CN117500842A - Cs-4 fermentation mycelium heteropolysaccharide and preparation method and application thereof - Google Patents
Cs-4 fermentation mycelium heteropolysaccharide and preparation method and application thereof Download PDFInfo
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- CN117500842A CN117500842A CN202280043095.8A CN202280043095A CN117500842A CN 117500842 A CN117500842 A CN 117500842A CN 202280043095 A CN202280043095 A CN 202280043095A CN 117500842 A CN117500842 A CN 117500842A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
A Cs-4 fermented mycelium heteropolysaccharide with the structure shown in 6-104 and its preparing process and application are disclosed.
Description
The present application claims priority from China patent application 2021110438722 with application date 2021/9/7. The present application refers to the entirety of the above-mentioned chinese patent application.
The invention belongs to the field of traditional Chinese medicine pharmacy, and in particular relates to a Cs-4 fermentation mycelium heteropolysaccharide prepared by taking paecilomyces hepiali (Cs-4) mycelium as a raw material, and a preparation method and application thereof.
Cordyceps sinensis is Cordyceps sinensis Cordyceps sinensis (Berk.) Sacc, a complex of stroma and larva corpse parasitic on larva of insect of hepialaceae, and is a rare Chinese medicinal material (hereinafter called Cordyceps sinensis). Because the natural cordyceps sinensis has special growth environment and higher price, the requirements of the market cannot be met. Therefore, people utilize modern biological fermentation technology to cultivate cordyceps mycelia to replace natural cordyceps sinensis. More than ten strains are separated from Cordyceps samples collected from Tibetan of Yunnan Diqing in 1982, and 4 strains are cultured and morphological researched, and identified as Paecilomyces hepiali (Paecilonyces hepialid chen & Dai). The Cs-4 strain is separated from fresh Cordyceps sinensis collected in Qinghai Hualong county in 1982, and identified as Paecilomyces hepiali. The fermented Cordyceps powder (Cs-4), hereinafter called Cs-4, is the dry powder of mycelium obtained by fermenting isolated Paecilomyces hepiali Cs-4 strain, and is used as raw material to make into medicine for successful marketing, and the medicine names are Jinshuibao capsule and Jinshuibao tablet.
The Jinshuibao is used as a representative variety of artificial cordyceps, has the effects of tonifying lung and kidney and replenishing vital essence and qi, and is widely applied to the adjuvant therapy of kidney system diseases, respiratory system diseases, metabolic diseases, cardiovascular and cerebrovascular system diseases, endocrine system diseases and tumors clinically, but the drug effect material basis is not clear.
Cordyceps polysaccharide is used as one of main active ingredients in cordyceps and is concerned by a plurality of researchers at home and abroad, but the current research is focused on the active screening of cordyceps crude polysaccharide, the polysaccharide structure research is still in a primary stage, and only monosaccharide composition and molecular weight information are covered, or the polysaccharide is simply classified and not completely purified, so that the chemical structure of the cordyceps polysaccharide cannot be deeply excavated, and the quality control and subsequent clinical transformation of the active cordyceps polysaccharide are severely limited.
Disclosure of Invention
In the prior art, researches on Cs-4 fermentation mycelium polysaccharide are few, the purity of the extracted Cs-4 fermentation mycelium polysaccharide in the prior art is low, and researches on the fine structure of the polysaccharide are few, so that the invention carries out intensive researches on the Cs-4 fermentation mycelium polysaccharide, the purity of the extracted polysaccharide is high, specific monosaccharide composition, relative molecular mass, glycosidic bond connection mode and the like of the polysaccharide are further researched, and a comparison test is carried out on animal models with the fermented cordyceps sinensis powder, so that the polysaccharide of the invention can be compared with the fermented cordyceps sinensis powder, and a better effect is achieved at a lower dosage.
In a first aspect of the present invention, the present invention provides a Cs-4 fermentation mycelium heteropolysaccharide. According to an embodiment of the present invention, the structural formula is as follows:
wherein Manp is mannopyranose, galp is galactopyranose, glcp is glucopyranose, and n is selected from 6-104. The inventor firstly extracts and identifies the polysaccharide, and 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, and obtains better effects at lower dosage compared with fermented cordyceps fungus powder.
According to an embodiment of the present invention, the above polysaccharide may further include one of the following additional technical features:
according to an embodiment of the invention, manp is mannose pyran, galp is galactose pyran, glcp is glucose pyran, and n is selected from 6-68. According to an embodiment of the invention, n is selected from 15-21.
According to an embodiment of the invention, n is selected from 15-104.
According to an embodiment of the invention, n is selected from 21-104.
According to an embodiment of the invention, n is selected from 6-15.
According to an embodiment of the invention, n is selected from 6-21.
According to an embodiment of the invention, n is selected from 15-68.
According to an embodiment of the invention, n is selected from 21-68.
According to an embodiment of the invention, n is selected from any integer between 6 and 104.
According to an embodiment of the invention, n is selected from any integer between 6 and 68.
According to an embodiment of the invention, n is selected from 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 invention, the relative molecular weight of the polysaccharide is selected from 10-152kDa. The relative molecular weight used in the present invention means a weight average molecular weight, which means a weight average value, that is, a weight-weighted average of molecular weight to molecular weight.
According to an embodiment of the invention, the relative molecular weight of the polysaccharide is selected from 10-100kDa.
According to an embodiment of the invention, the relative molecular weight of the polysaccharide is selected from 10-23.25kDa.
According to an embodiment of the invention, the relative molecular weight of the polysaccharide is selected from 10-30.12kDa.
According to an embodiment of the invention, the relative molecular weight of the polysaccharide is selected from 10-151.15kDa.
According to an embodiment of the invention, the relative molecular weight of the polysaccharide is selected from 23.25-30.12kDa.
According to an embodiment of the invention, the relative molecular weight of the polysaccharide is selected from 23.25-100kDa.
According to an embodiment of the invention, the relative molecular weight of the polysaccharide is selected from 30.12-100kDa.
According to an embodiment of the invention, the relative molecular weight of the polysaccharide is selected from 23.25-151.15kDa.
According to an embodiment of the invention, the relative molecular weight of the polysaccharide is selected from 30.12-151.15kDa.
According to an embodiment of the invention, the relative molecular weight of the polysaccharide is selected from 23.15kDa or 30.12kDa or 151.15kDa.
According to an embodiment of the invention, the polysaccharide composition comprises: glucose, galactose and mannose in a molar ratio of 1: (1.0-2.0): (1.5-2.5).
According to an embodiment of the invention, the polysaccharide composition comprises: glucose, galactose and mannose in a molar ratio of 1: (1.5-1.8): (2.0-2.5).
According to an embodiment of the invention, the polysaccharide composition comprises: glucose, galactose and mannose in a molar ratio of 1: (1.71-1.74): (2.09-2.44).
In a second aspect of the invention, the invention provides a method of extracting the Cs-4 fermentation mycelium heteropolysaccharide described above. According to an embodiment of the invention, the method comprises: degreasing Cs-4 bacterial powder with ethanol, discarding ethanol extract to obtain degreasing residues, extracting the residues in water to obtain water extract, 3) performing ethanol precipitation on the water extract, wherein the solvent of the ethanol precipitation is ethanol, and 4) purifying the precipitate obtained after the ethanol precipitation in the step 3) to obtain the fermentation mycelium heteropolysaccharide. The method provided by the embodiment of the invention is simple to operate, the extraction efficiency is high, and the purity of the extracted polysaccharide is high and can reach more than 94%.
