fig. 8 is a 1 H- 13 C HSQC chart according to an embodiment of the present invention.
  • Fig. 11 is a diagram of a pathological slice according to an embodiment of the present invention.

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Abstract

A Cs-4 fermented mycelium heteropolysaccharide, a preparation method therefor and a use thereof. The polysaccharide structure is shown below, where N is selected from 6-104. The polysaccharide can be used for preventing or treating chronic renal failure, inhibiting immune rejection, preventing or treating hyperlipidemia, and preventing or treating acute kidney injury.

Description

Cs-4发酵菌丝体杂聚多糖及其制备方法与用途Cs-4 fermented mycelia heteropolysaccharide and its preparation method and application
本申请要求申请日为2021/9/7的中国专利申请2021110438722的优先权。本申请引用上述中国专利申请的全文。This application claims the priority of the Chinese patent application 2021110438722 with the filing date of 2021/9/7. This application cites the full text of the above-mentioned Chinese patent application.
技术领域technical field
本发明属中药制药领域，具体涉及一种以蝙蝠蛾拟青霉(Cs-4)菌丝体为原料制备的Cs-4发酵菌丝体杂聚多糖及其制备方法与用途。The invention belongs to the field of traditional Chinese medicine pharmacy, and in particular relates to a Cs-4 fermented mycelia heteropolysaccharide prepared by using Paecilomyces mothii (Cs-4) mycelium as a raw material, a preparation method and application thereof.
背景技术Background technique
冬虫夏草为麦角菌科真菌冬虫夏草菌Cordyceps sinensis(Berk.)Sacc.寄生在蝙蝠蛾科昆虫幼虫上的子座及幼虫尸体的复合体，是名贵中药材(以下简称虫草)。由于天然虫草生长环境特殊，且价格较高，不能满足市场的需求。因此，人们利用现代生物发酵技术培育出虫草菌丝体来代替天然虫草。中国中医科学院中药研究所于1982年从云南迪庆藏族自治州采集的冬虫夏草样品上分离获得十几个菌株，并对其中4个菌株进行了培养和形态学的研究，经鉴定命名为蝙蝠蛾拟青霉(Paecilonyces hepialid chen&Dai)。中国医学科学院药物研究所1982年从青海化隆县采集的新鲜冬虫夏草中分离得到Cs-4菌株，经鉴定也是蝙蝠蛾拟青霉。发酵虫草菌粉(Cs-4)，以下简称Cs-4，即是通过分离的蝙蝠蛾拟青霉Cs-4菌株发酵而得的菌丝体的干燥粉末，并作为原料制成药品成功上市，药品名称为金水宝胶囊和金水宝片。Cordyceps sinensis is a complex of the subunits and larvae corpses of Cordyceps sinensis (Berk.) Sacc., a fungus of the family Ergotaceae, and is a complex of precious Chinese medicinal materials (hereinafter referred to as Cordyceps). Because the natural Cordyceps growth environment is special, and the price is higher, it cannot meet the demand of the market. Therefore, people use modern bio-fermentation technology to cultivate Cordyceps mycelium to replace natural Cordyceps. In 1982, the Institute of Chinese Materia Medica of the Chinese Academy of Chinese Medical Sciences isolated more than a dozen strains from Cordyceps sinensis samples collected in Diqing Tibetan Autonomous Prefecture, Yunnan, and carried out culture and morphological studies on 4 of the strains. Mold (Paecilonyces hepialid chen & Dai). The Institute of Materia Medica of the Chinese Academy of Medical Sciences isolated the Cs-4 strain from fresh Cordyceps sinensis collected in Hualong County, Qinghai in 1982, and was identified as Paecilomyces bat moth. Fermented Cordyceps fungus powder (Cs-4), hereinafter referred to as Cs-4, is the dry powder of mycelium obtained by fermenting the isolated Paecilomyces hematilis Cs-4 strain, and it is used as a raw material to make medicines and has been successfully marketed. The drug names are Jinshuibao Capsules and Jinshuibao Tablets.
金水宝作为人工虫草代表品种，具有补益肺肾、秘精益气功效，临床上已广泛应用于治疗肾脏系统疾病、呼吸系统疾病、代谢疾病、心脑血管系统疾病、内分泌系统疾病和肿瘤辅助治疗，但其药效物质基础并不明确。As a representative species of artificial Cordyceps, Jinshuibao has the functions of nourishing the lungs and kidneys, secreting essence and nourishing qi, and has been widely used clinically in the treatment of renal system diseases, respiratory system diseases, metabolic diseases, cardiovascular and cerebrovascular system diseases, endocrine system diseases and adjuvant therapy for tumors. However, the material basis of its medicinal effect is not clear.
虫草多糖作为虫草中主要的活性成分之一，备受国内外众多研究学者的关注，但是目前的研究偏重于虫草粗多糖的活性筛选，多糖结构研究尚处于初级阶段，多是只涵盖单糖组成和分子量信息，或对多糖只通过简单分级，未进行完全纯化从而无法准确对虫草多糖的化学结构进行深入的挖掘，严重限制了活性虫草多糖的质量控制和后续临床转化。Cordyceps polysaccharide, as one of the main active ingredients in Cordyceps sinensis, has attracted the attention of many researchers at home and abroad. However, the current research focuses on the activity screening of crude polysaccharides from Cordyceps sinensis. The research on polysaccharide structure is still in its infancy, and most of them only cover the composition of monosaccharides. and molecular weight information, or simply grading the polysaccharide without complete purification, so that the chemical structure of the polysaccharide cannot be accurately and deeply excavated, which severely limits the quality control and subsequent clinical transformation of the active polysaccharide.
发明内容Contents of the invention
现有技术中关于Cs-4发酵菌丝体多糖的研究很少，现有技术中提取的Cs-4发酵菌丝体多糖纯度低，对多糖精细结构研究较少，基于此，本发明对Cs-4发酵菌丝体多糖进行了深入的研究，提取出的多糖纯度高，且深入研究了多糖的具体单糖组成、相对分子质量、糖苷键连接方式等，并将其与发酵虫草菌粉在动物模型上开展了对比试验，可以看出本发明的多糖相对于发酵虫草菌粉，以更低的剂量取得了更佳的效果。There are few studies on Cs-4 fermented mycelia polysaccharides in the prior art, the purity of the Cs-4 fermented mycelia polysaccharides extracted in the prior art is low, and there are few studies on the fine structure of polysaccharides. -4 Fermented mycelia polysaccharides have been studied in depth, the extracted polysaccharides have high purity, and the specific monosaccharide composition, relative molecular weight, and glycosidic bond connection mode of the polysaccharides have been studied in depth, and they are combined with fermented Cordyceps powder in Comparative experiments have been carried out on animal models, and it can be seen that the polysaccharide of the present invention achieves a better effect at a lower dose than the fermented Cordyceps powder.
在本发明的第一方面，本发明提出了一种Cs-4发酵菌丝体杂聚多糖。根据本发明的实施例，结构式如下所示：In the first aspect of the present invention, the present invention proposes a Cs-4 fermented mycelia heteropolysaccharide. According to an embodiment of the present invention, the structural formula is as follows:
Figure PCTCN2022117637-appb-000001
Figure PCTCN2022117637-appb-000001
其中，Manp为吡喃型甘露糖、Galp为吡喃型半乳糖、Glcp为吡喃型葡萄糖，n选自6-104。发明人首次提取并鉴定出该多糖，该多糖具有较优的预防或治疗慢性肾衰、抑制免疫排斥反应、预防或治疗高血脂、预防或治疗急性肾损伤的效果，相对于发酵虫草菌粉而言，以更低的剂量获得更优的效果。Wherein, Manp is mannose pyranose, Galp is galactopyranose, Glcp is glucopyranose, and n is selected from 6-104. The inventor extracted and identified the polysaccharide for the first time. The polysaccharide has better effects of preventing or treating chronic renal failure, inhibiting immune rejection, preventing or treating hyperlipidemia, and preventing or treating acute kidney injury. Compared with fermented Cordyceps powder In other words, better results can be obtained with lower doses.
根据本发明的实施例，上述多糖还可以进一步包括如下附加技术特征之一：According to an embodiment of the present invention, the above polysaccharide may further include one of the following additional technical features:
根据本发明的实施例，Manp为吡喃型甘露糖、Galp为吡喃型半乳糖、Glcp为吡喃型葡萄糖，n选自6-68。根据本发明的实施例，n选自15-21。According to an embodiment of the present invention, Manp is mannose pyranose, Galp is galactopyranose, Glcp is glucopyranose, and n is selected from 6-68. According to an embodiment of the present invention, n is selected from 15-21.
根据本发明的实施例，n选自15-104。According to an embodiment of the present invention, n is selected from 15-104.
根据本发明的实施例，n选自21-104。According to an embodiment of the present invention, n is selected from 21-104.
根据本发明的实施例，n选自6-15。According to an embodiment of the present invention, n is selected from 6-15.
根据本发明的实施例，n选自6-21。According to an embodiment of the present invention, n is selected from 6-21.
根据本发明的实施例，n选自15-68。According to an embodiment of the present invention, n is selected from 15-68.
根据本发明的实施例，n选自21-68。According to an embodiment of the present invention, n is selected from 21-68.
根据本发明的实施例，n选自6-104之间的任意整数。According to an embodiment of the present invention, n is selected from any integer between 6-104.
根据本发明的实施例，n选自6-68之间的任意整数。According to an embodiment of the present invention, n is selected from any integer between 6-68.
根据本发明的实施例，n选自15、16、17、18、19、20、21、22、23、24、25、100、101、102、103、104、105、106、107。According to an embodiment of the present invention, n is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 100, 101, 102, 103, 104, 105, 106, 107.
根据本发明的实施例，所述多糖的相对分子量选自10-152kDa。需要说明的是，本发明所采用的相对分子量是指重均分子量，所述重均分子量是指重量的平均值，即分子量对分子的重量加权平均。According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 10-152kDa. It should be noted that the relative molecular weight used in the present invention refers to the weight average molecular weight, and the weight average molecular weight refers to the average weight of the weight, that is, the weighted average of molecular weight to molecular weight.
根据本发明的实施例，所述多糖的相对分子量选自10-100kDa。According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 10-100kDa.
根据本发明的实施例，所述多糖的相对分子量选自10-23.25kDa。According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 10-23.25kDa.
根据本发明的实施例，所述多糖的相对分子量选自10-30.12kDa。According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 10-30.12kDa.
根据本发明的实施例，所述多糖的相对分子量选自10-151.15kDa。According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 10-151.15kDa.
根据本发明的实施例，所述多糖的相对分子量选自23.25-30.12kDa。According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 23.25-30.12kDa.
根据本发明的实施例，所述多糖的相对分子量选自23.25-100kDa。According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 23.25-100kDa.
根据本发明的实施例，所述多糖的相对分子量选自30.12-100kDa。According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 30.12-100kDa.
根据本发明的实施例，所述多糖的相对分子量选自23.25-151.15kDa。According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 23.25-151.15kDa.
根据本发明的实施例，所述多糖的相对分子量选自30.12-151.15kDa。According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 30.12-151.15kDa.
根据本发明的实施例，所述多糖的相对分子量选自23.15kDa或30.12kDa或151.15kDa。According to an embodiment of the present invention, the relative molecular weight of the polysaccharide is selected from 23.15kDa or 30.12kDa or 151.15kDa.
根据本发明的实施例，所述多糖组成包括：葡萄糖、半乳糖和甘露糖，所述葡萄糖、半乳糖和甘露糖的摩尔比为1：(1.0-2.0):(1.5-2.5)。According to an embodiment of the present invention, the polysaccharide composition includes: glucose, galactose and mannose, and the molar ratio of the glucose, galactose and mannose is 1:(1.0-2.0):(1.5-2.5).
根据本发明的实施例，所述多糖组成包括：葡萄糖、半乳糖和甘露糖，所述葡萄糖、半乳糖和甘露糖的摩尔比为1：(1.5-1.8):(2.0-2.5)。According to an embodiment of the present invention, the polysaccharide composition includes: glucose, galactose and mannose, and the molar ratio of the glucose, galactose and mannose is 1:(1.5-1.8):(2.0-2.5).
根据本发明的实施例，所述多糖组成包括：葡萄糖、半乳糖和甘露糖，所述葡萄糖、半乳糖和甘露糖的摩尔比为1：(1.71-1.74)：(2.09-2.44)。According to an embodiment of the present invention, the polysaccharide composition includes: glucose, galactose and mannose, and the molar ratio of the glucose, galactose and mannose is 1:(1.71-1.74):(2.09-2.44).
在本发明的第二方面，本发明提出了一种提取前面所述Cs-4发酵菌丝体杂聚多糖的方法。根据本发明的实施例，所述方法包括：1)将Cs-4菌粉用乙醇进行脱脂处理，舍去乙醇提液，获得脱脂残渣，2)将所述残渣在水中进行提取处理，获得水提液，3)将所述水提液进行醇沉处理，所述醇沉处理的溶剂为乙醇，4)将步骤3)中醇沉处理后的沉淀进行纯化处理，获得发酵菌丝体杂聚多糖。根据本发明的实施例的方法操作简单，提取效率高，提取的多糖的纯度高，能达到94％以上。In the second aspect of the present invention, the present invention proposes a method for extracting heteropolysaccharides from the aforementioned Cs-4 fermented mycelia. According to an embodiment of the present invention, the method includes: 1) degreasing the Cs-4 bacterial powder with ethanol, discarding the ethanol extract to obtain a degreasing residue, 2) extracting the residue in water to obtain a water Extraction, 3) subjecting the water extract to alcohol precipitation treatment, the solvent of the alcohol precipitation treatment is ethanol, 4) purifying the precipitate after the alcohol precipitation treatment in step 3), to obtain the fermented mycelia heteropolymer polysaccharides. The method according to the embodiment of the present invention is simple in operation, high in extraction efficiency, and the purity of the extracted polysaccharide is high, which can reach more than 94%.