According to an embodiment of the present invention, the method may further comprise at least one of the following additional technical features:
according to an embodiment of the present invention, the degreasing treatment is performed at a temperature of 100 ℃.
According to an embodiment of the invention, the degreasing treatment is performed three times, each time for 1 hour.
According to an embodiment of the invention, the ethanol in the degreasing treatment is 10 times the amount of Cs-4.
According to the embodiment of the invention, the ethanol subjected to degreasing is 85% -100% ethanol.
According to an embodiment of the present invention, the extraction treatment is performed at a temperature of 75-78 ℃.
According to an embodiment of the invention, the extraction process is performed three times, each time for 1 hour.
According to an embodiment of the invention, the extraction treatment is performed with water in an amount of 10 times the amount of the residue.
According to an embodiment of the invention, the ethanol treated by the alcohol precipitation is 80% ethanol.
In yet another aspect of the present invention, a method of extracting the aforementioned Cs-4 fermentation mycelium heteropolysaccharide is provided. According to an embodiment of the invention, the method comprises: 1) Extracting Cs-4 with 10 times of 85% ethanol under heating for 3 times each for 1 hr, and removing ethanol extractive solution to obtain residue; 2) Extracting the residue with 10 times of water under heating for 3 times each for 1 hr, and mixing the water extracts; 3) Precipitating the water extract with 80% ethanol to obtain precipitate Cs-4-P; 4) Purifying the precipitate Cs-4-P to obtain fermented mycelium heteropolysaccharide Cs-4-P1, and continuing purifying to obtain 3 homogeneous polysaccharides named Cs-4-P1-1, cs-4-P1-2 and Cs-4-P1-3.
According to an embodiment of the present invention, the method may further comprise at least one of the following additional technical features:
according to an embodiment of the present invention, the purification treatment includes deproteinization treatment, decolorization treatment, column purification treatment. The deproteinizing treatment, decolorizing treatment, and column purifying treatment may be conventional treatment methods, and an appropriate operation method may be selected according to specific needs.
In yet another aspect of the invention, the invention also provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the Cs-4 fermented mycelium heteropolysaccharide described above or Cs-4 fermented mycelium heteropolysaccharide Cs-4-P1 obtained according to the method described above, and further purifying the resulting 3 homogeneous polysaccharides (Cs-4-P1-1, cs-4-P1-2, cs-4-P1-3).
In a further aspect of the invention, the invention also provides the use of the Cs-4 fermented mycelium heteropolysaccharide as described above or the Cs-4 fermented mycelium heteropolysaccharide obtained according to the method as described above or the pharmaceutical composition as described above for the preparation of a medicament for the prevention or treatment of chronic renal failure, for the inhibition of immune rejection, for the prevention or treatment of hyperlipidemia, for the prevention or treatment of acute renal injury. The inventor finds 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, and obtains better effects at lower dosage compared with Jinshuibao.
In one embodiment of the 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 Cs-4 fermented mycelium heteropolysaccharide described above or the Cs-4 fermented mycelium heteropolysaccharide obtained according to the method described above. The inventor finds that the polysaccharide has better effect of preventing or treating acute kidney injury, and the effect is equivalent to that of positive drugs amifostine and dexamethasone.
In yet another aspect of the present invention, the present invention also provides 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 above-described Cs-4 fermented mycelium heteropolysaccharide or the Cs-4 fermented mycelium heteropolysaccharide obtained according to the method described above. The inventor finds that the polysaccharide has better effect of preventing or treating hyperlipidemia, and better effect is obtained at lower dosage compared with Jinshuibao.
According to an embodiment of the present invention, the above pharmaceutical composition may further include at least one of the following additional technical features:
according to an embodiment of the invention, the polysaccharide concentration is 3.0. Mu.g/mL. The inventor finds that the effect of preventing or treating hyperlipidemia is better at the concentration of polysaccharide of 3.0 mug/mL, and the effect is obviously higher than that of Jinshuibao and the classical old medicine atorvastatin calcium.
In another aspect of the present invention, the present invention provides at least one of the following technical effects:
1) The invention extracts and identifies polysaccharide with repeated structural units (Cs-4-P1-1, cs-4-P1-2 and Cs-4-P1-3 for short) for the first time;
2) The extraction method is simple to operate;
3) The purity of the heteropolysaccharide of the invention is high and can reach more than 94 percent;
4) The heteropolysaccharide of the invention has better effect than the fermented Cordyceps powder Cs-4 (1600 mg/kg/day) and the okra capsule (1000 mg/kg/day) at low dose (16 mg/kg/day) when preventing or treating chronic renal failure, and has equivalent or better effect than the clinical classical dexamethasone, and the invention aims to provide a novel alternative medicament for preventing or treating chronic renal failure;
5) The heteropolysaccharide of the invention has better effect than the fermented cordyceps fungus powder Cs-4 in suppressing immune rejection reaction, has equal or better effect and better safety compared with clinical classical old medicine cyclosporin, and aims to provide a novel alternative medicine for suppressing immune rejection reaction;
6) The heteropolysaccharide of the invention can reduce triglyceride and total cholesterol at low dosage (1.0-10.0 mug/mL) when preventing or treating hyperlipidemia, has better effect than Jinshuibao, and has equivalent or better effect than classical old medicine atorvastatin calcium.
In the context of the present invention, the words "about" or "about" when used or whether or not are used mean within 10%, suitably within 5%, in particular within 1% of a given value or range. Alternatively, the term "about" or "approximately" means within an acceptable standard error of the average value to one of ordinary skill in the art. Whenever a number is disclosed having a value of N, any number within the values of N+/-1%, N+/-2%, N+/-3%, N+/-5%, N+/-7%, N+/-8% or N+/-10% will be explicitly disclosed, where "+/-" means plus or minus.
FIG. 1 is a HPGPC chart according to an embodiment of the invention.
FIG. 2 is an infrared spectrum of a sample after methylation according to an embodiment of the present invention.
FIG. 3 is a graph of the total ion flux of a Cs-4-P1-2 derivative according to an embodiment of the invention.
FIG. 4 is a diagram of Cs-4-P1-2 according to an embodiment of the invention 1 H NMR spectrum.
FIG. 5 is a diagram of Cs-4-P1-2 according to an embodiment of the invention 13 C NMR spectrum.
FIG. 6 is a diagram of Cs-4-P1-2 according to an embodiment of the invention 1 H- 1 H COSY pattern.
FIG. 7 is a diagram of Cs-4-P1-2 according to an embodiment of the invention 1 H- 1 H TOCSY profile.
FIG. 8 is a diagram of an embodiment according to the invention 1 H- 13 C HSQC spectrum.
FIG. 9 is a diagram of an embodiment according to the invention 1 H- 13 C HMBC profile.
FIG. 10 is a diagram of an embodiment according to the invention 1 H- 1 H NOESY profile.
Fig. 11 is a view of a pathological section according to an embodiment of the present invention.
The present application is described in detail below by way of examples, but is not meant to be limiting in any way. The present application has been described in detail herein, and specific embodiments thereof are also disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present application without departing from the spirit and scope of the application.
The raw materials used in the present invention are commercially available unless otherwise specified.
Dexamethasone acetate tablet (lot number: 015200406, shanghai pharmaceutical Co., ltd.), okra (lot number: 20110210, jiangsu Su pharmaceutical Co., ltd.), fermented Cordyceps powder (lot number: 190800810-1, jiangxi national pharmaceutical Co., ltd.), new Siping cyclosporin soft capsule (lot number: 200825, hangzhong Meihua east pharmaceutical Co., ltd.).
The abbreviations used in the present invention are as follows:
glcp represents glucopyranose, galp represents galactopyranose, mann represents mannopyranose, h represents hours, min represents minutes, W represents weeks, rpm represents the number of revolutions per minute of the device, DEAE represents diethylaminoethyl cellulose, seph represents dextran gel, HPGPC represents high performance gel chromatography, mw represents weight average molecular weight, mp represents peak molecular weight, t R Representing retention time, BUN represents blood for urea nitrogen detection, CREA represents creatinine, UA represents uric acid, csA represents cyclosporin, MTC represents minimal toxic concentration, and LPS represents lipopolysaccharide.
Example 1 Cs-4-P, cs-4-P1, cs-4-P1-2, cs-4-P1-3
1. Degreasing: the fermented cordyceps sinensis powder Cs-4 is heated to boiling (78 ℃) by 10 times of 85% ethanol, and is subjected to micro-boiling extraction for 3 times, each time for 1 hour, and the ethanol extract is removed.
2. Water extraction and alcohol precipitation: the residue was heated to boiling (100 ℃ C.) with 10 times of water and subjected to micro-boiling extraction for 3 times each for 1 hour. Mixing the water extracts, concentrating under reduced pressure at 70 ℃ to obtain fluid extract (density 1.05-1.07). Adding 95% ethanol to the fluid extract until ethanol content is 85%, stirring for 30min, standing for 24 hr, vacuum filtering/concentrating supernatant, collecting precipitate, drying under reduced pressure at 65deg.C, and pulverizing to obtain crude polysaccharide Cs-4-P (yield 21%).
3. Deproteinization and decoloration: extracting the crude polysaccharide aqueous solution with 2% water, shaking for 10min, 4000rpm and 10min with equal amount of Sevage reagent, decolorizing repeatedly for 3-4 times until no obvious white layer appears, and mixing the upper solutions.
4. Decoloring: adding 2% active carbon powder into the solution of the removal unit Bai Duotang, preserving the temperature at 50 ℃ for 30min, carrying out suction filtration on double-layer filter paper twice, passing through a 0.45 micrometer membrane, concentrating until no organic reagent smell exists, and preparing for the next purification.
5. DEAE-52 column chromatography: eluting with pure water to obtain neutral polysaccharide Cs-4-P1;
6. seph G-100 column chromatography separation and purification: eluting with pure water to obtain three homogeneous polysaccharides named Cs-4-P1-1, cs-4-P1-2 and Cs-4-P1-3, wherein the yield of Cs-4-P1-1 is low.
Example 2 Cs-4-P1-1, cs-4-P1-2, cs-4-P1-3 content, molecular weight and monosaccharide composition
Polysaccharide content determination:
reference is made to a method for measuring polysaccharide in SN/T4260-2015 of phenol-sulfuric acid method for measuring crude polysaccharide in export plant source food. The method utilizes polysaccharide to hydrolyze into monosaccharide under the action of concentrated acid, forms a furfural derivative through dehydration condensation, combines with phenol for color development, and calculates the total sugar content by measuring the absorbance value after color development. The method is adopted to determine the polysaccharide content, and a monosaccharide is selected as a reference substance to draw a standard curve.
Drawing a standard curve: weighing 5.417mg of glucose, precisely weighing, placing in a 50mL volumetric flask, adding water for dissolution and fixing the volume to obtain a glucose standard solution of 0.1 mg/mL. Respectively sucking the standard solutions 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL into a 20mL test tube with a plug, supplementing the test tube with distilled water to 1.0mL, adding 1.0mL of the prepared 5% phenol solution and 5.0mL of concentrated sulfuric acid, shaking uniformly, standing at room temperature for 10min, fully mixing the reaction solution by using a vortex oscillator, then placing the test tube into a water bath at 30 ℃ for reaction for 20min, and measuring absorbance at 490 nm. And (3) carrying out linear regression by taking the glucose concentration as an abscissa and the absorbance value as an ordinate to obtain a standard curve equation.
And (3) detecting a test article: weighing Cs-4-P1 mg, precisely weighing, placing in a 10mL measuring flask, ultrasonically dissolving, adding water to a certain volume to scale, and shaking to obtain the final product. Taking 0.2mL of each prepared solution into a 20mL test tube with a plug, and adding water to make up to 1.0mL. Adding distilled water to 1.0mL, adding 1.0mL of the existing 5% phenol solution and 5.0mL of concentrated sulfuric acid, shaking, standing at room temperature for 10min, fully mixing the reaction solution by using a vortex oscillator, then placing a test tube in a water bath at 30 ℃ for reaction for 20min, and measuring absorbance at 490 nm.
The polysaccharide content is measured, and the polysaccharide contents of Cs-4-P1-1, cs-4-P1-2 and Cs-4-P1-3 are as follows: 94.61%, 98.61% and 99.52%.
2. Determination of molecular weight
High performance gel chromatography (HPGPC) can be used to determine the relative molecular mass of macromolecular substances and their distribution, the preferred method of polysaccharide relative molecular weight. The principle is based on polysaccharide of different relative molecular masses on gel column and elution retention time (t R ) The characteristic of a certain relation is that standard curves are firstly prepared by standard samples with known relative molecular mass, and then the standard curves are prepared by t of the samples R The relative molecular mass was determined from the curve. The peak molecular weight Mp represents the molecular weight corresponding to the highest peak retention time, and the peak start and end molecular weights represent the molecular weights corresponding to the peak start time and the peak end time, respectively. The weight average molecular weight Mw is obtained by means of statistical averaging of the molecular weights and represents a molecular weight having an equal weight distributed on both sides.
2.1 preparation of reagents
0.71% sodium sulfate: 14.20g of sodium sulfate is weighed into a 2L beaker, 2000ml of purified water is added for dissolution and dilution, and the mixture is subjected to ultrasonic degassing for about 10min through a 0.22um microporous filter membrane.
Preparing a test solution: taking 50mg to 10ml measuring flask of each sample, precisely weighing, adding a proper amount of the mobile phase obtained by filtering, carrying out ultrasonic treatment for 10min, fixing the volume to the scale, and shaking uniformly to obtain the sample solution. The sample solution was stored at 4 ℃.
Preparing a reference substance solution: 10mg of dextran D0, D1, D2, D3, D4, D5, D6, D7, D8, D2000 and dextran 410000 are weighed into 1.5ml of disposable centrifuge tubes respectively, precisely weighed, 1ml of 0.71% sodium sulfate solution is precisely moved into each centrifuge tube, and uniformly shaken to obtain the novel dextran.
2.2 chromatographic conditions
Chromatographic column: ZRD-LC-53TOSOH TSKgel guardcolumn PWXL (6 x 40mm,12 um); ZRD-LC-54 TOSOH TSKgel G3000PWXL (7.8 x 300mm,7 um); ZRD-LC-55TOSOH TSKgel G4000 PWXL (7.8 x 300mm,10 um); ZRD-LC-62TOSOH TSKgel G5000PWXL (7.8X300 mm,10 um).