根据本发明的实施例，所述方法还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:
根据本发明的实施例，所述脱脂处理是在温度为100℃的条件下进行的。According to an embodiment of the present invention, the degreasing treatment is performed at a temperature of 100°C.
根据本发明的实施例，所述脱脂处理进行三次，每次时间为1小时。According to an embodiment of the present invention, the degreasing treatment is performed three times, and each time is 1 hour.
根据本发明的实施例，所述脱脂取处理中乙醇是Cs-4的10倍量。According to an embodiment of the present invention, the amount of ethanol in the degreasing process is 10 times that of Cs-4.
根据本发明的实施例，所述脱脂取处理的乙醇为85％-100％乙醇。According to an embodiment of the present invention, the degreasing ethanol is 85%-100% ethanol.
根据本发明的实施例，所述提取处理是在温度为75℃-78℃的条件下进行的。According to an embodiment of the present invention, the extraction treatment is carried out at a temperature of 75°C-78°C.
根据本发明的实施例，所述提取处理进行三次，每次时间为1小时。According to an embodiment of the present invention, the extraction process is carried out three times, and each time is 1 hour.
根据本发明的实施例，所述提取处理中水是残渣的10倍量。According to an embodiment of the present invention, the amount of water in the extraction process is 10 times that of the residue.
根据本发明的实施例，所述醇沉处理的乙醇为80％乙醇。According to an embodiment of the present invention, the alcohol precipitation treatment is 80% ethanol.
在本发明的再一方面，本发明提出了一种提取前面所述Cs-4发酵菌丝体杂聚多糖的方法。根据本发明的实施例，所述方法包括：1)将Cs-4在10倍量的85％乙醇加热提取3次，每次1小时，舍去乙醇提液，获得残渣；2)将所述残渣在10倍量水加热提取3次，每次1小时，合并水提液；3)将所述水提液经80％乙醇醇沉，获得沉淀Cs-4-P；4)将所述沉淀Cs-4-P进行纯化处理，获得发酵菌丝体杂聚多糖Cs-4-P1，继续纯化得3个均一多糖，命名为Cs-4-P1-1，Cs-4-P1-2，Cs-4-P1-3。In yet another aspect of the present invention, the present invention proposes a method for extracting heteropolysaccharides from the aforementioned Cs-4 fermented mycelia. According to an embodiment of the present invention, the method includes: 1) heating and extracting Cs-4 in 10 times the amount of 85% ethanol for 3 times, each time for 1 hour, discarding the ethanol extract to obtain a residue; 2) extracting the The residue was heated and extracted with 10 times the amount of water for 3 times, each time for 1 hour, and the water extracts were combined; 3) The water extract was subjected to 80% ethanol precipitation to obtain the precipitate Cs-4-P; 4) The precipitate was Purify Cs-4-P to obtain fermented mycelia heteropolysaccharide Cs-4-P1, and continue to purify to obtain 3 homogeneous polysaccharides, named Cs-4-P1-1, Cs-4-P1-2, Cs -4-P1-3.
根据本发明的实施例，所述方法还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:
根据本发明的实施例，所述纯化处理包括去蛋白处理、脱色处理、柱纯化处理。需要说明的是，可以去蛋白处理、脱色处理、柱纯化处理为常规的处理方法，可以根据具体需要，选择合适的操作方法。According to an embodiment of the present invention, the purification treatment includes protein removal treatment, decolorization treatment, and column purification treatment. It should be noted that protein removal treatment, decolorization treatment, and column purification treatment can be routine treatment methods, and an appropriate operation method can be selected according to specific needs.
在本发明的再一方面，本发明还提出了一种药物组合物。根据本发明的实施例，所述药物组合物包含前面所述的Cs-4发酵菌丝体杂聚多糖或根据前面所述的方法获得的Cs-4发酵菌丝体杂聚多糖Cs-4-P1，及其继续纯化所得的3个均一多糖(Cs-4-P1-1，Cs-4-P1-2，Cs-4-P1-3)。In another aspect of the present invention, the present invention also proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the aforementioned Cs-4 fermented mycelia heteropolysaccharide or the Cs-4 fermented mycelia heteropolysaccharide Cs-4- P1, and the three homogeneous polysaccharides (Cs-4-P1-1, Cs-4-P1-2, Cs-4-P1-3) obtained by further purification.
在本发明的再一方面，本发明还提出了前面所述的Cs-4发酵菌丝体杂聚多糖或根据前面所述的方法获得的Cs-4发酵菌丝体杂聚多糖或前面所述的药物组合物在制备药物中的用途，所述药物用于预防或治疗慢性肾衰、抑制免疫排斥反应、预防或治疗高血脂、预防或治疗急性肾损伤。发明人发现该多糖具有较优的预防或治疗慢性肾衰、抑制免疫排斥反应、预防或治疗高血脂的效果、预防或治疗急性肾损伤，相对于金水宝而言，以更低的剂量获得更优的效果。In another aspect of the present invention, the present invention also proposes the aforementioned Cs-4 fermented mycelia heteropolysaccharide or the Cs-4 fermented mycelia heteropolysaccharide obtained according to the method described above or the aforementioned The pharmaceutical composition of the invention is used in the preparation of medicines, and the medicines are used for preventing or treating chronic renal failure, suppressing immune rejection, preventing or treating hyperlipidemia, and preventing or treating acute kidney injury. The inventors found that the polysaccharide has better effects of preventing or treating chronic renal failure, inhibiting immune rejection, preventing or treating hyperlipidemia, and preventing or treating acute kidney injury. Excellent effect.
在本发明的一个方案中，所述急性肾损伤选自顺铂所致急性肾损伤和/或LPS所致急性肾损伤。根据本发明药物组合物包含前面所述的Cs-4发酵菌丝体杂聚多糖或根据前面所述的方法获得的Cs-4发酵菌丝体杂聚多糖。发明人发现，该多糖具有较优的预防或治疗急性肾损伤的效果，效果与阳性药氨磷汀、地塞米松相当。In one aspect of the present invention, the acute kidney injury is selected from cisplatin-induced acute kidney injury and/or LPS-induced acute kidney injury. The pharmaceutical composition according to the present invention comprises the aforementioned Cs-4 fermented mycelia heteropolysaccharide or the Cs-4 fermented mycelia heteropolysaccharide obtained according to the aforementioned method. The inventors found that the polysaccharide has a better effect of preventing or treating acute kidney injury, and the effect is equivalent to that of the positive drugs amifostine and dexamethasone.
在本发明的再一方面，本发明还提出了一种预防或治疗高血脂的药物组合物。根据本发明的实施例，所述药物组合物包含1.0-10.0μg/mL前面所述的Cs-4发酵菌丝体杂聚多糖或根据前面所述的方法获得的Cs-4发酵菌丝体杂聚多糖。发明人发现，该多糖具有较优的预防或治疗高血脂的效果，相对于金水宝而言，以更低的剂量获得更优的效果。In another aspect of the present invention, the present invention also proposes a pharmaceutical composition for preventing or treating hyperlipidemia. According to an embodiment of the present invention, the pharmaceutical composition comprises 1.0-10.0 μg/mL of the aforementioned Cs-4 fermented mycelia heteropolysaccharide or the Cs-4 fermented mycelia heteropolysaccharide obtained according to the aforementioned method. Polysaccharides. The inventors found that the polysaccharide has a better effect of preventing or treating hyperlipidemia, and compared with Jinshuibao, a lower dose can obtain a better effect.
根据本发明的实施例，上述药物组合物还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above pharmaceutical composition may further include at least one of the following additional technical features:
根据本发明的实施例，所述多糖的浓度为3.0μg/mL。发明人发现，在多糖的浓度为3.0μg/mL，其的预防或治疗高血脂的效果更佳，且显著高于金水宝的功效和经典老药阿托伐他汀钙。According to an embodiment of the present invention, the concentration of the polysaccharide is 3.0 μg/mL. The inventors found that at a polysaccharide concentration of 3.0 μg/mL, its effect on preventing or treating hyperlipidemia is better, and is significantly higher than that of Jinshuibao and the classic old drug atorvastatin calcium.
在本发明的另一方面，本发明至少提出了如下技术效果至少之一：In another aspect of the present invention, the present invention at least proposes at least one of the following technical effects:
1)本发明首次提取并鉴定出具有重复结构单元的多糖(简称Cs-4-P1-1、Cs-4-P1-2、Cs-4-P1-3)；1) The present invention extracts and identifies polysaccharides with repeating structural units for the first time (Cs-4-P1-1, Cs-4-P1-2, Cs-4-P1-3 for short);
2)本发明的提取方法操作简单；2) the extraction method of the present invention is simple to operate;
3)本发明的杂聚多糖的纯度高，能达到94％以上；3) The purity of the heteropolysaccharide of the present invention is high, which can reach more than 94%;
4)本发明的杂聚多糖在预防或治疗慢性肾衰时，能在低剂量(16mg/kg/天)比发酵虫草菌粉Cs-4(1600mg/kg/天)、黄葵胶囊(1000mg/kg/天)的效果更优，且与临床经典老药地塞米松相比，效果相当或更优，本发明旨在提供一种新的可供选择的预防或治疗慢性肾衰的药物；4) When the heteropolysaccharide of the present invention is preventing or treating chronic renal failure, it can be used in low doses (16mg/kg/day) than fermented Cordyceps fungus powder Cs-4 (1600mg/kg/day), Huangkui capsule (1000mg/kg/day) kg/day) is better, and compared with the clinical classic old drug dexamethasone, the effect is equivalent or better, and the present invention aims to provide a new alternative drug for the prevention or treatment of chronic renal failure;
5)本发明的杂聚多糖在抑制免疫排斥反应时，效果比发酵虫草菌粉Cs-4更优，且和临床经典老药环孢素相比，效果相当或更优，安全性更佳，本发明旨在提供一种新的可供选择的抑制免疫排斥反应的药物；5) The effect of the heteropolysaccharide of the present invention on inhibiting immune rejection is better than that of fermented Cordyceps powder Cs-4, and compared with the classic clinical drug cyclosporine, the effect is equivalent or better, and the safety is better. The present invention aims to provide a new alternative drug for suppressing immune rejection;
6)本发明的杂聚多糖在预防或治疗高血脂时，能在低剂量(1.0-10.0μg/mL)降甘油三酯、降总胆固醇功效效果比金水宝更优，且和经典老药阿托伐他汀钙相比，效果相当或更优，本发明旨在提供一种新的可供选择的预防或治疗高血脂的药物。6) The heteropolysaccharides of the present invention can lower triglycerides and total cholesterol at low doses (1.0-10.0 μg/mL) when preventing or treating hyperlipidemia. Compared with atorvastatin calcium, the effect is equivalent or better, and the present invention aims to provide a new alternative drug for preventing or treating hyperlipidemia.
需要说明的是，在本发明的上下文中，当使用或者无论是否使用“大约”或“约”等字眼时，表示在给定的值或范围的10％以内，适当地在5％以内，特别是在1％以内。或者，对于本领域普通技术人员而言，术语“大约”或“约”表示在平均值的可接受的标准误差范围内。每当公开一个具有N值的数字时，任何具有N+/-1％，N+/-2％，N+/-3％，N+/-5％，N+/-7％，N+/-8％或N+/-10％值以内的数字会被明确地公开，其中“+/-”是指加或减。It should be noted that, in the context of the present invention, when words such as "about" or "approximately" are used or whether they are used or not, it means within 10% of a given value or range, suitably within 5%, especially is within 1%. Alternatively, the term "about" or "approximately" means within an acceptable standard error range of the mean, as understood by those of ordinary skill in the art. Whenever a number with a value of N is disclosed, any number with N+/-1%, N+/-2%, N+/-3%, N+/-5%, N+/-7%, N+/-8%, or N+ Figures within /-10% of the value are explicitly disclosed, where "+/-" means plus or minus.
附图说明Description of drawings
图1是根据本发明实施例的HPGPC图谱。Fig. 1 is a HPGPC spectrum according to an embodiment of the present invention.
图2是根据本发明实施例的甲基化后样品红外图谱。Fig. 2 is an infrared spectrum of a sample after methylation according to an embodiment of the present invention.
图3是根据本发明实施例的Cs-4-P1-2衍生物的总离子流图谱。Fig. 3 is a total ion current spectrum of a Cs-4-P1-2 derivative according to an embodiment of the present invention.
图4是根据本发明实施例的Cs-4-P1-2的 1H NMR图谱。 Fig. 4 is a 1 H NMR spectrum of Cs-4-P1-2 according to an embodiment of the present invention.
图5是根据本发明实施例的Cs-4-P1-2的 13C NMR图谱。 Fig. 5 is a 13 C NMR spectrum of Cs-4-P1-2 according to an embodiment of the present invention.
图6是根据本发明实施例的Cs-4-P1-2的 1H- 1H COSY图谱。 Fig. 6 is a 1 H- 1 H COZY spectrum of Cs-4-P1-2 according to an embodiment of the present invention.