Elution conditions: 0.71% sodium sulfate solution, and isocratic elution.
RID detector, column temperature 35 ℃, flow rate 0.8mL/min, sample injection amount 20ul.
And drawing a standard curve by taking the retention time as an abscissa and the corresponding molecular weight logarithmic value as an ordinate, and calculating the relative molecular weights of the samples Cs-4-P1, cs-4-P1-2 and Cs-4-P1-3 according to a regression equation. As a result, the weight average molecular weights Mw of Cs-4-P1, cs-4-P1-2, cs-4-P1-3 were distributed at 23.25-151.15kDa, as shown in FIG. 1 and Table 1.
TABLE 1
3. Monosaccharide composition determination
2mg of the sample was weighed into a reaction flask, 3mL of 2mol/L trifluoroacetic acid (TFA) was added, the mixture was heated in an oil bath at 110℃for 3 hours, cooled to room temperature, dried with nitrogen at 40℃and 3mL of methanol was added thereto, and the drying was repeated 4 to 5 times to completely remove TFA. Dissolving the sample in the reaction flask with ultrapure water, fixing the volume to 100mL, taking 12000g of partial solution, centrifuging for 20min, taking supernatant, detecting monosaccharide composition by using high performance anion chromatography (HPAEC), and comparing with a monosaccharide mixed standard. As a result of Table 2, it was found that the monosaccharides of Cs-4-P1-1, cs-4-P1-2 and Cs-4-P1-3 had the same composition and the same constituent unit.
TABLE 2
| Sample of | Glcp | Galp | Manp |
| Cs-4-P1-1 | 1.00 | 1.71 | 2.44 |
| Cs-4-P1-2 | 1.00 | 1.73 | 2.34 |
| Cs-4-P1-3 | 1.00 | 1.74 | 2.09 |
Structural resolution of example 3 Cs-4-P1-2
1. Polysaccharide methylation
After methylation, the infrared spectrum of the polysaccharide is measured, and the polysaccharide is measured at 3100-3600cm -1 The disappearance of the hydroxyl absorption peak between indicates that the polysaccharide has been completely methylated (FIG. 1). Subjecting the completely methylated sample to hydrolysis, reduction and acetylation, and performing GC-MS analysis, wherein the total ion flow diagram of methylation analysis of Cs-4-P1-2 polysaccharide is shown in figure 2, AThe results of the glycosylation analysis are shown in Table 3.
TABLE 3 Table 3
| Methylated sugar | Connection mode | Molar ratio of |
| 2,3,4,6-Me 4 -Glcp | Terminal Glcp | 1.77 |
| 2,3,4,6-Me 4 -Galp | Terminal Galp | 1.00 |
| 2,3,6-Me 3 -Galp | 1,4-Linked Galp | 2.04 |
| 2,3,4-Me 3 -Manp | 1,6-Linked Manp | 2.33 |
| 2,3-Me 2 -Manp | 1,4,6-Linked Manp | 2.96 |
2. Spectrum analysis
From the slave 1 As can be seen in H NMR (FIG. 3), the hetero-head hydrogen region had 5 resonance signal peaks, and a total of 9 sugar residue signal peaks were determined in combination with the hetero-head hydrogen peak area ratio, and appeared at δ4.92ppm (3H), δ4.89ppm (2H), δ4.58ppm, δ4.46ppm (2H), δ4.40ppm, respectively, indicating that sugar residues were β -configuration, and the numbers of the respective sugar residues were A, B, C, D, E according to the descending order of chemical shift of hetero-head hydrogen. At the position of 13 In the C NMR (FIG. 4) spectrum, the signal of the anomeric carbon signal region was δ102.1ppm to δ107.1ppm, further explaining that the glucose residue was in the β configuration.
To further elucidate the chemical structure of the polysaccharide, two-dimensional nuclear magnetic spectrum (COSY, TOCSY, HMQC, HMBC) was analyzed and assigned to the corresponding chemical shift of each sugar residue.
Sugar residue a: according to 1 The H NMR-determined chemical shift of hydrogen (H-1) at position 1 of sugar residue A revealed that the H-2 and H-3 signals were assigned to delta 3.53ppm and delta 3.72ppm respectively for sugar residue A by COSY spectra (FIG. 5), and the chemical shifts of H-4 (delta 3.92 ppm), H-5 (delta 3.65 ppm) and H-6 (delta 3.95ppm, delta 3.75 ppm) signals were assigned in combination with COSY and TOSCY spectra (FIG. 6). After the chemical shift of H is completed, the chemical shift of each C on the sugar ring can be attributed by HSQC spectrum (FIG. 7). According to
The chemical shifts of C and H in Table 4 can be determined that sugar residue A is → 4, 6) - β -D-Manp- (1 →.
TABLE 4 Table 4
According to the method, the chemical shift of the carbon (C) and hydrogen (H) signals in the sugar residue B, C, D, E is attributed in turn, and the total attribution result is shown in
Table 4. The connection mode of the sugar residue B, C, D, E is determined to be 4-beta-D-Galp- (1-beta-Galp), 1-beta-D-Glcp- (1-beta-Galp) 6-beta-D-Manp- (1-beta-Galp) by comparison.
After completion of assignment, the positions and the order of the linkage between the sugar residues were further determined by using HMBC spectroscopy (fig. 9). From the HMBC signals, the signals of H-1 at residue A and C-4 at residue B and C-6 at residue E are significantly correlated, the signals of H-1 at residue B and C-6 at residue A are correlated, the signals of H-1 at residue C and C-4 at residue B are correlated, the signals of H-1 at residue D and C-4 at residue A are correlated, the signals of H-1 at residue E and C-4 at residue A are correlated, and the remote correlation data for each sugar residue are shown in Table 5.
TABLE 5
| Sugar residues | Hydrogen protons | Associated |
| A:→4,6)-β-D-Manp-(1→ | H-1 | 80.4(B；C-4),68.9(E；C-6) |
| B:→4)-β-D-Galp-(1→ | H-1 | 68.7(A；C-6) |
| C:β-D-Galp-(1→ | H-1 | 80.4(B；C-4) |
| D:β-D-Glcp-(1→ | H-1 | 79.7(A；C-4) |
| E:→6)-β-D-Manp-(1→ | H-1 | 79.3(A:C-4) |
From the NOESY spectra (FIG. 10) and the HMBC spectra, it was then also seen that the H-1 of residue A and the H-4 of residue B and the H-6 of residue D have correlation points, the H-1 of residue B and the H-6 of residue A have correlation points, the H-1 of residue D and the H-4 of residue A have correlation points, the H-1 of residue E and the H-4 of residue A have correlation points, and NOE data are shown in Table 6.
TABLE 6
| Sugar residues | Hydrogen protons | NOE association |
| A:→4,6)-β-D-Manp-(1→ | H-1 | 4.02(B；H-4),3.87(E；H-6) |
| B:→4)-β-D-Galp-(1→ | H-1 | 3.75(A；H-6) |
| D:β-D-Glcp-(1→ | H-1 | 3.90(A；H-4) |
| E:→6)-β-D-Manp-(1→ | H-1 | 3.92(A；H-4) |
Based on the results of the mutual verification of HMBC and NOESY spectra, the connection mode between sugar residues can be judged, the connection of the 1-position of the residue a with the 4-position of the residue B and the 6-position of the E can be deduced, the 1-position of the residue B is connected with the 6-position of the residue a, the 1-position of the residue C is connected with the 4-position of the residue B, the 1-position of the residue D and the 1-position of the residue E are respectively connected with the 4-position of the residue a, the ratio of each sugar residue in the combined methylation result is a: B: C: D: e=3:2:1:2:1, and the repeating units of the polysaccharide component are confirmed according to the connection mode:
The polysaccharide is a heteropolysaccharide which takes beta- (1-4) -D-mannose, beta- (1-6) -D-mannose and beta- (1-4) -D-galactose as main chains and is respectively connected with beta-D-galactose and beta-D-glucose branched chains at 6-position and 4-position of mannose. The repeat fragment was 9-sugar, whereby the n-values were calculated from the weight average molecular weights (Mw) of Cs-4-P1-1, cs-4-P1-2, cs-4-P1-3 in Table 1 to be about: 104. 21, 16.
EXAMPLE 4 study of the Effect of adenine-induced chronic renal failure in rats
The test method comprises the following steps: 70 SD rats, male and female halves, were randomly grouped by body weight, 10 per group: normal control group, model group, cs-4-P1-2 group, fermented Cordyceps powder group, abelmoschus manihot capsule group, and dexamethasone group. Before the test, rats were placed in a metabolic cage and fasted for 24 hours (water intake as usual), urine volume was recorded for 24 hours, and urinary proteins and urinary creatinine were detected. After weighing the rats, 1mL of blood is taken from the orbit, the rats are placed in a 1.5mL centrifuge tube, and centrifuged at 3000 rpm for 10min, and urea nitrogen (BUN), creatinine (Scr) and uric acid are detected by taking the blood as basic values. The rats in the other groups, except the normal control group, were given 200mg/kg of adenine (dissolved in physiological saline) by lavage once daily for 4 consecutive weeks, and the administration was not performed only for molding for the last week. Blood was collected from the orbits of rats at 7, 14, 21, 28 and 35 days, respectively, and centrifuged at 3000 rpm for 10min, serum was taken to test urea nitrogen (BUN), creatinine (Scr) and uric acid values, urine volume was recorded for 24 hours, urinary protein and urinary creatinine were detected, and creatinine clearance was calculated. The rats were dissected at the end of the experiment to remove kidneys, and the sebum of the kidneys were separated on ice, weighed, and the organ index was calculated. Then 4% neutral formaldehyde solution is used for fixation, paraffin embedding, hematoxylin-eosin (HE) staining, optical examination and film reading.
Test results: 1) The weight of the animals was varied as follows
Table 7 shows the results.
TABLE 7
Note that: the number of rats 0d is 10 rats/group, and the rats are respectively male and female, compared with the model group * P≤0.05， ** P≤0.01， *** P≤0.001
From the following components
Table 7 shows that: test 7d, 11d, 14d compared with the model group, the body weight of the rats of the normal control group was significantly increased, the body weight of the rats of the 18d, 21d and 28d normal control groups was significantly increased, and the body weight of the rats of the 25d and 32d normal control groups was significantly increased (P.ltoreq.0.001); the dexamethasone group had a weight loss from 11d compared to the model group, with significant loss at 18d and 28d, suggesting a hormonal side effect; the Cs-4-P1-2 group, the Cs-4-P group and the fermented Cordyceps sinensis powder group have no obvious influence on the weight.
2) The kidney weights and organ coefficients of the rats are shown in Table 8 below.
TABLE 8
| Group of | Weight of 5w | 5w kidney weight | 5w organ coefficient |
| Normal control group | 333.55±88.76 *** | 2.40±0.74 *** | 0.72±0.09 *** |
| Model group | 229.44±62.94 | 9.24±3.37 | 3.98±0.90 |
| Dexamethasone (0.1 mg/kg/day) | 175.08±14.54 | 5.72±0.68 * | 3.29±0.51 |
| Abelmoschus manihot (1 g/kg/day) | 266.80±45.23 | 9.89±4.33 | 3.66±1.52 |
| Cs-4-P1-2 (16 mg/kg/day) | 230.99±52.21 | 6.99±3.43 | 2.94±0.92 * |
| Cs-4-P (448 mg/kg/day) | 261.16±57.00 | 8.69±2.69 | 3.35±0.92 |
| Fermented Cordyceps sinensis bacteria powder (1600 mg/kg/day) | 233.04±57.48 | 7.02±2.54 | 2.97±0.60 * |
Note that: the male and female rats were combined and counted, and compared with the model group, * P≤0.05, ** P≤0.01, *** P≤0.001
as can be seen from the above Table 8, the model group had a weight lower than that of the normal control group, and the kidney weight and kidney organ coefficient were both significantly different (P.ltoreq.0.001) from those of the normal control group; compared with the model group, the kidney weight of rats in the dexamethasone group is obviously reduced (P is less than or equal to 0.05), and the kidney weight of rats in the Cs-4-P1-2, cs-4-P and fermented Cordyceps sinensis bacterial powder group has a reduced tendency; compared with the model group, the kidney coefficients of the normal control group, the Cs-4-P1-2 and the fermented cordyceps sinensis bacterial powder group are obviously reduced (P is less than or equal to 0.05), and the Cs-4-P group has a descending trend.
3) The changes in serum BUN of rats during the experiment are shown in table 9 below.
TABLE 9
Note that: and (5) merging and counting female and male rats. In comparison with the set of models, * P≤0.05, ** P≤0.01, *** P≤0.001
urea Nitrogen (BUN) in serum is an important indicator of the clinical response to glomerular filtration, and gastric lavage adenine can lead to elevated serum BUN in rats. As can be seen from Table 9, compared with the model group, the BUN level of the normal control group for molding 1 week is 5.22+ -1.16 mmol/L, the BUN level of the molding group is between 13.99+ -2.95 mmol/L and 17.00+ -6.31 mmol/L, and the two groups have very significant difference (P is less than or equal to 0.001); experiment 2W is that the BUN level of the okra group and the Cs-4-P1 group is obviously reduced (P is less than or equal to 0.05) after one week of administration; experiment 4W is that the administration is performed for three weeks, and BUN levels of the okra group, the Cs-4-P1-2 and the fermented cordyceps sinensis powder group are obviously reduced (P is less than or equal to 0.05); experiment 5W is that the BUN level of dexamethasone group and Cs-4-P1-2 group is obviously reduced (P is less than or equal to 0.05) after four weeks of administration; during the administration period, the BUN level of the Cs-4-P group and the fermented Cordyceps powder group is decreased. Therefore, the Cs-4-P1-2 has the effect of improving the serum BUN of the rat with chronic renal failure and has obvious treatment effect on the chronic renal failure.
4) The changes in serum CREA during the experiment are shown in table 10 below.