图7是根据本发明实施例的Cs-4-P1-2的 1H- 1H TOCSY图谱。 Fig. 7 is a 1 H- 1 H TOCSY spectrum of Cs-4-P1-2 according to an embodiment of the present invention.
图8是根据本发明实施例的 1H- 13C HSQC图谱。 Fig. 8 is a 1 H- 13 C HSQC chart according to an embodiment of the present invention.
图9是根据本发明实施例的 1H- 13C HMBC图谱。 Fig. 9 is a 1 H- 13 C HMBC spectrum according to an embodiment of the present invention.
图10是根据本发明实施例的 1H- 1H NOESY图谱。 Fig. 10 is a 1 H- 1 H NOESY spectrum according to an embodiment of the present invention.
图11是根据本发明实施例的病理切片图。Fig. 11 is a diagram of a pathological slice according to an embodiment of the present invention.
具体实施方式Detailed ways
下面通过实施例对本申请进行详细描述，但并不意味着存在对本申请而言任何不利的限制。本文已经详细地描述了本申请，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本申请精神和范围的情况下针对本申请具体实施方式进行各种变化和改进将是显而易见的。The following is a detailed description of the present application through examples, but it does not mean that there is any unfavorable limitation on the present application. The present application has been described in detail herein, and its specific embodiments are also disclosed. For those skilled in the art, various changes and improvements can be made to the specific embodiments of the application without departing from the spirit and scope of the application. will be obvious.
本发明所使用的原料如无特殊说明，均来自市售。The raw materials used in the present invention are commercially available unless otherwise specified.
醋酸地塞米松片(批号：015200406，上海上药信谊药厂有限公司)，黄葵(批号：20110210，江苏苏中药业集团股份有限公司)，发酵虫草菌粉(批号：190800810-1，江西国药有限公司)， 新赛斯平环孢素软胶囊(批号：200825，杭州中美华东制药有限公司)。Dexamethasone acetate tablets (batch number: 015200406, Shanghai Pharmaceutical Xinyi Pharmaceutical Co., Ltd.), ambrette (batch number: 20110210, Jiangsu Suzhong Pharmaceutical Group Co., Ltd.), fermented Cordyceps powder (batch number: 190800810-1, Jiangxi Sinopharm Co., Ltd.), Xinsaisiping cyclosporine soft capsules (batch number: 200825, Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd.).
本发明所采用的缩写如下所示：The abbreviations used in the present invention are as follows:
Glcp代表吡喃型葡萄糖，Galp代表吡喃型半乳糖，Manp代表吡喃型甘露糖，h代表小时，min代表分钟，W代表周，rpm代表设备每分钟旋转次数，DEAE代表二乙氨乙基纤维素，Seph代表葡聚糖凝胶，HPGPC代表高效凝胶色谱法，Mw代表重均分子量，Mp代表峰值分子量，t R代表保留时间，BUN代表血检测尿素氮，CREA代表血肌酐，UA代表尿酸，CsA代表环孢素,MTC代表最小中毒浓度，LPS代表脂多糖。 Glcp stands for glucopyranose, Galp stands for galactopyranose, Manp stands for mannose, h stands for hour, min stands for minute, W stands for week, rpm stands for the number of revolutions per minute of the device, DEAE stands for diethylaminoethyl Cellulose, Seph stands for dextran gel, HPGPC stands for high performance gel chromatography, Mw stands for weight average molecular weight, Mp stands for peak molecular weight, t R stands for retention time, BUN stands for blood urea nitrogen, CREA stands for blood creatinine, UA stands for Uric acid, CsA stands for cyclosporine, MTC stands for minimum toxic concentration, and LPS stands for lipopolysaccharide.
实施例1 Cs-4-P、Cs-4-P1、Cs-4-P1-1、Cs-4-P1-2、Cs-4-P1-3的制备方法The preparation method of embodiment 1 Cs-4-P, Cs-4-P1, Cs-4-P1-1, Cs-4-P1-2, Cs-4-P1-3
1、脱脂：发酵虫草菌粉Cs-4经10倍量85％乙醇加热至沸腾(78℃)，保持微沸提取3次，每次1小时，舍去醇提液.1. Degreasing: fermented Cordyceps fungus powder Cs-4 was heated to boiling (78°C) with 10 times the amount of 85% ethanol, kept slightly boiling and extracted 3 times, each time for 1 hour, and discarded the alcohol extract.
2、水提醇沉：剩余残渣经10倍量水加热至沸腾(100℃)，保持微沸提取3次，每次1小时。合并水提液，70℃减压浓缩成清膏(密度1.05～1.07)。清膏加95％乙醇至含醇量为85％，搅拌30分钟，静置24h，上清液抽滤/浓缩，收集沉淀，65℃减压干燥、粉碎，即得粗多糖Cs-4-P(得率21％)。2. Water extraction and alcohol precipitation: the remaining residue is heated to boiling (100°C) with 10 times the amount of water, and kept at slight boiling for 3 extractions, 1 hour each time. The combined water extracts were concentrated under reduced pressure at 70°C to form a clear paste (density 1.05-1.07). Add 95% ethanol to the clear paste until the alcohol content is 85%, stir for 30 minutes, let stand for 24 hours, filter/concentrate the supernatant, collect the precipitate, dry under reduced pressure at 65°C, and pulverize to obtain the crude polysaccharide Cs-4-P (Yield 21%).
3、脱蛋白脱色：2％水提粗多糖水溶液，等量Sevage试剂，每次剧烈震荡10分钟，4000rpm，10min，反复脱色3～4次，至无明显白色层出现，合并上层溶液。3. Deproteinization and decolorization: 2% crude polysaccharide aqueous solution extracted with water, equal amount of Sevage reagent, shake vigorously for 10 minutes each time, 4000rpm, 10min, repeat decolorization 3-4 times until no obvious white layer appears, and combine the upper solution.
4、脱色：脱单白多糖溶液中，加入2％活性炭粉，50℃保温30min，双层滤纸抽滤两次，过0.45微米膜，浓缩至无有机试剂味，备下一步纯化。4. Decolorization: Add 2% activated carbon powder to the white polysaccharide solution, keep warm at 50°C for 30 minutes, filter twice with double-layer filter paper, pass through a 0.45 micron membrane, concentrate until there is no organic reagent smell, and prepare for the next step of purification.
5、DEAE-52柱层析：纯水洗脱，得中性多糖Cs-4-P1；5. DEAE-52 column chromatography: eluted with pure water to obtain neutral polysaccharide Cs-4-P1;
6、Seph G-100柱层析分离纯化：纯水洗脱，获得三个均一多糖，命名为Cs-4-P1-1、Cs-4-P1-2、Cs-4-P1-3，其中Cs-4-P1-1得率低。6. Seph G-100 column chromatography separation and purification: elution with pure water, three homogeneous polysaccharides were obtained, named Cs-4-P1-1, Cs-4-P1-2, and Cs-4-P1-3, of which The yield of Cs-4-P1-1 was low.
实施例2 Cs-4-P1-1、Cs-4-P1-2、Cs-4-P1-3的含量、分子量和单糖组成Example 2 Cs-4-P1-1, Cs-4-P1-2, Cs-4-P1-3 content, molecular weight and monosaccharide composition
多糖含量测定：Determination of polysaccharide content:
参考《出口植物源食品中粗多糖的测定苯酚-硫酸法》SN/T 4260-2015中多糖测定方法。本方法利用多糖在浓酸的作用下水解成单糖，经脱水缩合形成糖醛衍生物，再与苯酚结合显色，通过测定显色后的吸光度值大小来计算总糖含量的方法。采用该方法测定多糖含量需选择一种单糖作为对照品绘制标准曲线。Refer to the determination method of polysaccharides in "Determination of Crude Polysaccharides in Foods of Plant Origin for Export" SN/T 4260-2015 by Phenol-sulfuric acid method. This method utilizes polysaccharides to be hydrolyzed into monosaccharides under the action of concentrated acid, dehydrated and condensed to form furfural derivatives, then combined with phenol to develop color, and the total sugar content is calculated by measuring the absorbance value after color development. Using this method to determine the content of polysaccharides requires selecting a monosaccharide as a reference substance to draw a standard curve.
标准曲线绘制：称取葡萄糖5.417mg，精密称定，置于50mL容量瓶中，加水溶解并定容，得0.1mg/mL的葡萄糖标准溶液。分别吸取标准溶液0、0.2、0.4、0.6、0.8、1.0mL于20mL具塞试管，用蒸馏水补至1.0mL，加入现配的5％苯酚溶液1.0mL及浓硫酸5.0mL，摇匀，室温放置10min后用涡旋振荡器使反应液充分混合，然后将试管置于30℃水浴中反应20min，于490nm测吸光度。以葡萄糖浓度为横坐标，吸光度值为纵坐标，进行线性回归得到标准曲线方程。Standard curve drawing: Weigh 5.417mg of glucose, weigh it accurately, place it in a 50mL volumetric flask, add water to dissolve and make it to volume, and obtain a glucose standard solution of 0.1mg/mL. Pipette standard solutions 0, 0.2, 0.4, 0.6, 0.8, 1.0mL into 20mL stoppered test tubes, make up to 1.0mL with distilled water, add 1.0mL of 5% phenol solution and 5.0mL of concentrated sulfuric acid, shake well, and place at room temperature After 10 minutes, use a vortex shaker to fully mix the reaction solution, then place the test tube in a water bath at 30°C for 20 minutes, and measure the absorbance at 490 nm. With the glucose concentration as the abscissa and the absorbance as the ordinate, perform linear regression to obtain the standard curve equation.
供试品检测：称取Cs-4-P1 50mg，精密称定，置于10mL量瓶中，超声溶解，加水定容至刻度，摇匀，即得。取上述配制溶液各0.2mL于20mL具塞试管，加水补至1.0mL。用蒸馏水补至1.0mL，加入现配的5％苯酚溶液1.0mL及浓硫酸5.0mL，摇匀，室温放置10min后用涡旋振荡器使反应液充分混合，然后将试管置于30℃水浴中反应20min，于490nm测吸光度。Testing of the test product: Weigh 50mg of Cs-4-P1, weigh it accurately, put it in a 10mL measuring bottle, dissolve it by ultrasonic, add water to make it up to the mark, shake well, and you get it. Take 0.2 mL each of the above prepared solutions in a 20 mL stoppered test tube, and add water to make up to 1.0 mL. Make up to 1.0mL with distilled water, add 1.0mL of 5% phenol solution and 5.0mL of concentrated sulfuric acid, shake well, leave at room temperature for 10min, mix the reaction solution thoroughly with a vortex oscillator, and then place the test tube in a 30°C water bath After reacting for 20min, measure the absorbance at 490nm.
测定多糖含量，Cs-4-P1-1、Cs-4-P1-2、Cs-4-P1-3多糖含量为：94.61％、98.61％、99.52％。Determination of polysaccharide content, Cs-4-P1-1, Cs-4-P1-2, Cs-4-P1-3 polysaccharide content: 94.61%, 98.61%, 99.52%.
2.分子量测定2. Molecular weight determination
高效凝胶色谱法(HPGPC)可用于测定大分子物质的相对分子质量及其分布，是多糖相对分子量的首选方法。其原理为根据在凝胶柱上不同相对分子质量的多糖与洗脱保留时间(t R)成一定关系的特性，先用已知相对分子质量的标样制成标准曲线，然后由样品的t R从曲线中求得相对分子质量。峰值分子量Mp表示最高峰保留时间对应的分子量，峰起始和峰结束分子量分别表示峰起始时间和峰结束时间对应分子量。重均分子量Mw为按分子重量统计平均而得，代表两侧分布有同等重量的分子。 High-performance gel chromatography (HPGPC) can be used to determine the relative molecular weight and distribution of macromolecular substances, and is the preferred method for the relative molecular weight of polysaccharides. The principle is that according to the characteristics that polysaccharides with different relative molecular masses have a certain relationship with the elution retention time (t R ) on the gel column, a standard curve is first prepared with a standard sample of known relative molecular mass, and then the t of the sample is R obtains the relative molecular mass from the curve. The peak molecular weight Mp represents the molecular weight corresponding to the highest peak retention time, and the peak start and peak end molecular weights represent the molecular weight corresponding to the peak start time and peak end time, respectively. The weight-average molecular weight Mw is obtained based on the statistical average of the molecular weight, which means that molecules of the same weight are distributed on both sides.
2.1试剂配制2.1 Reagent preparation
0.71％硫酸钠配制：称取硫酸钠14.20g于2L烧杯中，加纯化水2000ml溶解并稀释，过0.22um微孔滤膜，超声脱气约10min即得。Preparation of 0.71% sodium sulfate: Weigh 14.20g of sodium sulfate into a 2L beaker, add 2000ml of purified water to dissolve and dilute, pass through a 0.22um microporous filter membrane, and ultrasonically degas for about 10min.
供试品溶液配制：取各样品50mg至10ml量瓶，精密称定，加适量上述过滤所得流动相，超声10min后，定容至刻度，摇匀即得供试品溶液。将供试品溶液置于4℃条件下保存。Preparation of the test solution: Take 50mg to 10ml measuring bottles of each sample, weigh them accurately, add an appropriate amount of mobile phase obtained from the above filtration, and after ultrasonication for 10min, set the volume to the mark and shake well to obtain the test solution. The test solution was stored at 4°C.