Table 10
Note that: and (5) merging and counting female and male rats. In comparison with the set of models, * P≤0.05, ** P≤0.01, *** P≤0.001
Serum Creatinine (CREA) is an important indicator of kidney function, elevated CREA means impairment of kidney function. As can be seen from Table 10, compared with the model group, the CREA level of the normal control group for molding 1 week was 27.20.+ -. 5.73. Mu. Mol/L, the model group was between 58.28.+ -. 13.40. Mu. Mol/L and 65.86.+ -. 11.13. Mu. Mol/L, and there was a very significant difference (P.ltoreq.0.001) between the two groups; experiment 2W is that the administration is carried out for one week, the CREA level of a dexamethasone group is obviously reduced (P is less than or equal to 0.001), and the CREA level of a okra group and a Cs-4-P1-2 group is obviously reduced (P is less than or equal to 0.05); experiment 3W is that dexamethasone groups are administered for two weeks, CREA level is obviously reduced (P is less than or equal to 0.01), and other groups have a reduction trend; experiment 4W was administered for three weeks, and there was a trend in decreasing CREA levels in each group; four weeks after the administration of experiment 5W, the levels of CREA were very significantly reduced in the dexamethasone group, and the remaining groups had a tendency to decrease. Therefore, the Cs-4-P1-2 has the effect of improving the serum CREA of the rats with chronic renal failure and has obvious treatment effect on the chronic renal failure.
5) The changes in UA during the experiment are shown in Table 11 below
TABLE 11
Note that: and (5) merging and counting female and male rats. In comparison with the set of models, * P≤0.05, ** P≤0.01, *** P≤0.001
as can be seen from Table 11, compared with the model group, the UA level of the normal control group for molding 1 week was 91.00.+ -. 12.51. Mu. Mol/L, the model group was between 113.75.+ -. 24.84. Mu. Mol/L and 133.29.+ -. 18.50. Mu. Mol/L, and there was a very significant difference (P.ltoreq.0.001) between the two groups; the normal control group is obviously lower, the UA level of the dexamethasone group is obviously reduced (P is less than or equal to 0.001), the UA level of the okra group and the Cs-4-P group is obviously reduced (P is less than or equal to 0.05), and the UA level of the Cs-4-P1-2 and the UA level of the fermented cordyceps sinensis powder group are obviously reduced (P is less than or equal to 0.01); experiment 3W is that the administration is carried out for two weeks, the UA level of the dexamethasone group is obviously reduced, and the rest groups have a reducing trend; the UA level of the fermented cordyceps sinensis powder group is obviously trend after the experiment is carried out for three weeks at 4W, and the rest groups have a reduction trend; experiment 5W is four weeks after administration, the UA level of the dexamethasone group is obviously increased, the Cs-4-P1-2 is obviously increased, and the other groups have an increasing trend.
6) Kidney pathology.
Adenine-induced chronic renal failure can cause severe renal tissue lesions, mimicking the end-stage state of renal failure. The results were seen by HE staining the kidneys for pathological sections, and observing them under a microscope. The normal control group rat has normal kidney tissue structure, the glomerular vascular loop is thin and clear, the endothelial cells and the mesangial cells have normal numbers, and the peripheral renal tubules are normal. Compared with the normal control group, the kidney tissue of the model group is found to have glomerular fibrosis, tubular atrophy or expansion, foreign matter filling in the glomerulus of the tubular, massive fibrous tissue proliferation in the interstitium and inflammatory cell infiltration. Compared with the model group, the residual glomerulus unit of the Cs-4-P1-2 administration group is increased, the pathology score is obviously reduced (P is less than or equal to 0.01), and the pathology scores of the okra and the fermented cordyceps fungus powder group are obviously reduced (P is less than or equal to 0.05). The result shows that the Cs-4-P1-2 and the fermented cordyceps fungus powder have the effects of improving and recovering the kidney pathological changes caused by long-term adenine gastric lavage, wherein the Cs-4-P1-2 is optimal.
Table 12
| Group of | Pathology scoring |
| Normal control group | 0.00±0.00 *** |
| Model group | 9.00±0.00 |
| Dexamethasone (0.1 mg/kg) | 8.60±0.55 |
| Abelmoschus manihot (1 g/kg/day) | 8.43±0.45 * |
| Cs-4-P1-2 (16 mg/kg/day) | 8.31±0.37 ** |
| Cs-4-P | 8.58±0.49 |
| Fermented Cordyceps sinensis bacteria powder group (1600 mg/kg/day) | 8.31±0.88 * |
Injection comparison with model set * P≤0.05， ** P≤0.01， *** P≤0.001
The effect of Cs-4-P1 on the kidney was judged from the remaining glomeruli and nephron. As can be seen from Table 12, compared with the model group, the pathological score of the normal control group is 0, the pathological scores of rats in the okra group and the fermented Cordyceps sinensis powder group are reduced, the Cs-4-P group has a reduction trend, and the pathological scores of rats in the Cs-4-P1-2 group are obviously reduced.
7) The mortality of the rats at the end of the test is shown in table 13 below.
TABLE 13
| Group of | Original number of | Survival count | Mortality (%) |
| Normal control group | 10 | 10 | 0.00 |
| Model group | 10 | 7 | 30.00 |
| Dexamethasone (0.1 mg/kg) | 10 | 5 | 50.00 |
| Abelmoschus manihot (1 g/kg/day) | 10 | 7 | 30.00 |
| Cs-4-P1-2 (16 mg/kg/day) | 10 | 8 | 20.00 |
| Cs-4-P | 10 | 6 | 30.00 |
| Fermented Cordyceps sinensis bacteria powder group (1600 mg/kg/day) | 10 | 8 | 20.00 |
As can be seen from Table 13, the model groups all had deaths during the test, with the dexamethasone group mortality rate being up to 50% and the analysis being associated with hormonal side effects. The mortality rate of the Cs-4-P1-2 group and the fermented cordyceps sinensis powder group is 20 percent, and is reduced compared with that of a model group.
In conclusion, under the test condition, the effect study of the fermented cordyceps fungus powder component on adenine-induced chronic renal failure is carried out from several aspects of observation, weight, serum biochemical indexes, pathological total and the like during the test period of rats, and test results show that the model is successfully constructed, and compared with the model, the detection of the Cs-4-P1-2 serum biochemical indexes is relatively effective, the death rate is reduced, and the effect of adenine-induced chronic renal failure can be reduced. And the effect of the Cs-4-P1-2 on adenine-induced chronic renal failure is obviously better than that of dexamethasone, okra, fermented cordyceps fungus powder and crude polysaccharide (Cs-4-P) thereof.
EXAMPLE 5 pharmacodynamic studies of the inhibition of immune rejection
The test method comprises the following steps:
(1) Construction of skin graft model
According to reference (Cai Chunxiao, ma Chunmei, et al) establishment of a xenograft model in mice and evaluation of the efficacy of immunosuppressive drugs Chinese pharmacological report 2016, 32 (11): 1613-1619.), BALB/C mice and C57/BL6 mice were anesthetized with sodium pentobarbital (40 mg/kg) and the back skin of donor mice and recipient mice were sterilized with 70% alcohol. The ears of the donor mice were removed from the roots and placed in ice-cold sterile PBS for later use. The skin on one side of the back of the recipient mouse is cut off (the diameter is about 1 cm), the inner skin and the outer skin of the ear of the donor mouse are separated, the inner skin is taken out, the separation surface is downward, the skin is attached to the back of the recipient mouse, the skin which is not completely anastomosed is removed and repaired by scissors, then the skin is wrapped by a sterile wound plaster, and the wound plaster is sewn and fixed by silk threads. After 2 days, the adhesive bandage was gently cut off to expose the graft site, and the graft rejection was evaluated. Xenograft (Allograft) in this experiment C57/BL6 mice were skin-transplanted to BALB/C mice and allogenic skin-transplanted (isoraft) BALB/C mice were skin-transplanted to BALB/C mice.