对照品溶液配制：称取右旋糖酐D0、D1、D2、D3、D4、D5、D6、D7、D8、D2000、右旋糖酐410000各10mg于各1.5ml一次性离心管中，精密称定，精密移取1ml 0.71％硫酸钠溶液1ml于各离心管，摇匀即得。Preparation of reference solution: Weigh 10 mg each of dextran D0, D1, D2, D3, D4, D5, D6, D7, D8, D2000, and dextran 410,000 into each 1.5ml disposable centrifuge tube, accurately weigh and pipette 1ml Put 1ml of 0.71% sodium sulfate solution in each centrifuge tube and shake well.
2.2色谱条件2.2 Chromatographic conditions
色谱柱：ZRD-LC-53TOSOH TSKgel guardcolumn PWXL(6*40mm,12um)；ZRD-LC-54 TOSOH TSKgel G3000PWXL(7.8*300mm,7um)；ZRD-LC-55TOSOH TSKgel G4000 PWXL(7.8*300mm,10um)；ZRD-LC-62TOSOH TSKgel G5000PWXL(7.8*300mm,10um).Chromatographic column: ZRD-LC-53TOSOH TSKgel guardcolumn PWXL(6*40mm,12um); ZRD-LC-54 TOSOH TSKgel G3000PWXL(7.8*300mm,7um); ZRD-LC-55TOSOH TSKgel G4000 PWXL(7.8*300mm,10um) ;ZRD-LC-62TOSOH TSKgel G5000PWXL(7.8*300mm,10um).
洗脱条件：0.71％硫酸钠溶液，等度洗脱.Elution conditions: 0.71% sodium sulfate solution, isocratic elution.
RID检测器，柱温为35℃，流速为0.8mL/min，进样量为20ul.RID detector, the column temperature is 35°C, the flow rate is 0.8mL/min, and the injection volume is 20ul.
以保留时间为横坐标，对应分子量对数值为纵坐标，绘制标准曲线，根据回归方程计算样品Cs-4-P1、Cs-4-P1-1、Cs-4-P1-2、Cs-4-P1-3相对分子量。结果如图1和表1所示，Cs-4-P1、Cs-4-P1-1、Cs-4-P1-2、Cs-4-P1-3的重均分子量Mw分布在23.25-151.15kDa。Take the retention time as the abscissa, and the corresponding molecular weight logarithm as the ordinate, draw a standard curve, and calculate the samples Cs-4-P1, Cs-4-P1-1, Cs-4-P1-2, Cs-4- P1-3 relative molecular weight. The results are shown in Figure 1 and Table 1, the weight average molecular weight Mw of Cs-4-P1, Cs-4-P1-1, Cs-4-P1-2, Cs-4-P1-3 is distributed in 23.25-151.15kDa .
表1Table 1
Figure PCTCN2022117637-appb-000002
Figure PCTCN2022117637-appb-000002
3、单糖组成测定3. Determination of monosaccharide composition
称取2mg样品于反应瓶中，加3mL 2mol/L三氟乙酸(TFA)，110℃油浴加热3h，冷却至室温，用氮气在40℃下吹干，加入3mL甲醇，吹干，重复4～5次以完全除去TFA。用超纯水将反应瓶中的样品溶解，并定容至100mL，取部分溶液12000g离心20min，取上清液，运用高效阴离子色谱法(HPAEC)检测其单糖组成，以单糖混合标品对照。由表2结果，可见Cs-4-P1-1、Cs-4-P1-2、Cs-4-P1-3的单糖组成一致，具有相同的组成单元。Weigh 2mg of the sample into a reaction bottle, add 3mL of 2mol/L trifluoroacetic acid (TFA), heat in an oil bath at 110°C for 3h, cool to room temperature, blow dry with nitrogen at 40°C, add 3mL of methanol, blow dry, repeat 4 ~5 times to completely remove TFA. Dissolve the sample in the reaction bottle with ultrapure water, and set the volume to 100mL, take part of the solution and centrifuge at 12,000g for 20min, take the supernatant, and use high-performance anion chromatography (HPAEC) to detect the composition of monosaccharides. control. From the results in Table 2, it can be seen that the monosaccharide compositions of Cs-4-P1-1, Cs-4-P1-2, and Cs-4-P1-3 are consistent and have the same constituent units.
表2Table 2
样品sample GlcpGlcp GalpGalp ManpManp
Cs-4-P1-1Cs-4-P1-1 1.001.00 1.711.71 2.442.44
Cs-4-P1-2Cs-4-P1-2 1.001.00 1.731.73 2.342.34
Cs-4-P1-3Cs-4-P1-3 1.001.00 1.741.74 2.092.09
实施例3 Cs-4-P1-2的结构解析Example 3 Structural Analysis of Cs-4-P1-2
1、多糖甲基化1. Polysaccharide methylation
多糖经过甲基化后，测定其红外图谱，在3100-3600cm -1间的羟基吸收峰消失表明多糖已经完全甲基化(图1)。完全甲基化的样品经过水解、还原和乙酰化处理，进行GC-MS分析，其中Cs-4-P1-2多糖甲基化分析的总离子流图谱见图2，甲基化分析结果见表3。 After the polysaccharide is methylated, its infrared spectrum is measured, and the hydroxyl absorption peak between 3100-3600 cm -1 disappears, indicating that the polysaccharide has been completely methylated (Figure 1). The fully methylated samples were processed by hydrolysis, reduction and acetylation, and then analyzed by GC-MS. The total ion current spectrum of the methylation analysis of Cs-4-P1-2 polysaccharide is shown in Figure 2, and the results of the methylation analysis are shown in the table 3.
表3table 3
甲基化糖methylated sugar 连接方式connection method 摩尔比The molar ratio of
2,3,4,6-Me 4-Glcp 2,3,4,6-Me 4 -Glcp Terminal GlcpTerminal Glcp 1.771.77
2,3,4,6-Me 4-Galp 2,3,4,6-Me 4 -Galp Terminal GalpTerminal Galp 1.001.00
2,3,6-Me 3-Galp 2,3,6-Me 3 -Galp 1,4-Linked Galp1,4-Linked Galp 2.042.04
2,3,4-Me 3-Manp 2,3,4-Me 3 -Manp 1,6-Linked Manp1,6-Linked Manp 2.332.33
2,3-Me 2-Manp 2,3-Me 2 - Manp 1,4,6-Linked Manp1,4,6-Linked Manp 2.962.96
2、波谱解析2. Spectrum analysis
从 1H NMR(图3)中可以看出，异头氢区域有5个共振信号峰，结合异头氢峰面积比例确定共有9个糖残基信号峰，分别在δ4.92ppm(3个H)、δ4.89ppm(2个H)、δ4.58ppm、δ4.46ppm(2个H)、δ4.40ppm处出现，说明糖残基是β构型，根据异头氢的化学位移递减顺序，设定各糖残基的编号为A、B、C、D、E。在 13C NMR(图4)图谱中，异头碳信号区域的信号为δ102.1ppm～δ107.1ppm，进一步说明葡萄糖残基为β构型。 It can be seen from 1 H NMR (Fig. 3) that there are 5 resonance signal peaks in the anomeric hydrogen region, and there are 9 sugar residue signal peaks determined in combination with the area ratio of the anomeric hydrogen, respectively at δ4.92ppm (3 H ), δ4.89ppm (2 H), δ4.58ppm, δ4.46ppm (2 H), and δ4.40ppm, indicating that the sugar residue is in the β configuration. According to the order of decreasing chemical shift of the anomeric hydrogen, set The sugar residues are numbered A, B, C, D, E. In the 13 C NMR (Fig. 4) spectrum, the signal in the anomeric carbon signal region is δ102.1ppm-δ107.1ppm, further indicating that the glucose residue is in the β configuration.
为了进一步阐明多糖的化学结构，对其进行了二维核磁谱(COSY、TOCSY、HMQC、HMBC)的分析，并归属了各糖残基对应的化学位移。In order to further elucidate the chemical structure of the polysaccharide, two-dimensional nuclear magnetic spectrum (COSY, TOCSY, HMQC, HMBC) analysis was carried out on it, and the chemical shift corresponding to each sugar residue was assigned.
糖残基A：根据 1H NMR确定的糖残基A的1位氢(H-1)化学位移，通过COSY谱(图5)明确了H-2和H-3信号，糖残基A的H-2和H-3化学位移分别归属为δ3.53ppm和δ3.72ppm，结合COSY和TOSCY谱(图6)对H-4(δ3.92ppm)、H-5(δ3.65ppm)和H-6(δ3.95ppm，δ3.75ppm)信号的化学位移进行了归属。在归属完H的化学位移后，可通过HSQC谱(图7)归属该糖环上各C的化学位移。根据 Sugar residue A: According to the 1-position hydrogen (H-1) chemical shift of sugar residue A determined by 1 H NMR, the H-2 and H-3 signals were clarified by COZY spectrum (Figure 5). The chemical shifts of H-2 and H-3 are assigned to δ3.53ppm and δ3.72ppm, respectively, combined with COZY and TOSCY spectra (Figure 6) for H-4 (δ3.92ppm), H-5 (δ3.65ppm) and H- The chemical shifts of the 6 (δ3.95ppm, δ3.75ppm) signals were assigned. After assigning the chemical shift of H, the chemical shift of each C on the sugar ring can be assigned by HSQC spectrum (Figure 7). according to
表4中C和H的化学位移，可确定糖残基A是→4,6)-β-D-Manp-(1→。From the chemical shifts of C and H in Table 4, it can be determined that sugar residue A is →4,6)-β-D-Manp-(1→.
表4Table 4
Figure PCTCN2022117637-appb-000003
Figure PCTCN2022117637-appb-000003
根据上述方法，依次对糖残基B、C、D、E中的碳(C)、氢(H)信号化学位移进行归属，全归属结果见According to the above method, the chemical shifts of the carbon (C) and hydrogen (H) signals in sugar residues B, C, D, and E were assigned sequentially. For the full assignment results, see
表4。通过比对，确定了糖残基B、C、D、E的连接方式分别为→4)-β-D-Galp-(1→、β-D-Galp-(1→、β-D-Glcp-(1→、→6)-β-D-Manp-(1→。Table 4. Through comparison, it was determined that the sugar residues B, C, D, and E were connected in the following ways: →4)-β-D-Galp-(1→, β-D-Galp-(1→, β-D-Glcp -(1→,→6)-β-D-Manp-(1→.
在完成归属后，进一步利用HMBC谱(图9)明确各糖残基间连接的位点及顺序。从HMBC信号可知，残基A的H-1和残基B的C-4和残基E的C-6有明显相关信号，残基B 的H-1和残基A的C-6有相关信号，残基C的H-1和残基B的C-4有相关信号，残基D的H-1和残基A的C-4有相关信号，残基E的H-1和残基A的C-4有相关信号，各糖残基的远程相关数据见表5。After the assignment was completed, the HMBC spectrum (Fig. 9) was further used to clarify the position and sequence of connections between sugar residues. From the HMBC signal, it can be seen that H-1 of residue A and C-4 of residue B and C-6 of residue E have obvious correlation signals, and H-1 of residue B has correlation with C-6 of residue A Signals, H-1 of residue C and C-4 of residue B have related signals, H-1 of residue D and C-4 of residue A have related signals, H-1 of residue E and residues There is a correlation signal at C-4 of A, and the long-range correlation data of each sugar residue is shown in Table 5.
表5table 5
糖残基sugar residue 氢质子hydrogen proton 关联associate
A:→4,6)-β-D-Manp-(1→A:→4,6)-β-D-Manp-(1→ H-1H-1 80.4(B；C-4),68.9(E；C-6)80.4 (B; C-4), 68.9 (E; C-6)
B:→4)-β-D-Galp-(1→B:→4)-β-D-Galp-(1→ H-1H-1 68.7(A；C-6)68.7 (A; C-6)
C:β-D-Galp-(1→C:β-D-Galp-(1→ H-1H-1 80.4(B；C-4)80.4 (B; C-4)
D:β-D-Glcp-(1→D:β-D-Glcp-(1→ H-1H-1 79.7(A；C-4)79.7 (A; C-4)
E:→6)-β-D-Manp-(1→E:→6)-β-D-Manp-(1→ H-1H-1 79.3(A:C-4)79.3 (A:C-4)
之后根据NOESY谱(图10)和HMBC谱相互验证，从NOESY谱也可看出残基A的H-1和残基B的H-4和残基D的H-6有相关点，残基B的H-1和残基A的H-6有相关点，残基D的H-1和残基A的H-4有相关点，残基E的H-1和残基A的H-4有相关点，NOE数据见表6。Afterwards, according to the mutual verification of the NOESY spectrum (Figure 10) and the HMBC spectrum, it can also be seen from the NOESY spectrum that H-1 of residue A, H-4 of residue B and H-6 of residue D have related points, and residue H-1 of B and H-6 of residue A have correlation points, H-1 of residue D and H-4 of residue A have correlation points, H-1 of residue E and H- 4 has relevant points, see Table 6 for NOE data.
表6Table 6
糖残基sugar residue 氢质子hydrogen proton NOE关联NOE association
A:→4,6)-β-D-Manp-(1→A:→4,6)-β-D-Manp-(1→ H-1H-1 4.02(B；H-4),3.87(E；H-6)4.02 (B; H-4), 3.87 (E; H-6)
B:→4)-β-D-Galp-(1→B:→4)-β-D-Galp-(1→ H-1H-1 3.75(A；H-6)3.75 (A; H-6)
D:β-D-Glcp-(1→D:β-D-Glcp-(1→ H-1H-1 3.90(A；H-4)3.90 (A; H-4)
E:→6)-β-D-Manp-(1→E:→6)-β-D-Manp-(1→ H-1H-1 3.92(A；H-4)3.92 (A; H-4)
根据HMBC和NOESY谱相互验证的结果，可以判断糖残基之间的连接方式，可以推断残基A的1位与残基B的4位和E的6位相连接，残基B的1位与残基A的6位相连接，残基C的1位与残基B的4位相连，残基D与残基E的1位分别与残基A的4位相连接，结合甲基化结果中各糖残基的比例为A:B:C:D:E＝3:2:1:2:1，据此确认多糖组分的重复单元为：According to the results of mutual verification of HMBC and NOESY spectra, the connection mode between sugar residues can be judged, and it can be inferred that the 1st position of residue A is connected with the 4th position of residue B and the 6th position of E, and the 1st position of residue B is connected with The 6th position of residue A is connected, the 1st position of residue C is connected with the 4th position of residue B, and the 1st position of residue D and residue E are respectively connected with the 4th position of residue A. The ratio of sugar residues is A:B:C:D:E=3:2:1:2:1, so it is confirmed that the repeating unit of the polysaccharide component is:
Figure PCTCN2022117637-appb-000004
Figure PCTCN2022117637-appb-000004
该多糖是以β-(1→4)-D-甘露糖、β-(1→6)-D-甘露糖和β-(1→4)-D-半乳糖交替连接为主链，在甘露糖的6位和4位分别连接β-D-半乳糖和β-D-葡萄糖支链的杂多糖。该重复片段为9糖，据此根据表1中Cs-4-P1-1、Cs-4-P1-2、Cs-4-P1-3的重均分子量(Mw)计算其n值分别约为：104、21、16。The polysaccharide is β-(1→4)-D-mannose, β-(1→6)-D-mannose and β-(1→4)-D-galactose are alternately linked as the main chain, and in mannose The 6th and 4th positions of the sugar are respectively linked to β-D-galactose and β-D-glucose branched heteropolysaccharides. This repeat segment is 9 sugars, according to which according to the weight average molecular weight (Mw) of Cs-4-P1-1, Cs-4-P1-2, Cs-4-P1-3 in Table 1, its n value is calculated to be about : 104, 21, 16.
实施例4对腺嘌呤诱导的大鼠慢性肾衰的作用研究Example 4 Effect Research on Chronic Renal Failure in Rats Induced by Adenine
试验方法：70只SD大鼠，雌雄各半，按体重随机分组，每组10只：正常对照组、模型组、Cs-4-P1-2组、发酵虫草菌粉组、黄葵胶囊组、地塞米松组。试验前，大鼠放入代谢笼，禁食24h(照常进水)，记录24h的尿量，检测尿蛋白和尿肌酐。大鼠称重后眼眶取血1mL，置于1.5mL离心管中，以3000转/min离心10min，取血检测尿素氮(BUN)，肌酐(Scr)和尿酸，作为基础值。除正常对照组外，其余组别大鼠灌胃给予腺嘌呤(生理盐水溶解)200mg/kg，每天一次，连续4周，最后一周不造模只给药。分别于7、14、21、28天、35天大鼠眼眶取血，以3000转/min离心10min，取血清检验尿素氮(BUN)，肌酐(Scr)和尿酸值，记录24h的尿量，检测尿蛋白和尿肌酐，计算肌酐清除率。实验终点解剖大鼠取出肾脏，在冰上将肾皮脂分离，称重，计算脏器指数。然后4％中性甲醛溶液固定，石蜡包埋，苏木素-伊红(HE)染色，光镜检查，读片。Test method: 70 SD rats, half male and half male, were randomly divided into groups according to body weight, 10 rats in each group: normal control group, model group, Cs-4-P1-2 group, fermented Cordyceps powder group, Huangkui capsule group, Dexamethasone group. Before the test, the rats were put into metabolic cages, fasted for 24 hours (water intake as usual), recorded the urine output for 24 hours, and detected urine protein and urine creatinine. After the rats were weighed, 1 mL of blood was taken from the orbit, placed in a 1.5 mL centrifuge tube, centrifuged at 3000 rpm for 10 min, and the blood was taken to detect urea nitrogen (BUN), creatinine (Scr) and uric acid, as the basic value. Except for the normal control group, the rats in the other groups were given 200 mg/kg of adenine (dissolved in normal saline) by intragastric administration, once a day, for 4 consecutive weeks, and in the last week, only administration was performed without modeling. On days 7, 14, 21, 28, and 35, blood was collected from the orbits of the rats, centrifuged at 3000 rpm for 10 minutes, serum was taken to test urea nitrogen (BUN), creatinine (Scr) and uric acid values, and the urine volume of 24 hours was recorded. Urine protein and creatinine were detected, and creatinine clearance was calculated. At the end of the experiment, the rats were dissected to take out the kidneys, the renal sebum was separated on ice, weighed, and the organ index was calculated. Then fixed in 4% neutral formaldehyde solution, embedded in paraffin, stained with hematoxylin-eosin (HE), examined with light microscope, and read the slides.
试验结果：1)动物体重变化如下Test results: 1) The animal body weight changes as follows
表7所示。Table 7 shows.
表7Table 7
Figure PCTCN2022117637-appb-000005
Figure PCTCN2022117637-appb-000005
Figure PCTCN2022117637-appb-000006
Figure PCTCN2022117637-appb-000006
注：大鼠数量0d为10只/组，雌雄各半，与模型组比较 *P≤0.05， **P≤0.01， ***P≤0.001 Note: The number of rats 0d is 10/group, half male and half male, compared with the model group * P≤0.05, ** P≤0.01, *** P≤0.001
由Depend on
表7可知：试验第7d、11d、14d与模型组比较，正常对照组的大鼠体重明显增加，第18d、21d和28d正常对照组的大鼠体重显著增加，第25d和32d正常对照组的大鼠体重非常显著地增加(P≤0.001)；地塞米松组从第11d开始体重较模型组有所减轻，其中第18d和28d显著减轻，提示与激素副作用有关；Cs-4-P1-2组、Cs-4-P组、发酵虫草菌粉组未见对体重有显著影响。Table 7 shows that: compared with the model group on the 7th, 11d, and 14d of the test, the body weight of the rats in the normal control group increased significantly; The body weight of the rats increased significantly (P≤0.001); the body weight of the dexamethasone group was lower than that of the model group from the 11th day, and the body weight was significantly reduced on the 18th and 28th days, suggesting that it was related to the side effects of hormones; Cs-4-P1-2 group, Cs-4-P group, and fermented Cordyceps powder group had no significant effect on body weight.
2)大鼠肾重及脏器系数如下表8所示。2) The rat kidney weight and organ coefficients are shown in Table 8 below.
表8Table 8
组别group 5w体重5w weight 5w肾重5w kidney weight 5w脏器系数5w organ coefficient
正常对照组normal control group 333.55±88.76 *** 333.55±88.76 *** 2.40±0.74 *** 2.40±0.74 *** 0.72±0.09 *** 0.72±0.09 ***
模型组model group 229.44±62.94229.44±62.94 9.24±3.379.24±3.37 3.98±0.903.98±0.90
地塞米松(0.1mg/kg/天)Dexamethasone (0.1mg/kg/day) 175.08±14.54175.08±14.54 5.72±0.68 * 5.72±0.68 * 3.29±0.513.29±0.51
黄葵(1g/kg/天)Ambrette (1g/kg/day) 266.80±45.23266.80±45.23 9.89±4.339.89±4.33 3.66±1.523.66±1.52
Cs-4-P1-2(16mg/kg/天)Cs-4-P1-2 (16mg/kg/day) 230.99±52.21230.99±52.21 6.99±3.436.99±3.43 2.94±0.92 * 2.94±0.92 *
Cs-4-P(448mg/kg/天)Cs-4-P (448mg/kg/day) 261.16±57.00261.16±57.00 8.69±2.698.69±2.69 3.35±0.923.35±0.92
发酵虫草菌粉(1600mg/kg/天)Fermented Cordyceps powder (1600mg/kg/day) 233.04±57.48233.04±57.48 7.02±2.547.02±2.54 2.97±0.60 * 2.97±0.60 *
注：雌雄大鼠合并统计，与模型组比较， *P≤0.05, **P≤0.01, ***P≤0.001 Note: Combined statistics of male and female rats, compared with model group, * P≤0.05, ** P≤0.01, *** P≤0.001
由上表8可知，模型组体重低于正常对照组，肾重和肾脏器系数高于正常对照组，均有极显著差异(P≤0.001)；与模型组比较，地塞米松组的大鼠肾重明显下降(P≤0.05)，Cs-4-P1-2、Cs-4-P和发酵虫草菌粉组大鼠肾重有下降趋势；与模型组比较，正常对照组、Cs-4-P1-2和发酵虫草菌粉组肾脏系数明显降低(P≤0.05)，Cs-4-P组有下降趋势。As can be seen from the above table 8, the body weight of the model group is lower than that of the normal control group, and the kidney weight and kidney organ coefficient are higher than that of the normal control group, all of which have extremely significant differences (P≤0.001); compared with the model group, the rats in the dexamethasone group The kidney weight decreased significantly (P≤0.05), and the kidney weight of rats in the Cs-4-P1-2, Cs-4-P and fermented Cordyceps powder groups showed a downward trend; compared with the model group, the normal control group, Cs-4- The kidney coefficient of P1-2 and fermented Cordyceps powder group decreased significantly (P≤0.05), and Cs-4-P group showed a downward trend.
3)实验期间大鼠血清BUN的变化如下表9所示。3) The changes of rat serum BUN during the experiment are shown in Table 9 below.
表9Table 9
Figure PCTCN2022117637-appb-000007
Figure PCTCN2022117637-appb-000007
注：雌雄大鼠合并统计。与模型组比较， *P≤0.05, **P≤0.01, ***P≤0.001 Note: Combined statistics of male and female rats. Compared with the model group, * P≤0.05, ** P≤0.01, *** P≤0.001
血清中的尿素氮(BUN)是临床中反应肾小球滤过功能的重要指标，灌胃腺嘌呤可导致大鼠血清BUN升高。由表9可知，与模型组比较，造模1周正常对照组BUN水平为5.22±1.16mmol/L，造模组BUN水平在13.99±2.95mmol/L和17.00±6.31mmol/L之间，两组间有非常显著的差别(P≤0.001)；实验2W即给药一周，黄葵组、Cs-4-P1组BUN水平明显降低(P≤0.05)；实验4W即给药三周，黄葵组、Cs-4-P1-2和发酵虫草菌粉组BUN水平明显降低(P≤0.05)；实验5W即给药四周，地塞米松组和Cs-4-P1-2组BUN水平明显降低(P≤0.05)；给药期间，Cs-4-P组和发酵虫草菌粉组BUN水平有下降趋势。由此可知，Cs-4-P1-2具有改善慢性肾衰大鼠血清BUN的作用，对慢性肾衰竭具有明显治疗作用。Blood urea nitrogen (BUN) in serum is an important indicator of glomerular filtration function in clinical practice, and intragastric administration of adenine can lead to elevated serum BUN in rats. It can be seen from Table 9 that compared with the model group, the BUN level of the normal control group was 5.22±1.16mmol/L at 1 week after modeling, and the BUN level of the model group was between 13.99±2.95mmol/L and 17.00±6.31mmol/L. There is a very significant difference between the groups (P≤0.001); in the experiment 2W, the BUN levels of the ambrette group and the Cs-4-P1 group were significantly reduced (P≤0.05); in the experiment 4W, the administration was three weeks, and the ambrette group, Cs-4-P1-2 and fermented Cordyceps powder group BUN levels were significantly reduced (P≤0.05); experiment 5W that administration for four weeks, dexamethasone group and Cs-4-P1-2 group BUN levels were significantly reduced ( P≤0.05); During the administration period, the BUN level of the Cs-4-P group and the fermented Cordyceps powder group had a downward trend. It can be seen that Cs-4-P1-2 has the effect of improving serum BUN in rats with chronic renal failure, and has obvious therapeutic effect on chronic renal failure.
4)实验期间血清CREA的变化如下表10所示。4) The changes of serum CREA during the experiment are shown in Table 10 below.