(2) Graft rejection score (Graft Rejection Score)
According to literature, rejection of transplanted skin was classified on a scale of 0-5 (as shown in Table 14).
TABLE 14
| Level of | Graft rejection reaction |
| 0 | Intact skin, no rejection reaction |
| 1 | Just rejection phenomenon occurs |
| 2 | >Skin necrosis of 25% |
| 3 | >50% skin necrosis |
| 4 | >75% of skin necrosis |
| 5 | Complete necrosis of skin>95％) |
Note that: graft rejection is expressed as a percentage of necrotic skin over the total skin, and skin damage due to suturing, suturing material, skin displacement, and other trauma is not counted.
When the skin graft rejection level reaches level 5, it can be defined as the death of the transplanted skin.
(3) Dose design and basis
The clinical dose of cyclosporin used as immunosuppressant for human is 15 mg/kg/day, the mouse dose is 150mg/kg, the human administration dose of the fermented Cordyceps sinensis powder is 3 g-6 g/day, the fermented Cordyceps sinensis powder dose is 3000 mg/kg/day of the mouse, and the Cs-4-P1-2 dose is 30 mg/kg/day of the mouse according to the yield.
TABLE 15
* P <0.01vs model P <0.05vs model # # P <0.01vs blank #p <0.05vs blank
Scoring the skin from day four
Table 15 shows that the blank (allograft) group had a lower score than the model group after transplantation, and that there was a very significant difference (P < 0.01) between the model group after 14 days; the scores of the cyclosporine group were consistently lower than the model group for 6-13 days, with very significant differences (P < 0.01), large areas of focal spot starting from transplanted skin after fourteenth day, and no differences in model; the scores of the fermented cordyceps fungus powder groups are always lower than those of the model groups within 6-12 days, the scores are extremely obviously different, the transplanted skin starts to be burnt up in a large area after thirteenth day, and the models are not different; the Cs-4-P1-2 group was lower than the model group within 10-13 days, with very significant differences (P < 0.01), large areas of focal spot started in the transplanted skin after the fourteenth day, and no differences in the model. The result shows that cyclosporin, cs-4-P1-2 and fermented Cordyceps powder have obvious inhibition effect on the immune rejection reaction of mice skin transplantation, wherein Cs-4-P1-2 has dosage advantage.
2) Comparison of the weight of each group
Table 16
* P <0.01vs model P <0.05vs model # # P <0.01vs blank #p <0.05vs blank
As can be seen from table 16, there was always no difference in body weight between the blank (allograft) group, cs-4-P1-2 and the model group after transplantation; mice in the cyclosporine group showed a slower weight gain after transplantation administration, consistently lower than the model group, with a very significant difference (P < 0.01); the weight of mice in the fermented Cordyceps powder group is lower than that in the model group within 7-12 days, and the weight is obviously different. The side effect of Cs-4-P1-2 is small compared with cyclosporine, and the safety is high.
EXAMPLE 6 Studies of the effects of hyperlipemic zebra fish
The test method comprises the following steps:
(1) Experimental animal
Zebra fish are all cultivated in water for fish cultivation at 28 deg.C (water quality: 200mg instant sea salt is added into 1L reverse osmosis water, conductivity is 450-550 mu S/cm, pH is 6.5-8.5, hardness is 50-100 mg/L CaCO) 3 ) The experimental animal use license number is provided by the breeding of the fish culture center of the company: SYXK (Zhe) 2012-0171, the feeding management meets the requirements of International AAALAC authentication (authentication number: 001458).
The melanin allele mutant semitransparent Albino strain zebra fish is carried out in a natural pairing mating propagation mode. Zebra fish aged 7 days after fertilization (7 dpf) were used for maximum detection concentration (MTC) determination of hypolipidemic efficacy.
The melanin allele mutant semitransparent Albino strain zebra fish is carried out in a natural pairing mating propagation mode. Zebra fish with the age of 5dpf is used for evaluating the efficacy of reducing blood fat.
(2) Detection method
MTC assay
7dpf melanin allele mutant translucent Albino strain zebra fish were randomly selected in 6-well plates, and 30 zebra fish were treated per well (experimental group). Samples were given water-soluble (concentrations are shown in Table 17) respectively, while normal controls were set at a capacity of 3mL per well. After 48h of treatment at 28 ℃, the samples were assayed for MTC from normal zebra fish.
b. Evaluation of hypolipidemic efficacy
Randomly selecting 5dpf melanin allele mutant semitransparent Albino strain zebra fish, and water-soluble administration of egg yolk powder to establish a zebra fish hyperlipidemia model. Zebra fish were randomly distributed into 6-well plates 16h after feeding, and 30 zebra fish were treated per well. Samples were given water-soluble (concentrations shown in tables 17-18) respectively, and the positive control atorvastatin calcium at a concentration of 0.240 μg/mL, while setting the normal control group and the model control group at a capacity of 3mL per well. After 48 hours of treatment at 28 ℃, the zebra fish homogenate is taken to obtain supernatant, triglyceride and total cholesterol reagent kit are used for reaction, the OD value of the triglyceride and the total cholesterol of each experimental group is respectively measured by a multifunctional enzyme-labeling instrument, the triglyceride content (C1) and the total cholesterol content (C2) are analyzed, and the blood lipid reducing efficacy of the sample is evaluated according to the statistical analysis results of the indexes. Statistical treatment results are expressed in mean+ -SE. The blood lipid lowering efficacy calculation formula is as follows:
1) Triglyceride lowering efficacy:
2) Total cholesterol lowering effect:
statistical analysis was performed with SPSS 26.0 software, p <0.05 indicated that the differences were statistically significant.
Detection result
Under the experimental conditions, the MTC (minimum poisoning concentration) of the fermented cordyceps sinensis bacterial powder and the Cs-4-P1-2 on the normal zebra fish is respectively 300 and 10.0 mug/mL. See Table 17 for details.
TABLE 17
Evaluation of hypolipidemic efficacy
Under the experimental condition, the fermented cordyceps sinensis bacterial powder and Cs-4-P1-2 have the effect of reducing triglyceride; cs-4-P1-2 has the effect of reducing total cholesterol, and is shown in Table 18.
TABLE 18
P <0.05, p <0.01, p <0.001 compared to model control group
TABLE 19
P <0.05, p <0.01, p <0.001 compared to model control group
As is clear from Table 19 above, the concentration of Cs-4-P1-2 was found to be excellent in triglyceride lowering effect at 1.0-10.0. Mu.g/mL, and excellent in triglyceride lowering effect at 3.00. Mu.g/mL, with triglyceride lowering activity superior to atorvastatin calcium.
Table 20
Compared with the model control group, p <0.05
Table 21
P <0.05, < p <0.01 compared to model control group
As shown in Table 21, the concentration of Cs-4-P1-2 is 1.0-10.0 mug/mL, which has better total cholesterol effect, and the total cholesterol reducing effect is 1.00-3.00 mug/mL, which is obviously better than the fermented Cordyceps powder and atorvastatin calcium.