表10Table 10
Figure PCTCN2022117637-appb-000008
Figure PCTCN2022117637-appb-000008
注：雌雄大鼠合并统计。与模型组比较， *P≤0.05, **P≤0.01, ***P≤0.001 Note: Combined statistics of male and female rats. Compared with the model group, * P≤0.05, ** P≤0.01, *** P≤0.001
血清肌酐(CREA)是肾脏功能的重要指标，CREA升高意味着肾功能的损害。由表10可知，与模型组比较，造模1周正常对照组CREA水平为27.20±5.73μmol/L，造模组在58.28±13.40μmol/L和65.86±11.13μmol/L之间，两组间有非常显著的差别(P≤0.001)；实验2W即给药一周，地塞米松组CREA水平非常显著降低(P≤0.001)，黄葵组和Cs-4-P1-2组CREA水平明显降低(P≤0.05)；实验3W即给药两周，地塞米松组CREA水平显著降低(P≤0.01)，其余各组有降低趋势；实验4W即给药三周，各组CREA水平有降低趋势；实验5W即给药四周，地塞米松组CREA水平非常显著降低，其余各组有降低趋势。由此可知，Cs-4-P1-2具有改善慢性肾衰大鼠血清CREA的作用，对慢性肾衰竭具有明显治疗作用。Serum creatinine (CREA) is an important indicator of kidney function, and an increase in CREA means damage to kidney function. It can be seen from Table 10 that compared with the model group, the CREA level of the normal control group was 27.20±5.73 μmol/L at 1 week after modeling, and that of the model group was between 58.28±13.40 μmol/L and 65.86±11.13 μmol/L. Very significant difference (P≤0.001) is arranged; Experiment 2W promptly administers a week, and dexamethasone group CREA level reduces very significantly (P≤0.001), and ambrette group and Cs-4-P1-2 group CREA level obviously reduces ( P≤0.05); Experiment 3W was given for two weeks, and the CREA level in the dexamethasone group was significantly reduced (P≤0.01), and the rest of the groups had a downward trend; Experiment 4W was given for three weeks, and the CREA levels in each group had a downward trend; In experiment 5W, namely administration for four weeks, the CREA level of the dexamethasone group was significantly reduced, and all the other groups had a downward trend. It can be seen that Cs-4-P1-2 has the effect of improving serum CREA in rats with chronic renal failure, and has obvious therapeutic effect on chronic renal failure.
5)实验期间UA的变化如下表11所示5) The changes of UA during the experiment are shown in Table 11 below
表11Table 11
Figure PCTCN2022117637-appb-000009
Figure PCTCN2022117637-appb-000009
注：雌雄大鼠合并统计。与模型组比较， *P≤0.05, **P≤0.01, ***P≤0.001 Note: Combined statistics of male and female rats. Compared with the model group, * P≤0.05, ** P≤0.01, *** P≤0.001
由表11可知，与模型组比较，造模1周正常对照组UA水平为91.00±12.51μmol/L，造模组在113.75±24.84μmol/L和133.29±18.50μmol/L之间，两组间有非常显著的差别(P≤0.001)；实验2W即给药一周，正常对照组显著较低，地塞米松组UA水平非常显著降低(P≤0.001)，黄葵组和Cs-4-P组明显降低(P≤0.05)，Cs-4-P1-2和发酵虫草菌粉组UA水平显著降低(P≤0.01)；实验3W即给药两周，地塞米松组UA水平显著降低，其余各组有降低趋势；实验4W即给药三周，发酵虫草菌粉组UA水平显著趋势，其余各组有降低趋势；实验5W即给药四周，地塞米松组UA水平显著升高，Cs-4-P1-2明显升高，其余各组有升高趋势。It can be seen from Table 11 that compared with the model group, the UA level of the normal control group was 91.00±12.51 μmol/L at 1 week after modeling, and that of the model group was between 113.75±24.84 μmol/L and 133.29±18.50 μmol/L. There is a very significant difference (P≤0.001); in experiment 2W, that is, administration for one week, the normal control group was significantly lower, and the UA level of the dexamethasone group was very significantly reduced (P≤0.001), and the ambrette group and the Cs-4-P group significantly decreased (P≤0.05), and UA levels in Cs-4-P1-2 and fermented Cordyceps powder groups significantly decreased (P≤0.01); in experiment 3W, two weeks after administration, the UA levels in the dexamethasone group decreased significantly, and the rest In experiment 4W, that is, administration for three weeks, the level of UA in the fermented Cordyceps powder group had a significant trend, and the rest of the groups had a tendency to decrease; in experiment 5W, that is, administration for four weeks, the level of UA in the dexamethasone group increased significantly, and Cs-4 -P1-2 increased significantly, and the rest of the groups had a rising trend.
6)肾脏病理。6) Renal pathology.
腺嘌呤致慢性肾衰能能引起严重的肾脏组织病变，模拟肾衰终末期状态。通过对肾脏进行HE染色病理切片，在光镜下观察，其结果见。正常对照组大鼠肾脏组织结构正常，肾小球 血管袢薄而清晰，内皮细胞和系膜细胞数码正常，周围肾小管正常。与正常对照组相比，模型组大鼠肾组织发现肾小球纤维化，肾小管萎缩或扩张，肾小管肾小球内见异物充塞，间质内大量纤维组织增生及炎细胞浸润。与模型组相比，Cs-4-P1-2给药组残存肾小球单位增加，病理评分显著降低(P≤0.01)，黄葵和发酵虫草菌粉组病理评分明显降低(P≤0.05)。说明Cs-4-P1-2、发酵虫草菌粉对长期腺嘌呤灌胃导致的肾脏病理变化有所改善和恢复作用，其中Cs-4-P1-2最优。Adenine-induced chronic renal failure can cause severe renal tissue lesions, simulating the end-stage state of renal failure. Through the HE staining pathological section of the kidney, observed under the light microscope, see the results. In the normal control group, the renal tissue structure was normal, the glomerular vascular loops were thin and clear, the numbers of endothelial cells and mesangial cells were normal, and the surrounding renal tubules were normal. Compared with the normal control group, glomerular fibrosis, renal tubule atrophy or dilation, foreign body filling in the renal tubule and glomerulus, a large amount of fibrous tissue proliferation and inflammatory cell infiltration in the interstitium were found in the renal tissue of rats in the model group. Compared with the model group, the residual glomerular units in the Cs-4-P1-2 administration group increased significantly, and the pathological score decreased significantly (P≤0.01), and the pathological score in the ambrette and fermented Cordyceps powder group decreased significantly (P≤0.05) . It shows that Cs-4-P1-2 and fermented Cordyceps powder can improve and restore the renal pathological changes caused by long-term adenine gavage, and Cs-4-P1-2 is the best.
表12Table 12
组别group 病理评分pathological score
正常对照组normal control group 0.00±0.00 *** 0.00±0.00 ***
模型组model group 9.00±0.009.00±0.00
地塞米松(0.1mg/kg)Dexamethasone (0.1mg/kg) 8.60±0.558.60±0.55
黄葵(1g/kg/天)Ambrette (1g/kg/day) 8.43±0.45 * 8.43±0.45 *
Cs-4-P1-2(16mg/kg/天)Cs-4-P1-2 (16mg/kg/day) 8.31±0.37 ** 8.31±0.37 **
Cs-4-PCs-4-P 8.58±0.498.58±0.49
发酵虫草菌粉组(1600mg/kg/天)Fermented Cordyceps powder group (1600mg/kg/day) 8.31±0.88 * 8.31±0.88 *
注:与模型组比较 *P≤0.05， **P≤0.01， ***P≤0.001 Note: Compared with the model group * P≤0.05, ** P≤0.01, *** P≤0.001
从残存的肾小球和肾单位来判断Cs-4-P1对肾脏的作用。由表12可知，与模型组比较，正常对照组病理评分为0，黄葵组、发酵虫草菌粉组大鼠病理评分有所降低，Cs-4-P组有降低趋势，Cs-4-P1-2组大鼠病理评分明显降低。The effect of Cs-4-P1 on the kidney was judged from the remaining glomeruli and nephrons. It can be seen from Table 12 that, compared with the model group, the pathological score of the normal control group was 0, the pathological score of the rats in the ambrette group and the fermented Cordyceps powder group decreased, and the Cs-4-P group had a downward trend, and the Cs-4-P1 The pathological score of the rats in the -2 group was significantly reduced.
7)试验终点大鼠死亡率如下表13所示。7) The death rate of rats at the end of the test is shown in Table 13 below.
表13Table 13
组别group 原有只数original number 存活只数number of survivors 死亡率(％)mortality rate(%)
正常对照组 normal control group 1010 1010 0.000.00
模型组 model group 1010 77 30.0030.00
地塞米松(0.1mg/kg)Dexamethasone (0.1mg/kg) 1010 55 50.0050.00
黄葵(1g/kg/天)Ambrette (1g/kg/day) 1010 77 30.0030.00
Cs-4-P1-2(16mg/kg/天)Cs-4-P1-2 (16mg/kg/day) 1010 88 20.0020.00
Cs-4-PCs-4-P 1010 66 30.0030.00
发酵虫草菌粉组(1600mg/kg/天)Fermented Cordyceps powder group (1600mg/kg/day) 1010 88 20.0020.00
由表13可知，试验期间造模组均有死亡，其中地塞米松组死亡率最高为50％，分析与激素副作用有关。Cs-4-P1-2组和发酵虫草菌粉组死亡率均为20％，较模型组降低。It can be seen from Table 13 that all the modeling groups died during the experiment, and the mortality rate in the dexamethasone group was the highest at 50%, which was related to the side effects of hormones. The mortality rate of the Cs-4-P1-2 group and the fermented Cordyceps powder group was 20%, which was lower than that of the model group.
综上，本试验条件下，从大鼠试验期间观察、体重、血清生化指标和病理总分等几个方 面进行发酵虫草菌粉组分对腺嘌呤致慢性肾衰的作用研究，试验结果显示模型组造模成功，与模型组比较，Cs-4-P1-2组血清生化指标检测相对有效，死亡率降低，能减轻腺嘌呤所致的慢性肾衰的作用。且可以看出本发明的Cs-4-P1-2对腺嘌呤致慢性肾衰的效果显著优于地塞米松、黄葵和发酵虫草菌粉和其粗多糖(Cs-4-P)。In summary, under the conditions of this experiment, the effects of fermented Cordyceps powder components on adenine-induced chronic renal failure were studied from the aspects of observation during the experiment, body weight, serum biochemical indicators, and pathological total scores of rats. The test results showed that the model The Cs-4-P1-2 group was successfully modeled. Compared with the model group, the detection of serum biochemical indicators in the Cs-4-P1-2 group was relatively effective, the mortality rate was reduced, and the effect of chronic renal failure caused by adenine could be alleviated. And it can be seen that the effect of Cs-4-P1-2 of the present invention on adenine-induced chronic renal failure is significantly better than that of dexamethasone, yellow sunflower, fermented Cordyceps fungus powder and its crude polysaccharide (Cs-4-P).
实施例5抑制免疫排斥反应的药效学研究Example 5 Pharmacodynamics Research on Inhibiting Immune Rejection
试验方法：experiment method:
(1)皮肤移植模型的建立(1) Establishment of skin graft model
根据参考文献(蔡春晓，马春梅,等.小鼠异种皮肤移植模型的建立及对免疫抑制药物的药效评价.中国药理学通报，2016，32(11):1613-1619.)，BALB/c小鼠和C57/BL6小鼠经戊巴比妥钠(40mg/kg)麻醉后，用70％酒精对供体小鼠的耳朵和受体小鼠的背部皮肤进行消毒。将供体小鼠的耳朵从根部取下，置于冰冷的无菌PBS中备用。将受体小鼠背部一侧皮肤的表皮剪下(直径约1cm)，将供体小鼠的耳朵内外侧皮肤分离，取内侧皮肤，分离面朝下，贴合到受体小鼠背部，用剪刀将未完全吻合的皮肤进行剔除修理后，用无菌创口贴包扎，用丝线将创口贴缝合固定。待2天后，将创口贴轻轻剪下，暴露移植部位，对移植排斥反应进行评价。在本实验中异种皮肤移植(Allograft)将C57/BL6小鼠皮肤移植到BALB/c小鼠身上，同种皮肤移植(Isograft)将BALB/c小鼠皮肤移植到BALB/c小鼠身上。According to references (Cai Chunxiao, Ma Chunmei, et al. Establishment of mouse xenograft model and evaluation of efficacy of immunosuppressive drugs. Chinese Pharmacology Bulletin, 2016, 32(11):1613-1619.), BALB/c After the mice and C57/BL6 mice were anesthetized with sodium pentobarbital (40 mg/kg), the ears of the donor mice and the back skin of the recipient mice were disinfected with 70% alcohol. Ears of donor mice were removed from the base and placed in ice-cold sterile PBS for later use. Cut off the epidermis of the skin on the back side of the recipient mouse (about 1 cm in diameter), separate the inner and outer skin of the ear of the donor mouse, take the inner skin, and attach it to the back of the recipient mouse with the separated side facing down. Scissors remove and repair the incompletely matched skin, then wrap it with a sterile wound dressing, and suture and fix the wound with silk thread. After 2 days, the wound was gently cut off to expose the graft site, and the graft rejection was evaluated. In this experiment, the skin of C57/BL6 mice was transplanted to BALB/c mice by Allograft, and the skin of BALB/c mice was transplanted to BALB/c mice by Isograft.
(2)移植排斥评分(Graft Rejection Score)(2) Graft Rejection Score
根据文献，将移植后皮肤的排斥反应分为0-5级(如表14所示)。According to the literature, the rejection reaction of the transplanted skin was divided into 0-5 grades (as shown in Table 14).
表14Table 14
级别 level 移植排斥反应transplant rejection
00 皮肤完好，无排斥反应Intact skin, no rejection
11 刚刚出现排斥现象Rejection just happened
22 >25％的皮肤坏死>25% skin necrosis
33 >50％的皮肤坏死>50% skin necrosis
44 >75％的皮肤坏死>75% skin necrosis
55 皮肤完全坏死(>95％)Complete necrosis (>95%) of the skin
注：移植排斥以坏死皮肤占总皮肤的百分比表示，因缝合、缝合材料、皮肤移位以及其他外伤等引起的皮肤损伤不计入统计。Note: Transplant rejection is expressed as the percentage of necrotic skin in total skin, and skin damage caused by sutures, suture materials, skin displacement, and other trauma is not included in the statistics.
当皮肤移植排斥级别达到5级时，可定义为移植的皮肤死亡。When the grade of skin graft rejection reaches grade 5, it can be defined as the death of the grafted skin.
(3)剂量设计及依据(3) Dose design and basis
临床人用环孢素作为免疫抑制剂的剂量为15mg/kg/天，小鼠剂量为150mg/kg，发酵虫草菌粉人体给药剂量为人体3g～6g/天，发酵虫草菌粉剂量设置为小鼠3000mg/kg/天，根据得率设置Cs-4-P1-2剂量为小鼠30mg/kg/天。The clinical dose of cyclosporine used as an immunosuppressant for humans is 15mg/kg/day, the dose for mice is 150mg/kg, the dosage of fermented Cordyceps powder for humans is 3g~6g/day for humans, and the dose of fermented Cordyceps powder is set to 3000mg/kg/day for mice, and set the dose of Cs-4-P1-2 as 30mg/kg/day for mice according to the yield.
表15Table 15
Figure PCTCN2022117637-appb-000010
Figure PCTCN2022117637-appb-000010
**P<0.01vs模型*P<0.05vs模型##P<0.01vs空白#P<0.05vs空白**P<0.01vs model *P<0.05vs model ##P<0.01vs blank #P<0.05vs blank
从第四天起对皮肤进行评分，从The skin was scored from the fourth day, from
表15可以看到，移植后，空白(同种皮肤移植)组和模型组相比评分较低，14天后和模型组有极显著差异(P<0.01)；环孢素组的评分在6-13天内始终低于模型组，有极显著差异(P<0.01)，第十四天后移植皮肤开始大面积焦枯，和模型没有差异；发酵虫草菌粉组的评分在6-12天内始终低于模型组，有极显著差异，第十三天后移植皮肤开始大面积焦枯，和模型没有差异；Cs-4-P1-2组在10-13天内低于模型组，有极显著差异(P<0.01)，第十四天后移植皮肤开始大面积焦枯，和模型没有差异。表明环孢素、Cs-4-P1-2和发酵虫草菌粉对小鼠皮肤移植的免疫排斥反应有明显抑制作用，其中Cs-4-P1-2具有剂量优势。As can be seen in Table 15, after transplantation, the score of the blank (syngeneic skin graft) group was lower than that of the model group, and there was a very significant difference (P<0.01) with the model group after 14 days; the score of the cyclosporine group was between 6- It was always lower than the model group within 13 days, with a very significant difference (P<0.01). After the 14th day, the transplanted skin began to scorch in large areas, and there was no difference from the model; the score of the fermented Cordyceps powder group was always lower than the model within 6-12 days In the Cs-4-P1-2 group, there was a very significant difference. After the 13th day, the transplanted skin began to scorch in a large area, and there was no difference from the model group; the Cs-4-P1-2 group was lower than the model group within 10-13 days, and there was a very significant difference (P<0.01) , After the fourteenth day, the transplanted skin began to scorch in large areas, and there was no difference with the model. It shows that cyclosporine, Cs-4-P1-2 and fermented Cordyceps powder have obvious inhibitory effect on immune rejection of mouse skin transplantation, and Cs-4-P1-2 has dose advantage.
2)各组体重比较2) Comparison of body weight in each group
表16Table 16
Figure PCTCN2022117637-appb-000011
Figure PCTCN2022117637-appb-000011
Figure PCTCN2022117637-appb-000012
Figure PCTCN2022117637-appb-000012
**P<0.01vs模型*P<0.05vs模型##P<0.01vs空白#P<0.05vs空白**P<0.01vs model *P<0.05vs model ##P<0.01vs blank #P<0.05vs blank
从表16可以看到，移植后，空白(同种皮肤移植)组、Cs-4-P1-2和模型组相比体重始终没有差异；环孢素组的小鼠移植给药后体重增长较缓，始终低于模型组，有极显著差异(P<0.01)；发酵虫草菌粉组的小鼠体重在7-12天内低于模型组，有显著差异。提示相比环孢素，Cs-4-P1-2副作用小，安全性高。As can be seen from Table 16, after transplantation, there is no difference in body weight compared with the blank (syngeneic skin transplantation) group, Cs-4-P1-2 and model group all the time; The body weight of the mice in the fermented Cordyceps powder group was lower than that of the model group within 7-12 days, and there was a significant difference. It is suggested that compared with cyclosporine, Cs-4-P1-2 has less side effects and higher safety.
实施例6对高血脂斑马鱼作用研究Example 6 Effect research on hyperlipidemia zebrafish
试验方法：experiment method:
(1)实验动物(1) Experimental animals
斑马鱼均饲养于28℃的养鱼用水中(水质：每1L反渗透水中加入200mg速溶海盐，电导率为450～550μS/cm；pH为6.5～8.5；硬度为50～100mg/L CaCO 3)，由本公司养鱼中心繁殖提供，实验动物使用许可证号为：SYXK(浙)2012-0171，饲养管理符合国际AAALAC认证(认证编号：001458)的要求。 All zebrafish were raised in fish culture water at 28°C (water quality: 200mg of instant sea salt was added to 1L of reverse osmosis water, conductivity was 450-550μS/cm; pH was 6.5-8.5; hardness was 50-100mg/L CaCO 3 ) , provided by the company's fish breeding center, the license number for the use of experimental animals is: SYXK (Zhejiang) 2012-0171, and the feeding management meets the requirements of the international AAALAC certification (certification number: 001458).
黑色素等位基因突变型半透明Albino品系斑马鱼，以自然成对交配繁殖方式进行。年龄为受精后7天(7dpf)的斑马鱼用于降血脂功效最大检测浓度(MTC)测定。The melanin allele mutant translucent Albino strain zebrafish reproduces by natural pair mating. Zebrafish aged 7 days after fertilization (7dpf) were used for the determination of maximum detectable concentration (MTC) of hypolipidemic efficacy.
黑色素等位基因突变型半透明Albino品系斑马鱼，以自然成对交配繁殖方式进行。年龄为5dpf的斑马鱼用于降血脂功效评价。The melanin allele mutant translucent Albino strain zebrafish reproduces by natural pair mating. Zebrafish aged 5dpf were used for the evaluation of blood lipid-lowering efficacy.
(2)检测方法(2) Detection method
a.MTC测定a. MTC determination
随机选取7dpf黑色素等位基因突变型半透明Albino品系斑马鱼于6孔板中，每孔(实验组)均处理30尾斑马鱼。分别水溶给予样品(浓度见表17)，同时设置正常对照组，每 孔容量为3mL。28℃处理48h后，测定样品对正常斑马鱼的MTC。The 7dpf melanin allele mutant translucent Albino strain zebrafish was randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). The samples were dissolved in water respectively (see Table 17 for concentration), and a normal control group was set at the same time, and the volume of each well was 3 mL. After treatment at 28°C for 48 hours, the MTC of the samples against normal zebrafish was measured.
b.降血脂功效评价b. Efficacy evaluation of blood lipid lowering
随机选取5dpf黑色素等位基因突变型半透明Albino品系斑马鱼，水溶给予蛋黄粉建立斑马鱼高血脂模型。喂饲16h后将斑马鱼随机分配至6孔板中，每孔均处理30尾斑马鱼。分别水溶给予样品(浓度见表17-18)，阳性对照阿托伐他汀钙0.240μg/mL浓度，同时设置正常对照组和模型对照组，每孔容量为3mL。28℃处理48h后，将斑马鱼匀浆取上清，用甘油三酯和总胆固醇试剂盒进行反应，应用多功能酶标仪分别测定各实验组甘油三酯和总胆固醇OD值，分析甘油三酯含量(C1)和总胆固醇含量(C2)，以上述指标的统计学分析结果评价样品降血脂功效。统计学处理结果采用mean±SE表示。降血脂功效计算公式如下：The zebrafish of the 5dpf melanin allele mutant translucent Albino strain were randomly selected, and given egg yolk powder in water to establish a hyperlipidemia model of the zebrafish. After 16 hours of feeding, the zebrafish were randomly assigned to 6-well plates, and 30 zebrafish were treated in each well. The samples were dissolved in water (see Table 17-18 for concentration), and the positive control was atorvastatin calcium at a concentration of 0.240 μg/mL. A normal control group and a model control group were set at the same time, and the volume of each well was 3 mL. After treatment at 28°C for 48 hours, the zebrafish was homogenized and the supernatant was taken, and reacted with a triglyceride and total cholesterol kit, and the OD value of triglyceride and total cholesterol in each experimental group was measured by a multifunctional microplate reader, and the triglyceride was analyzed. Ester content (C1) and total cholesterol content (C2), the blood lipid-lowering efficacy of the sample was evaluated by the statistical analysis results of the above indicators. Statistical results are expressed as mean±SE. The formula for calculating the lipid-lowering effect is as follows:
1)降甘油三酯功效：1) Efficacy of lowering triglycerides:
Figure PCTCN2022117637-appb-000013
Figure PCTCN2022117637-appb-000013
2)降总胆固醇功效：2) Efficacy of lowering total cholesterol:
Figure PCTCN2022117637-appb-000014
Figure PCTCN2022117637-appb-000014
用SPSS 26.0软件进行统计学分析，p<0.05表明差异具有统计学意义。Statistical analysis was performed with SPSS 26.0 software, and p<0.05 indicated that the difference was statistically significant.
检测结果Test results
在本实验条件下，发酵虫草菌粉、Cs-4-P1-2对正常斑马鱼的MTC(最小中毒浓度)分别为300和10.0μg/mL。详见表17。Under the conditions of this experiment, the MTC (minimum toxic concentration) of fermented Cordyceps powder and Cs-4-P1-2 to normal zebrafish were 300 and 10.0 μg/mL, respectively. See Table 17 for details.
表17Table 17
Figure PCTCN2022117637-appb-000015
Figure PCTCN2022117637-appb-000015
降血脂功效评价Evaluation of blood lipid lowering efficacy
在本实验条件下，发酵虫草菌粉、Cs-4-P1-2具有降甘油三酯功效；Cs-4-P1-2具有降总胆固醇功效，详见表18。Under the conditions of this experiment, the fermented Cordyceps powder and Cs-4-P1-2 have the effect of lowering triglyceride; Cs-4-P1-2 has the effect of lowering total cholesterol, see Table 18 for details.
表18Table 18
Figure PCTCN2022117637-appb-000016
Figure PCTCN2022117637-appb-000016
与模型对照组比较，*p<0.05，**p<0.01，***p<0.001Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001
表19Table 19
Figure PCTCN2022117637-appb-000017
Figure PCTCN2022117637-appb-000017
与模型对照组比较，*p<0.05，**p<0.01，***p<0.001Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001
由上表19可知，Cs-4-P1-2浓度在1.0-10.0μg/mL时，其具有较优降甘油三酯功效，且在3.00μg/mL时，降甘油三酯功效最优，降甘油三脂活性优于阿托伐他汀钙。It can be seen from the above table 19 that when the concentration of Cs-4-P1-2 is 1.0-10.0 μg/mL, it has a better triglyceride-lowering effect, and when it is 3.00 μg/mL, the triglyceride-lowering effect is the best. Triglyceride activity is superior to atorvastatin calcium.
表20Table 20
Figure PCTCN2022117637-appb-000018
Figure PCTCN2022117637-appb-000018
与模型对照组比较，*p<0.05Compared with the model control group, *p<0.05
表21Table 21
Figure PCTCN2022117637-appb-000019
Figure PCTCN2022117637-appb-000019
与模型对照组比较，*p<0.05，**p<0.01Compared with the model control group, *p<0.05, **p<0.01
由上表21可知，Cs-4-P1-2浓度在1.0-10.0μg/mL时，其具有较优总胆固醇功效，且在1.00-3.00μg/mL时，降总胆固醇功效最优，显著优于发酵虫草菌粉和阿托伐他汀钙。It can be seen from the above table 21 that when the concentration of Cs-4-P1-2 is 1.0-10.0 μg/mL, it has better total cholesterol effect, and when it is 1.00-3.00 μg/mL, the effect of lowering total cholesterol is the best, significantly superior For fermented Cordyceps powder and atorvastatin calcium.
实施例7对顺铂所致急性肾损伤小鼠作用研究Example 7 Study on the Action of Acute Kidney Injury Mice Caused by Cisplatin
试验方法：experiment method:
小鼠术前禁食12h，自由饮水。除对照组外，其余各组腹腔单次注射剂量为10mg/kg的顺铂溶液，对照组注射等体积的生理盐水。15min后分别按分组给予氨磷汀，Cs-4-P1和Cs-4-P1-2。分别于给药后第1，3和5天采血，测血清中尿素氮(BUN)和肌酐(Scr)。Mice were fasted for 12 h before operation and had free access to water. Except for the control group, the other groups received a single intraperitoneal injection of cisplatin solution at a dose of 10 mg/kg, and the control group was injected with an equal volume of normal saline. After 15 minutes, amifostine, Cs-4-P1 and Cs-4-P1-2 were administered in groups. Blood was collected on the 1st, 3rd and 5th days after the administration, and blood urea nitrogen (BUN) and creatinine (Scr) in serum were measured.