EXAMPLE 7 study of the Effect of cisplatin on acute kidney injury mice
The test method comprises the following steps:
mice were fasted for 12h preoperatively and were free to drink water. Except for the control group, the other groups of abdominal cavities are respectively injected with cisplatin solution with single injection dosage of 10mg/kg, and the control group is injected with normal saline with equal volume. After 15min, amifostine, cs-4-P1 and Cs-4-P1-2 were administered separately in groups. Blood was collected on days 1,3 and 5 after administration, and serum urea nitrogen (BUN) and creatinine (Scr) were measured.
Test results: the results are shown in table 22, where the control group serum urea nitrogen and creatinine tended to plateau on days 1,3 and 5, the model group serum urea nitrogen and creatinine began to rise at day 3 and remained high on day 5, and the model group serum urea nitrogen and creatinine on days 3 and 5 both exhibited statistical differences (p <0.05, p < 0.01) compared to the control group. The serum urea nitrogen and creatinine content of mice is obviously reduced on days 3 and 5 after the administration of the positive drug amifostine 300mg/kg group, and compared with a model group, the positive drug amifostine shows statistical differences (p <0.05, p < 0.01); 3 and 5 days after administration of the test substance Cs-4-P1 (160 mg/kg), the serum urea nitrogen and creatinine content of the mice is obviously reduced, and compared with the model group, the statistical difference is shown (P <0.05, P < 0.01); after administration of Cs-4-P1-2 (160 mg/kg and 80 mg/kg), respectively, serum urea nitrogen and creatinine levels were significantly reduced on days 3 and 5, and showed statistical differences (P <0.05, P < 0.01) compared to the model group. As can be seen, the improvement effect of Cs-4-P1-2 on serum urea nitrogen and creatinine at a dose of 160mg/kg is consistent with the positive drug amifostine (300 mg/kg). It can be seen that Cs-4-P1-2 has therapeutic and ameliorating effects on acute kidney injury in mice.
Table 22
In comparison with the control group, ## p<0.01; comparison with model group, p<0.05,**p<0.01
EXAMPLE 8 investigation of the Effect of LPS-induced acute kidney injury in mice
The test method comprises the following steps:
mice were fasted for 12h preoperatively and were free to drink water. Except for the control group, the other groups were injected with 20mg/kg LPS solution in single abdominal cavity, and the control group was injected with an equal volume of physiological saline. Dexamethasone and Cs-4-P1-2 were administered separately in groups after 15 min. After 36 hours the animals were sacrificed and blood was taken to measure serum creatinine and urea nitrogen.
Test results:
the results are shown in Table 23, and the serum urea nitrogen and creatinine levels of the model mice are 17.69.+ -. 1.13mmol/L and 45.35.+ -. 5.85. Mu. Mol/L, respectively, and exhibit statistical differences (p < 0.01) compared to the control. The positive drug dexamethasone is respectively given at the dosage of 10mg/kg, the serum urea nitrogen and creatinine levels of mice in the positive drug group are respectively 12.29+/-4.42 mmol/L and 24.03+/-13.81 mu mol/L, and the mice show significant differences (p < 0.05) compared with the mice in the model group; serum urea nitrogen levels of Cs-4-P1-2 low, medium and high dose mice were reduced to 14.41±1.73, 13.20±1.72 and 12.72±0.55mmol/L, respectively; serum creatinine levels decreased to 35.15 + -26.22, 26.34+ -2.75, 24.33+ -9.65 μmol/L, respectively, and both serum urea nitrogen and creatinine showed statistical differences in the Cs-4-P1-2 neutralized high dose group compared to the model group (P <0.05, P <0.01, P < 0.001). It can be seen that the effect of the Cs-4-P1-2 high dose group on serum urea nitrogen and creatinine of mice with acute kidney injury induced by LPS is equivalent to that of positive drug dexamethasone. It can be seen that Cs-4-P1-2 has therapeutic and ameliorating effects on acute kidney injury in mice.
Table 23
FIG. 8 is a diagram of an embodiment according to the invention 1 H- 13 C HSQC spectrum. Fig. 11 is a view of a pathological section according to an embodiment of the present invention.
Claims (11)
- The Cs-4 fermentation mycelium heteropolysaccharide is characterized by having the following structural formula:wherein Manp is mannopyranose, galp is galactopyranose, glcp is glucopyranose, and n is selected from 6-104.
- The Cs-4 fermentation mycelium heteropolysaccharide of claim 1, wherein the relative molecular weight of the polysaccharide is selected from the group consisting of-10-152 kDa.
- The Cs-4 fermentation mycelium heteropolysaccharide of claim 1 or 2, wherein the polysaccharide composition comprises: glucose, galactose and mannose in a molar ratio of 1: (1.0-2.0): (1.5-2.5).
- A process for extracting the heteropolysaccharide from Cs-4 fermented mycelia according to any one of claim 1 to 3,1) Degreasing Cs-4 bacteria powder with ethanol, discarding ethanol extract to obtain degreasing residue,2) Extracting the residue in water to obtain water extract,3) Carrying out alcohol precipitation treatment on the water extract, wherein the solvent for the alcohol precipitation treatment is ethanol,4) Purifying the precipitate obtained after the alcohol precipitation treatment in the step 3) to obtain the fermentation mycelium heteropolysaccharide.
- The method according to claim 4, wherein the degreasing treatment is performed at a temperature of 100 ℃;optionally, the degreasing treatment is performed three times, each time for 1 hour;optionally, the ethanol is 10 times the amount of Cs-4 in the degreasing treatment;optionally, the degreasing ethanol is 85% -100% ethanol;optionally, the extraction treatment is carried out at a temperature of 75 ℃ to 78 ℃;optionally, the extraction treatment is performed three times, each time for 1 hour;optionally, the extraction treatment is performed with 10 times the amount of water as the residue;optionally, the ethanol treated by the ethanol precipitation is 80% ethanol.
- A process for extracting the heteropolysaccharide from Cs-4 fermented mycelia according to any one of claim 1 to 3,1) Extracting Cs-4 with 10 times of 85% ethanol under heating for 3 times each for 1 hr, and removing ethanol extractive solution to obtain residue;2) Extracting the residue with 10 times of water under heating for 3 times each for 1 hr, and mixing the water extracts;3) Precipitating the water extract with 80% ethanol to obtain precipitate;4) And (3) purifying the precipitate to obtain the fermentation mycelium heteropolysaccharide.
- The method according to any one of claims 4 to 6, wherein the purification treatment comprises deproteinization treatment, decolorization treatment, column purification treatment.
- A pharmaceutical composition comprising the Cs-4 fermented mycelium heteropolysaccharide according to any one of claims 1 to 3 or obtained according to the method of any one of claims 4 to 7.
- Use of a Cs-4 fermented mycelium heteropolysaccharide according to any one of claims 1-3 or obtained according to the method of any one of claims 4-7 or a pharmaceutical composition according to claim 8 for the manufacture of a medicament for the prevention or treatment of chronic renal failure, for the inhibition of immune rejection, for the prevention or treatment of hyperlipidemia and/or for the prevention or treatment of acute renal injury.
- A pharmaceutical composition for preventing or treating hyperlipidemia, characterized by comprising 1.0-10.0 μg/mL of Cs-4 fermented mycelium heteropolysaccharide according to any one of claims 1-3 or Cs-4 fermented mycelium heteropolysaccharide obtained according to the method of any one of claims 4-7.
- The pharmaceutical composition of claim 10, wherein the polysaccharide is at a concentration of 3.0 μg/mL.
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