试验结果：结果如表22所示，对照组血清尿素氮和肌酐第1，3和5天均趋于平稳，模型组血清尿素氮和肌酐于第3开始升高并在第5天保持高水平，与对照组比较，模型组第3和5天的血清尿素氮和肌酐均表现出统计学差异(p<0.05，p<0.01)。阳性药氨磷汀300mg/kg组给药后第3和5天，小鼠血清尿素氮和肌酐含量明显下降，与模型组比较，表现出统计学差异(p<0.05，p<0.01)；给予受试物Cs-4-P1(160mg/kg)第3和5天，小鼠血清尿素氮和肌 酐含量明显下降，与模型组比较，表现出统计学差异(p<0.05，p<0.01)；分别给予Cs-4-P1-2(160mg/kg和80mg/kg)后，第3天和5天小鼠血清尿素氮和肌酐含量明显下降，与模型组比较均表现出统计学差异(p<0.05，p<0.01)。可见，Cs-4-P1-2在160mg/kg剂量下对血清尿素氮和肌酐的改善作用与阳性药氨磷汀(300mg/kg)一致。可见Cs-4-P1-2对小鼠急性肾损伤有治疗和改善作用。Test results: The results are shown in Table 22. The serum urea nitrogen and creatinine in the control group tended to be stable on the 1st, 3rd and 5th day, and the serum urea nitrogen and creatinine in the model group began to increase on the 3rd day and remained at a high level on the 5th day , Compared with the control group, the serum urea nitrogen and creatinine of the model group on the 3rd and 5th days showed statistical differences (p<0.05, p<0.01). On the 3rd and 5th days after administration of the positive drug amifostine 300mg/kg group, the contents of serum urea nitrogen and creatinine in the mice decreased significantly, and compared with the model group, there was a statistical difference (p<0.05, p<0.01); On the 3rd and 5th day of the test substance Cs-4-P1 (160mg/kg), the serum urea nitrogen and creatinine levels of the mice decreased significantly, and compared with the model group, there was a statistical difference (p<0.05, p<0.01); After administration of Cs-4-P1-2 (160mg/kg and 80mg/kg) respectively, the serum urea nitrogen and creatinine levels of the mice decreased significantly on the 3rd and 5th day, showing statistical difference compared with the model group (p< 0.05, p<0.01). It can be seen that the improvement effect of Cs-4-P1-2 on serum urea nitrogen and creatinine at a dose of 160 mg/kg is consistent with that of the positive drug amifostine (300 mg/kg). It can be seen that Cs-4-P1-2 can treat and improve acute kidney injury in mice.
表22Table 22
Figure PCTCN2022117637-appb-000020
Figure PCTCN2022117637-appb-000020
与对照组比较， ##p<0.01；与模型组比较，*p<0.05,**p<0.01 Compared with the control group, ## p<0.01; compared with the model group, *p<0.05,**p<0.01
实施例8对LPS所致急性肾损伤小鼠作用研究Example 8 Study on Effects on LPS-induced Acute Kidney Injury Mice
试验方法：experiment method:
小鼠术前禁食12h，自由饮水。除对照组外，其余各组腹腔单次注射剂量为20mg/kg的LPS溶液，对照组注射等体积的生理盐水。15min后分别按分组给予地塞米松和Cs-4-P1-2。36小时后处死动物，取血测血清肌酐和尿素氮。Mice were fasted for 12 h before operation and had free access to water. Except for the control group, the other groups received a single intraperitoneal injection of 20 mg/kg of LPS solution, and the control group was injected with an equal volume of normal saline. After 15 minutes, dexamethasone and Cs-4-P1-2 were administered in groups respectively. After 36 hours, the animals were sacrificed, and blood was collected to measure serum creatinine and urea nitrogen.
试验结果：test results:
结果如表23所示，与对照比较，模型组小鼠的血清尿素氮和肌酐水平分别为17.69±1.13mmol/L和45.35±5.85μmol/L，并表现出统计学差异(p<0.01)。分别给予10mg/kg剂量的阳性药地塞米松，阳性药组小鼠的血清尿素氮和肌酐水平分别为12.29±4.42mmol/L和24.03±13.81μmol/L，与模型组比较均表现出显著性差异(p<0.05)；Cs-4-P1-2低、中、高剂量组小鼠的血清尿素氮水平分别下降至14.41±1.73、13.20±1.72和12.72±0.55mmol/L；血清肌酐水平分别下降至35.15±26.22、26.34±2.75、24.33±9.65μmol/L，与模型组比较，Cs-4-P1-2中和高剂量组小鼠血清尿素氮和肌酐均表现出统计学差异(p<0.05，p<0.01，p<0.001)。可见，Cs-4-P1-2高剂量组对LPS诱导的急性肾损伤小鼠血清尿素氮和肌酐的影响与阳性药地塞米松效果相当。可见Cs-4-P1-2对小鼠急性肾损伤有治疗和改善作用。The results are shown in Table 23. Compared with the control group, the serum urea nitrogen and creatinine levels of the mice in the model group were 17.69±1.13mmol/L and 45.35±5.85μmol/L, respectively, showing a statistical difference (p<0.01). The positive drug dexamethasone was administered at a dose of 10mg/kg, and the serum urea nitrogen and creatinine levels of the mice in the positive drug group were 12.29±4.42mmol/L and 24.03±13.81μmol/L, respectively, which showed significant differences compared with the model group difference (p<0.05); the serum urea nitrogen levels of the mice in the Cs-4-P1-2 low, medium and high dose groups decreased to 14.41±1.73, 13.20±1.72 and 12.72±0.55mmol/L respectively; the serum creatinine levels were respectively decreased to 35.15±26.22, 26.34±2.75, 24.33±9.65μmol/L, and compared with the model group, the serum urea nitrogen and creatinine of the mice in the Cs-4-P1-2 middle and high dose groups showed statistical differences (p< 0.05, p<0.01, p<0.001). It can be seen that the effect of high-dose Cs-4-P1-2 group on serum urea nitrogen and creatinine in mice with acute kidney injury induced by LPS is equivalent to that of the positive drug dexamethasone. It can be seen that Cs-4-P1-2 can treat and improve acute kidney injury in mice.
表23Table 23
Figure PCTCN2022117637-appb-000021
Figure PCTCN2022117637-appb-000021
图8是根据本发明实施例的 1H- 13C HSQC图谱。图11是根据本发明实施例的病理切片图。 Fig. 8 is a 1 H- 13 C HSQC chart according to an embodiment of the present invention. Fig. 11 is a diagram of a pathological slice according to an embodiment of the present invention.

Claims (11)

  1. 一种Cs-4发酵菌丝体杂聚多糖，其特征在于，结构式如下所示：A Cs-4 fermentation mycelia heteropolysaccharide is characterized in that the structural formula is as follows:
    Figure PCTCN2022117637-appb-100001
    Figure PCTCN2022117637-appb-100001
    其中，Manp为吡喃型甘露糖、Galp为吡喃型半乳糖、Glcp为吡喃型葡萄糖，n选自6-104。Wherein, Manp is mannose pyranose, Galp is galactopyranose, Glcp is glucopyranose, and n is selected from 6-104.
  2. 根据权利要求1所述的Cs-4发酵菌丝体杂聚多糖，其特征在于，所述多糖的相对分子量选自-10-152kDa。The Cs-4 fermented mycelia heteropolysaccharide according to claim 1, characterized in that the relative molecular weight of the polysaccharide is selected from -10-152kDa.
  3. 根据权利要求1或2所述的Cs-4发酵菌丝体杂聚多糖，其特征在于，所述多糖组成包括：葡萄糖、半乳糖和甘露糖，所述葡萄糖、半乳糖和甘露糖的摩尔比为1：(1.0-2.0)：(1.5-2.5)。Cs-4 fermentation mycelia heteropolysaccharide according to claim 1 or 2, is characterized in that, described polysaccharide composition comprises: glucose, galactose and mannose, the molar ratio of described glucose, galactose and mannose is 1:(1.0-2.0):(1.5-2.5).
  4. 一种提取权利要求1-3任一项所述Cs-4发酵菌丝体杂聚多糖的方法，其特征在于，A method for extracting heteropolysaccharides from Cs-4 fermented mycelium described in any one of claims 1-3, characterized in that,
    1)将Cs-4菌粉用乙醇进行脱脂处理，舍去乙醇提液，获得脱脂残渣，1) Degreasing the Cs-4 bacterial powder with ethanol, discarding the ethanol extract to obtain a degreasing residue,
    2)将所述残渣在水中进行提取处理，获得水提液，2) extracting the residue in water to obtain a water extract,
    3)将所述水提液进行醇沉处理，所述醇沉处理的溶剂为乙醇，3) subjecting the water extract to alcohol precipitation treatment, the solvent of the alcohol precipitation treatment is ethanol,
    4)将步骤3)中醇沉处理后的沉淀进行纯化处理，获得发酵菌丝体杂聚多糖。4) Purifying the precipitate after the alcohol precipitation treatment in step 3) to obtain the fermented mycelia heteropolysaccharide.
  5. 根据权利要求4所述的方法，其特征在于，所述脱脂处理是在温度为100℃的条件下进行的；The method according to claim 4, wherein the degreasing treatment is carried out at a temperature of 100°C;
    任选地，所述脱脂处理进行三次，每次时间为1小时；Optionally, the degreasing treatment is carried out three times, each time being 1 hour;
    任选地，所述脱脂处理中乙醇是Cs-4的10倍量；Optionally, ethanol is 10 times the amount of Cs-4 in the degreasing treatment;
    任选地，所述脱脂处理的乙醇为85％-100％乙醇；Optionally, the degreasing ethanol is 85%-100% ethanol;
    任选地，所述提取处理是在温度为75℃-78℃的条件下进行的；Optionally, the extraction treatment is carried out at a temperature of 75°C-78°C;
    任选地，所述提取处理进行三次，每次时间为1小时；Optionally, the extraction treatment is carried out three times, each time being 1 hour;
    任选地，所述提取处理中水是残渣的10倍量；Optionally, the amount of water in the extraction process is 10 times that of the residue;
    任选地，所述醇沉处理的乙醇为80％乙醇。Optionally, the alcohol precipitation treatment is 80% ethanol.
  6. 一种提取权利要求1-3任一项所述Cs-4发酵菌丝体杂聚多糖的方法，其特征在于，A method for extracting heteropolysaccharides from Cs-4 fermented mycelium described in any one of claims 1-3, characterized in that,
    1)将Cs-4在10倍量的85％乙醇加热提取3次，每次1小时，舍去乙醇提液，获得残 渣；1) Heat and extract Cs-4 in 10 times the amount of 85% ethanol for 3 times, each time for 1 hour, and discard the ethanol extract to obtain a residue;
    2)将所述残渣在10倍量水加热提取3次，每次1小时，合并水提液；2) Heat and extract the residue in 10 times the amount of water for 3 times, each time for 1 hour, and combine the water extracts;
    3)将所述水提液经80％乙醇醇沉，获得沉淀；3) Precipitating the water extract with 80% ethanol to obtain a precipitate;
    4)将所述沉淀进行纯化处理，获得发酵菌丝体杂聚多糖。4) Purifying the precipitate to obtain fermented mycelia heteropolysaccharides.
  7. 根据权利要求4-6任一项所述的提取方法，其特征在于，所述纯化处理包括去蛋白处理、脱色处理、柱纯化处理。The extraction method according to any one of claims 4-6, characterized in that the purification treatment includes protein removal treatment, decolorization treatment, and column purification treatment.
  8. 一种药物组合物，其特征在于，包含权利要求1-3任一项所述的Cs-4发酵菌丝体杂聚多糖或根据权利要求4-7任一项所述的方法获得的Cs-4发酵菌丝体杂聚多糖。A pharmaceutical composition, characterized in that it comprises the Cs-4 fermented mycelia heteropolysaccharide according to any one of claims 1-3 or the Cs-4 obtained by the method according to any one of claims 4-7. 4 Fermentation of mycelia heteropolysaccharides.
  9. 权利要求1-3任一项所述的Cs-4发酵菌丝体杂聚多糖或根据权利要求4-7任一项所述的方法获得的Cs-4发酵菌丝体杂聚多糖或权利要求8所述的药物组合物在制备药物中的用途，所述药物用于预防或治疗慢性肾衰、抑制免疫排斥反应、预防或治疗高血脂和/或预防或治疗急性肾损伤。The Cs-4 fermented mycelia heteropolysaccharide described in any one of claims 1-3 or the Cs-4 fermented mycelia heteropolysaccharide obtained according to the method described in any one of claims 4-7 or claims 8. Use of the pharmaceutical composition in the preparation of medicaments for preventing or treating chronic renal failure, suppressing immune rejection, preventing or treating hyperlipidemia and/or preventing or treating acute kidney injury.
  10. 一种预防或治疗高血脂的药物组合物，其特征在于，包含1.0-10.0μg/mL权利要求1-3任一项所述的Cs-4发酵菌丝体杂聚多糖或根据权利要求4-7任一项所述的方法获得的Cs-4发酵菌丝体杂聚多糖。A pharmaceutical composition for preventing or treating hyperlipidemia, characterized in that it comprises 1.0-10.0 μg/mL of the Cs-4 fermented mycelia heteropolysaccharide according to any one of claims 1-3 or according to claim 4- 7. The Cs-4 fermented mycelia heteropolysaccharide obtained by any one of the methods.
  11. 根据权利要求10所述的药物组合物，其特征在于，所述多糖的浓度为3.0μg/mL。The pharmaceutical composition according to claim 10, characterized in that the concentration of the polysaccharide is 3.0 μg/mL.
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